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WO2025212789A1 - Methods for predicting genomic antibiotic susceptibility using antibiotic-induced mutational signatures - Google Patents

Methods for predicting genomic antibiotic susceptibility using antibiotic-induced mutational signatures

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Publication number
WO2025212789A1
WO2025212789A1 PCT/US2025/022769 US2025022769W WO2025212789A1 WO 2025212789 A1 WO2025212789 A1 WO 2025212789A1 US 2025022769 W US2025022769 W US 2025022769W WO 2025212789 A1 WO2025212789 A1 WO 2025212789A1
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WIPO (PCT)
Prior art keywords
pseudomonas
contexts
nucleic acid
acid sample
antibiotic
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PCT/US2025/022769
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French (fr)
Inventor
Kalen HALL
Leonard Williams
Zachary Pursell
Lisa MORICI
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Tulane University
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Tulane University
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Publication of WO2025212789A1 publication Critical patent/WO2025212789A1/en
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Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present technology relates to methods for predicting drug resistance in a subject diagnosed with or suffering from a Pseudomonas infection based on the detection of antibiotic- induced mutational signatures of Pseudomonas species.
  • the present disclosure provides a method for predicting the risk of antibiotic resistance in a subject diagnosed with or suffering from a Pseudomonas infection comprising sequencing Pseudomonas nucleic acids isolated from a biological sample obtained from the subject; generating a mutation spectrum from the sequenced Pseudomonas nucleic acids; and determining that the subject is at risk for antibiotic resistance when a Pseudomonas antibiotic-induced mutational signature is detected in the mutation spectrum.
  • the present disclosure provides a method for selecting a subject infected with a Pseudomonas species for treatment with an antibiotic therapy comprising sequencing Pseudomonas nucleic acids isolated from a biological sample obtained from the subject; generating a mutation spectrum of the Pseudomonas nucleic acid sequences; detecting a Pseudomonas antibiotic-induced mutational signature in the mutation spectrum; and selecting an antibiotic therapy based on the detected Pseudomonas antibiotic-induced mutational signature.
  • generating the mutation spectrum comprises generating a plurality of single base substitution (SBS) contexts and a plurality of insertion/deletion (indel) profiles from the sequenced Pseudomonas nucleic acids.
  • each indel profile of the plurality of indel profiles comprise an indel size, a number of sequence unit repeats at an indel location, and the presence or absence of flanking microhomology sequences at an indel location.
  • the plurality of SBS contexts comprise a plurality of single base changes in a trinucleotide context, wherein the plurality of single base changes in the trinucleotide context comprises one or more transitions/substitutions selected from the group consisting of OA, OG, C>T, T>A, T>C, T>G, G>T, G>C, G>A, A>T, A>G, and A>C.
  • the Pseudomonas antibiotic-induced mutational signature comprises one or more of: an increase in OT transitions in NCG and GCN contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>A transitions in CGN and NGC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in T>C transitions in ATC, ATG, CTC, and CTG contexts relative to a reference Pseudomonas nucleic acid sample, an increase in A>G transitions in GAT, CAT, GAG, and CAG contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertions in non-repeat regions of the sequenced Pseudomonas nucleic acids relative to a reference Pseudomonas nucleic acid sample.
  • Mutant frequencies of all subject isolates were measured using rifampicin reversion (rpoB mutants resistant to rifampicin per 10 8 viable cells). Hypermutators are defined as those having mutant frequencies above the dotted line, which represents 20-fold higher than the mutant frequency of the WT MPAO1 parent strain.
  • Clinical isolates predicted based on mutation spectra to be MMR-deficient and hypermutant are shown in dark grey. Clinical isolates predicted to be WT are shown in light grey. Lab strains are shown in white. For each isolate, the presence (+) or absence (-) of nonsynonymous mutations in the indicated key MMR genes are denoted below.
  • FIGs. 5A-5B Predicted MMR-deficient isolates acquire drug resistance in vitro.
  • FIGS. 5A-5B Representative resistance acquisition shown for subject isolates. Predicted MMR- deficient subject isolates rapidly acquired resistance to (FIG. 5A) aztreonam (AZ) and (FIG. 5B) colistin (COL), while WT isolates did not.
  • FIGs. 6A-6F Mutational signature predicts MMR-deficient isolates and is correlated with MDR in a large independent pwCF cohort.
  • FIG. 6A Representative trinucleotide mutation spectra of isolates predicted as MMR-deficient from pwCF.
  • FIG. 6B Clustering of trinucleotide mutation spectra based on cosine similarity from patient isolates using PaA/iw/A', HumanAMMR, and each individual COSMIC SBS associated with MMR-deficiency.
  • FIG. 6C Percentage of predicted MMR-deficient and WT isolates containing nonsynonymous mutations in MMR genes.
  • FIG. 6D Comparison of the mutator status for each isolate via rifampicin reversion reported by Lopez-Causape et al. (Normal vs Hypermutator) to the MMR status predicted by mutational signature (MMR-deficient vs WT) using the same dataset.
  • FIGs. 7A-7G Mutational signature analysis reveals occurrence of MMR- deficiency in several acute disease contexts.
  • FIG. 7A Trinucleotide mutation spectra of those patient isolates predicted as MMR-deficient from three different patient cohorts: cystic fibrosis (CF); respiratory tract infection (RTI); urinary tract infection (UTI); and intra-abdominal infection (IAI).
  • FIG. 7B Trinucleotide mutation spectra from patient isolates were clustered based on cosine similarity to Pa mutS, Human AMMR, and each individual COSMIC SBS associated with MMR-deficiency.
  • FIGs. 8A-8B 10 trinucleotide mutation spectra in VAP isolates resemble MMR- deficiency.
  • FIGs. 8A, 8B Trinucleotide mutation spectra of those patient isolates predicted as (FIG. 8A) MMR-deficient or (FIG. 8B) right below threshold.
  • FIG. 9 Predicted MMR-deficient isolates cluster with Pa nmtS. Trinucleotide mutation spectra from patient isolates were clustered based on cosine similarity to PaXmulS, Human AMMR, and each individual COSMIC SBS associated with MMR-deficiency. Red stars represent clusters of isolates with cosine similarity to PaXmulS above 0.78, whereas orange stars represent those with cosine similarity between 0.75 and 0.77.
  • FIGs. 11A-11B Aztreonam- and ciprofloxacin-treatment induces mutagenesis in P. aeruginosa.
  • FIG. 11 A Viable cells recovered on nonselective CAMHB agar in untreated (WT), ciprofloxacin-treated (+CIPRO), or aztreonam-treated (+AZ) groups.
  • FIG. 13 Clinical isolates of P. aeruginosa show enrichment in aztreonam- and ciprofloxacin-induced signatures. Red stars represent clusters of predicted MMR-deficient isolates. Orange star represents cluster with SBS6 and antibiotic-induced signature enrichment.
  • FIG. 15 Indel mutation spectra of all sample collapsed by treatment group.
  • Treatment groups are clustered and annotated on figure by drug class, structural similarities, or mechanism of action similarities to illustrate differences in mutation spectra despite these characteristics.
  • FIG. 16 Classification of small deletions and insertions for mutational signature analysis as represented by exemplary SEQ ID NOs: 19-109.
  • the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.
  • nucleic acid amplification refers to methods that increase the representation of a population of nucleic acid sequences in a sample. Nucleic acid amplification methods, such as PCR, isothermal methods, rolling circle methods, etc., are well known to the skilled artisan. Copies of a particular nucleic acid sequence generated in vitro in an amplification reaction are called “amplicons” or “amplification products”.
  • complementarity refers to the base-pairing rules.
  • control is an alternative sample used in an experiment for comparison purpose.
  • a control can be "positive” or “negative.”
  • the reference or control nucleic acid sample is a wild type or a non-mutated DNA or RNA sequence.
  • the reference nucleic acid sample is purified or isolated (e.g., it is removed from its natural state).
  • sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al., Nat. Biotechnol, 16:381-384 (1998)), and sequencing by hybridization.
  • MALDI-TOF/MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein.
  • a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated.
  • a therapeutically effective amount can be given in one or more administrations.
  • the terms “individual”, “patient”, or “subject” are used interchangeably and refer to an individual organism, a vertebrate, a mammal, or a human. In a preferred embodiment, the individual, patient or subject is a human.
  • a mutation can have an altered nucleotide sequence (e.g., a mutation), amino acid sequence, expression level, protein level, protein activity, in an infected tissue or infected cell, as compared to a normal, healthy tissue or cell.
  • exemplary mutations include, but are not limited to, point mutations (e.g., silent, missense, or nonsense), deletions, insertions, inversions, linking mutations, duplications, translocations, inter- and intra-chromosomal rearrangements. Mutations can be present in the coding or non-coding region of the gene.
  • the mutations are associated with a phenotype, e.g., an antibiotic-resistant or antibiotic-susceptibility phenotype.
  • microhomology is defined as one or more base pairs (bp) of perfectly matching sequence shared between the proximal and distal reference sequences surrounding a breakpoint (e.g., an indel location).
  • next-generation sequencing or NGS refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a high throughput parallel fashion (e.g., greater than 10 3 , 10 4 , 10 5 or more molecules are sequenced simultaneously).
  • the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment.
  • Next generation sequencing methods are known in the art, and are described, e.g. in Metzker, M. Nature Biotechnology Reviews 11 :31-46 (2010).
  • oligonucleotide refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals.
  • the nucleic acid bases are arranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide.
  • the most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position.
  • Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group.
  • Oligonucleotides of the method which function as primers or probes are generally at least about 10-15 nucleotides long and more preferably at least about 15 to 25 nucleotides long, although shorter or longer oligonucleotides may be used in the method. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide.
  • polynucleotide or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • a single repeating sequence unit [X] may comprise from about 1 to about 8 individual nucleotides. Additionally or alternatively, in some embodiments, the individual nucleotides present in a single repeating sequence unit [X] may be the same or distinct.
  • a “non-repeat region” refers to a region that lacks a repeating sequence unit in the context of a directly adjacent Pseudomonas nucleic acid sequence. [0071] As used herein, the term “sample” refers to clinical samples obtained from a patient or isolated microorganisms.
  • Exemplary sensitivities include at least 50, 60, 70, 80, 90, 95, 98, and 99%.
  • the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
  • sequential therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
  • a method has a specificity of 90% if, when applied to a sample set of 1,000 sequences, in which 500 sequences are truly variant and 500 are not truly variant, the method selects 90% of the 500 not truly variant sequences as not variant.
  • Exemplary specificities include at least 50, 60, 70, 80, 90, 95, 98, and 99%.
  • Treating” or “treatment” as used herein covers the treatment of a disease or condition described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or condition, i.e., arresting its development; (ii) relieving a disease or condition, i.e., causing regression of the condition; (iii) slowing progression of the condition; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or condition.
  • Therapeutic effects of treatment include, without limitation, inhibiting recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • Quinolones are a class of antibiotics that all share a 4-quinolone bactericidal core structure. Fluoroquinolones are a sub-class of quinolone antibiotics that are known to interfere with bacterial DNA replication. Fluoroquinolones further incorporate fluorine into the structure, and are broad spectrum antibiotics. Quinolones include, but are not limited to, Enrofloxacin (Baytril), Ciprofloxacin (i.e., Cipro and Proquin), Enoxacin (i.e., Penetrex), Gatifloxacin (i.e., Gatiflo, Tequin and Zymar), Gemifloxacin (i.e,.
  • the methods of the present technology comprise selecting for administration or administering a fluroquinolone. In some embodiments, the methods of the present technology comprise selecting for administration or administering one or more of Enrofloxacin (Baytril), Ciprofloxacin (i.e., Cipro and Proquin), Enoxacin (i.e., Penetrex), Gatifloxacin (i.e., Gatiflo, Tequin and Zymar), Gemifloxacin (i.e,.
  • Enrofloxacin Boytril
  • Ciprofloxacin i.e., Cipro and Proquin
  • Enoxacin i.e., Penetrex
  • Gatifloxacin i.e., Gatiflo, Tequin and Zymar
  • Gemifloxacin i.e,.
  • Levofloxacin i.e., Levaquin
  • Lomefloxacin i.e., Maxaquin
  • Moxifloxacin i.e., Avelox
  • Norfloxacin i.e., Noroxin
  • Ofloxacin i.e., Floxin
  • Prulifloxacin Sparfloxacin (i.e., Zagam)
  • Trovafloxacin/Altrofloxacin i.e., Trovan
  • Danofloxacin i.e., A180
  • Difloxacin i.e., Dicural
  • Marbofloxacin i.e., Orbax
  • Orbifloxacin i.e., Zeniquin
  • Cinoxacin i.e., Cinobac
  • Rosoxacin Fleroxacin
  • Pefloxacin Rosoxacin
  • Rosoxacin Fleroxacin
  • Pefloxacin Rosa
  • the methods of the present technology comprise selecting for administration or administering one or more of Thienamycin, imipenem, meropenem, doripenem, ertapenem, biapenem, tebipenem, Meropenem-vaborbactam, Imipenem-relobactam, and conjugates thereof.
  • Cephalosporins are bactericidal P-lactam antibiotics that inhibit enzymes in the cell wall of susceptible bacteria, disrupting cell wall synthesis. Cephalosporins are broad spectrum, acting against Gram-negative and Gram-positive bacteria. Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin, Cephalexin, Cephradine, Cefaclor, Cefotetan, Cefoxitin, Cefprozil, Cefuroxime, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftazidime-avibactam, Ceftibuten, Ceftriaxone, Cefepime, Ceftaroline-fosamil, Ceftobiprole/medocaril, Cefiderocol, Ceftolozane-
  • the methods of the present technology comprise selecting for administration or administering a cephalosporin. In some embodiments, the methods of the present technology comprise selecting for administration or administering one or more of Cefadroxil, Cefazolin, Cephalexin, Cephradine, Cefaclor, Cefotetan, Cefoxitin, Cefprozil, Cefuroxime, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefoperazone/sulbactam, Cefotaxime, Cefpodoxime, Ceftazidime/avibactam, Ceftibuten, Ceftriaxone, Cefepime, Ceftaroline/fosamil, Ceftobiprole/medocaril, Cefiderocol Ceftolozane/tazobactam, moxalactam, and conjugates thereof.
  • Penicillin antibiotics are a broad-spectrum class of bactericidal P-lactam antibiotics. Penicillin antibiotics interrupt bacterial cell-wall formation by binding to essential penicillin- binding proteins (PBPs). Penicillin antibiotics include, but are not limited to, amoxicillin, amoxicillin/clavulanic acid, ampicillin, benzylpenicillin, benzathine benzylpenicillin, dicloxacillin, flucloxacillin, oxacillin, cioxacillin, nafcillin, carbenicillin, ticarcillin, temocillin, mecillinam, phenoxymethylpenicillin, mezlocillin, piperacillin, piperacillin-tazobactam, and conjugates thereof.
  • PBPs essential penicillin- binding proteins
  • the methods of the present technology comprise selecting for administration or administering a penicillin antibiotic. In some embodiments, the methods of the present technology comprise selecting for administration or administering one or more of amoxicillin, amoxicillin/clavulanic acid, ampicillin, benzylpenicillin, benzathine benzylpenicillin, dicloxacillin, flucioxacillin, oxacillin, cioxacillin, nafcillin, carbenicillin, ticarcillin, temocillin, mecillinam, phenoxymethylpenicillin, piperacillin, piperacillin- tazobactam, and conjugates thereof.
  • Antimicrobial Peptides are a class of small peptides with antimicrobial activity, including against bacteria, viruses, fungi, and protozoa.
  • Antimicrobial peptides include aniconic peptides, linear cationic a-helical peptides, cationic peptides enriched for specific amino acids, and aniconic/cationic peptides that form disulfide bonds.
  • AMPs are numerous, and can include those described in Malak Pirtskhalava, et al., DBAASP v3: database of antimicrobial/cytotoxic activity and structure of peptides as a resource for development of new therapeutics, Nucleic Acids Research, Volume 49, Issue DI, 8 January 2021, Pages D288-D297.
  • AMPs having anti-bacterial activity include, but are not limited to, Colistin, Dalbavancin, Daptomycin, Oritavancin, Polymyxin b, Teicoplanin, Telavancin, Vancomycin, Murepavadin, PMX-30063, Friulimicin B, PLG0206, IDR1, Omiganan, LTX-109, OP-145, DPK-060, NP101, NP108, Novexatin, Pl 13, Ctx(Ile21)-Ha, and conjugates thereof.
  • the methods of the present technology comprise selecting for administration or administering an antimicrobial peptide.
  • the methods of the present technology comprise selecting for administration or administering one or more of Colistin, Dalbavancin, Daptomycin, Oritavancin, Polymyxin b, Teicoplanin, Telavancin, Vancomycin, Murepavadin, PMX-30063, Friulimicin B, PLG0206, IDR1, Omiganan, LTX-109, OP-145, DPK-060, NP101, NP108, Novexatin, Pl 13, Ctx(Ile21)-Ha, and conjugates thereof.
  • Aminoglycosides aminoglycosides
  • P-lactamase enzymes confer resistance to P-lactam antibiotics, including penicillin antibiotics, by digesting the P-lactams.
  • P-lactamase inhibitors are a class of compounds that inhibit P-lactamase enzymes, and can be used synergistically with P-lactam antibiotics.
  • P- lactamase inhibitors include, but are not limited to, clavulanic acid, sulbactam, tazobactam, avibactam, relebactam, RG06080, RPX7009, and conjugates thereof.
  • a penicillin antibiotic and a P-lactamase inhibitor are administered simultaneously, sequentially, or separately, including as part of the same or different compositions.
  • the methods of the present technology further comprise sequentially, simultaneously, or separately administering a therapeutically effective amount of a P-lactamase inhibitor.
  • the P-lactamase inhibitor is selected from the group consisting of clavulanic acid, sulbactam, tazobactam, avibactam, relebactam, RG06080, RPX7009, and conjugates thereof.
  • Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • the compositions disclosed herein are formulated for administration to a mammal, such as a human.
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • Compositions formulated for parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion.
  • the rate of compound release can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and g
  • Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner.
  • Examples of embedding compositions that can be used include polymeric substances and waxes.
  • the active compounds can also be in micro-encapsulated form with one or more excipients as noted above.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents.
  • opacifying agents may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions include polymeric substances and waxes.
  • high throughput, massively parallel sequencing employs sequencing-by-synthesis with reversible dye terminators.
  • sequencing is performed via sequencing-by-ligation.
  • sequencing is single molecule sequencing. Examples of Next Generation Sequencing techniques include, but are not limited to pyrosequencing, Reversible dye-terminator sequencing, SOLiD sequencing, Ion semiconductor sequencing, Helioscope single molecule sequencing etc.
  • the Ion TorrentTM (Life Technologies, Carlsbad, CA) amplicon sequencing system employs a flow-based approach that detects pH changes caused by the release of hydrogen ions during incorporation of unmodified nucleotides in DNA replication.
  • a sequencing library is initially produced by generating DNA fragments flanked by sequencing adapters. In some embodiments, these fragments can be clonally amplified on particles by emulsion PCR. The particles with the amplified template are then placed in a silicon semiconductor sequencing chip. During replication, the chip is flooded with one nucleotide after another, and if a nucleotide complements the DNA molecule in a particular microwell of the chip, then it will be incorporated.
  • a proton is naturally released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH.
  • the pH of the solution then changes in that well and is detected by the ion sensor. If homopolymer repeats are present in the template sequence, multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.
  • the 454TM GS FLX TM sequencing system (Roche, Germany), employs a light-based detection methodology in a large-scale parallel pyrosequencing system.
  • Pyrosequencing uses DNA polymerization, adding one nucleotide species at a time and detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates.
  • adapter-ligated DNA fragments are fixed to small DNA-capture beads in a water-in-oil emulsion and amplified by PCR (emulsion PCR).
  • PCR emulsion PCR
  • Each DNA-bound bead is placed into a well on a picotiter plate and sequencing reagents are delivered across the wells of the plate.
  • the four DNA nucleotides are added sequentially in a fixed order across the picotiter plate device during a sequencing run.
  • Helicos's single-molecule sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface. At each cycle, DNA polymerase and a single species of fluorescently labeled nucleotide are added, resulting in template-dependent extension of the surface-immobilized primer-template duplexes. The reads are performed by the Helioscope sequencer. After acquisition of images tiling the full array, chemical cleavage and release of the fluorescent label permits the subsequent cycle of extension and imaging.
  • Sequencing by synthesis like the "old style" dye-termination electrophoretic sequencing, relies on incorporation of nucleotides by a DNA polymerase to determine the base sequence.
  • a DNA library with affixed adapters is denatured into single strands and grafted to a flow cell, followed by bridge amplification to form a high-density array of spots onto a glass chip.
  • Reversible terminator methods use reversible versions of dye-terminators, adding one nucleotide at a time, detecting fluorescence at each position by repeated removal of the blocking group to allow polymerization of another nucleotide.
  • the sequencing by ligation method uses a DNA ligase to determine the target sequence.
  • This sequencing method relies on enzymatic ligation of oligonucleotides that are adjacent through local complementarity on a template DNA strand.
  • This technology employs a partition of all possible oligonucleotides of a fixed length, labeled according to the sequenced position.
  • Oligonucleotides are annealed and ligated and the preferential ligation by DNA ligase for matching sequences results in a dinucleotide encoded color space signal at that position (through the release of a fluorescently labeled probe that corresponds to a known nucleotide at a known position along the oligo).
  • This method is primarily used by Life Technologies’ SOLiDTM sequencers.
  • the DNA is amplified by emulsion PCR.
  • the resulting beads, each containing only copies of the same DNA molecule, are deposited on a solid planar substrate.
  • SMRTTM sequencing is based on the sequencing by synthesis approach.
  • the DNA is synthesized in zero-mode wave-guides (ZMWs)-small well-like containers with the capturing tools located at the bottom of the well.
  • the sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labeled nucleotides flowing freely in the solution.
  • the wells are constructed in a way that only the fluorescence occurring at the bottom of the well is detected.
  • the fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.
  • SBS trinucleotide mutation
  • indel spectra to predict antibiotic susceptibility in a patient diagnosed with or suffering from a Pseudomonas infection.
  • SBS signatures result from recurring trinucleotide patterns of the transit! on/transversi on types of somatic single nucleotide variants (SNVs) and their flanking nucleotides, whereas indel signatures are defined according to size, nucleotides affected, and the presence of repetitive/microhomology regions.
  • the present disclosure provides a method for predicting the risk of antibiotic resistance in a subject diagnosed with or suffering from a Pseudomonas infection comprising sequencing Pseudomonas nucleic acids isolated from a biological sample obtained from the subject; generating a mutation spectrum from the sequenced Pseudomonas nucleic acids; and determining that the subject is at risk for antibiotic resistance when a Pseudomonas antibiotic-induced mutational signature is detected in the mutation spectrum.
  • the Pseudomonas infection is caused by a Pseudomonas species selected from among P. aeruginosa, P fluorescens, P putida, P cepacia, P stutzeri, P maltophilia, and P putrefaciens.
  • the Pseudomonas species has been previously exposed to an antibiotic, whereas in other embodiments the Pseudomonas species has not been previously exposed to an antibiotic.
  • generating the mutation spectrum comprises generating a plurality of single base substitution (SBS) contexts and a plurality of insertion/deletion (indel) profiles from the sequenced Pseudomonas nucleic acids.
  • SBS single base substitution
  • Indel insertion/deletion
  • each indel profile of the plurality of indel profiles comprise an indel size, a number of sequence unit repeats at an indel location, and the presence or absence of flanking microhomology sequences at an indel location.
  • the indel size is about 1 base pair to about 10 base pairs. In some embodiments, the indel size is 1 base pair. In some embodiments, the indel size is 2 base pairs. In some embodiments, the indel size is 3 base pairs. In some embodiments, the indel size is 4 base pairs. In some embodiments, the indel size is 5 base pairs. In some embodiments, the indel size is 6 base pairs. In some embodiments, the indel size is 7 base pairs. In some embodiments, the indel size is 8 base pairs. In some embodiments, the indel size is 9 base pairs. In some embodiments, the indel size is 10 base pairs.
  • the number of sequence unit repeats at an indel location is about 0 to about 10. In some embodiments, the number of sequence unit repeats at an indel location is 0. In some embodiments, the number of sequence unit repeats at an indel location is 1. In some embodiments, the number of sequence unit repeats at an indel location is 2. In some embodiments, the number of sequence unit repeats at an indel location is 3. In some embodiments, the number of sequence unit repeats at an indel location is 4. In some embodiments, the number of sequence unit repeats at an indel location is 5. In some embodiments, the number of sequence unit repeats at an indel location is 6. In some embodiments, the number of sequence unit repeats at an indel location is 7. In some embodiments, the number of sequence unit repeats at an indel location is 8. In some embodiments, the number of sequence unit repeats at an indel location is 9. In some embodiments, the number of sequence unit repeats at an indel location is 10.
  • the length of the flanking microhomology sequences at an indel location is 6 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 7 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 8 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 9 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 10 base pairs.
  • the plurality of SBS contexts comprise a plurality of single base changes in a trinucleotide context, wherein the plurality of single base changes in the trinucleotide context comprises one or more transitions/transversions/substitutions selected from the group consisting of OA, OG, OT, T>A, T>C, T>G, G>T, G>C, G>A, A>T, A>G, and A>C.
  • the Pseudomonas antibiotic-induced mutational signature comprises one or more of: an increase in OT transitions in NCG and GCN contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>A transitions in CGN and NGC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in T>C transitions in ATC, ATG, CTC, and CTG contexts relative to a reference Pseudomonas nucleic acid sample, an increase in A>G transitions in GAT, CAT, GAG, and CAG contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertions in non-repeat regions of the sequenced Pseudomonas nucleic acids relative to a reference Pseudomonas nucleic acid sample.
  • the increase in C>T transitions in NCG and GCN contexts relative to a reference Pseudomonas nucleic acid sample are increases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the sequenced Pseudomonas nucleic acids relative to a reference Pseudomonas nucleic acid sample are increases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the
  • the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in OG transversions in ACG and GCC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>C transversions in CGT and GGC contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertion in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample.
  • the increase in C>G transversions in ACG and GCC contexts relative to a reference Pseudomonas nucleic acid sample, the increase in G>C transversions in CGT and GGC contexts relative to a reference Pseudomonas nucleic acid sample, and the increase in cytosine or guanine insertion in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample are increases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample.
  • the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, a carbapenem, an AMP, or an aminoglycoside.
  • the increase in T>C and A>G substitutions relative to a reference Pseudomonas nucleic acid sample, the increase in OG transversions in ACG and GCC contexts relative to a reference Pseudomonas nucleic acid sample, the increase in G>C transversions in CGT and GGC contexts relative to a reference Pseudomonas nucleic acid sample, and the increase in cytosine or guanine insertion in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample are increases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample.
  • the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, a carbapenem, an AMP
  • the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase of 5+ base pair insertions in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample, OG transversions in ACG and GCC contexts that are comparable to a reference Pseudomonas nucleic acid sample, and G>C transversions in CGT and GGC contexts that are comparable to a reference Pseudomonas nucleic acid sample.
  • the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in OA substitutions in SCA contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>T substitutions in TGS contexts relative to a reference Pseudomonas nucleic acid sample, an increase in T>G and A>C substitutions relative to a reference Pseudomonas nucleic acid sample, a decrease in C >T substitutions in ACG contexts and G>A substitutions in CGT contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in at least one of (a) single thymine or adenine deletions, (b) single thymine or adenine insertions, (c) 5+ base pair deletions, (d) 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units
  • the increase in OA substitutions in SCA contexts relative to a reference Pseudomonas nucleic acid sample, the increase in G>T substitutions in TGS contexts relative to a reference Pseudomonas nucleic acid sample, the increase in T>G and A>C substitutions relative to a reference Pseudomonas nucleic acid sample, and the increase in at least one of (a) single thymine or adenine deletions, (b) single thymine or adenine insertions, (c) 5+ base pair deletions, (d) 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units, and (e) 5+ base pair deletions in regions with microhomology, relative to a reference Pseudomonas nucleic acid sample are increases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or
  • the decrease in C >T substitutions in ACG contexts and G>A substitutions in CGT contexts relative to a reference Pseudomonas nucleic acid sample is a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample.
  • the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, an AMP, or an aminoglycoside.
  • the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: a decrease in 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertions in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample.
  • the decrease in 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units relative to a reference Pseudomonas nucleic acid sample is a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample.
  • the increase in cytosine or guanine insertions in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample is an increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample.
  • the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a monobactam, a quinolone, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, or an antimicrobial peptide (AMP).
  • the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in single nucleotide deletions relative to a reference Pseudomonas nucleic acid sample, an increase in 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units relative to a reference Pseudomonas nucleic acid sample, an increase in G transitions in GCT contexts and G>C transitions in AGC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in OA transitions in CCA, CCG, and GCA contexts and G>T transitions in TGG, CGG and TGC contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in T>C and/or T>G substitutions relative to a reference Pseudomonas nucleic acid sample.
  • the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, a monobactam, or an antimicrobial peptide (AMP).
  • one or more antibiotics selected from the group consisting of an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, a monobactam, or an antimicrobial peptide (AMP).
  • the increase in OG transitions in GCT contexts and G>C transitions in AGC contexts relative to a reference Pseudomonas nucleic acid sample the increase in C>A transitions in CCA, CCG, and GCA contexts and G>T transitions in TGG, CGG and TGC contexts relative to a reference Pseudomonas nucleic acid sample, the increase in T>C and/or T>G substitutions relative to a reference Pseudomonas nucleic acid sample, the increase in at least one of : (a) C >A substitutions in VNR contexts, (b) G>T substitutions in YNB contexts, (c) C >G substitutions in a GCG contexts, (d) G>C substitutions in CGC contexts, (e) T > G substitutions in ATN contexts, (f) A>C substitutions in NAT contexts, (g) T>A substitutions in GTT contexts, and (h) A>T substitutions
  • the reference Pseudomonas nucleic acid sample is obtained from a Pseudomonas control strain that is susceptible to antibiotic therapy, is a wild type MPA01 strain, and/or is a non-hypermutator strain.
  • the Pseudomonas control strain is susceptible to antibiotic therapy.
  • the Pseudomonas control strain is a wild type MPAO1 strain.
  • the Pseudomonas control strain is a non-hypermutator strain.
  • the reference Pseudomonas nucleic acid sample is obtained from a Pseudomonas control strain that is MMR proficient (pMMR).
  • pMMR MMR proficient
  • the reference Pseudomonas nucleic acid sample is obtained from a Pseudomonas control strain that is MMR deficient (dMMR).
  • the biological sample comprises skin tissue, throat swabs, stool, urine, blood, lung tissue, stomach tissue, or urinary tract tissue. Samples can be collected using any appropriate means to ensure nucleic acid stability for further processing and analysis.
  • the antibiotic therapy comprises one or more antibiotics selected from the group consisting of a monobactam, a quinolone, an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, or an antimicrobial peptide (AMP).
  • the monobactam is selected from among aztreonam, azactam, tigemonam, nocardicin A, BAL30072, and tabtoxin.
  • the quinolone is selected from among Enrofloxacin (Baytril), Ciprofloxacin (i.e., Cipro and Proquin), Enoxacin (i.e., Penetrex), Gatifloxacin (i.e., Gatiflo, Tequin and Zymar), Gemifloxacin (i.e,.
  • the carbapenem is selected from among Thienamycin, doripenem, ertapenem, biapenem, tebipenem, Meropenem, Meropenem-vaborbactam, Imipenem, and Imipenem-relobactam.
  • the cephalosporin is selected from among Cefadroxil, Cefazolin, Cephalexin, Cephradine, Cefaclor, Cefotetan, Cefoxitin, Cefprozil, Cefuroxime, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftazidime-avibactam, Ceftibuten, Ceftriaxone, Cefepime, Ceftaroline-fosamil, Ceftobiprole/medocaril, Cefiderocol, Ceftolozane-tazobactam, and moxalactam.
  • the penicillin-based beta-lactam is selected from among amoxicillin, amoxicillin/clavulanic acid, ampicillin, benzylpenicillin, benzathine benzylpenicillin, dicloxacillin, flucioxacillin, oxacillin, cioxacillin, nafcillin, carbenicillin, ticarcillin, temocillin, mecillinam, phenoxymethylpenicillin, mezlocillin, piperacillin, and piperacillin-tazobactam.
  • the Pseudomonas nucleic acids are sequenced via whole genome sequencing (WGS).
  • antibiotics e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based betalactams, antimicrobial peptides, or aminoglycosides
  • fluoroquinolones e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based betalactams, antimicrobial peptides, or aminoglycosides
  • carbapenems e.g., cephalosporins, penicillin-based betalactams, antimicrobial peptides, or aminoglycosides
  • the effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians.
  • An effective amount of one or more antibiotics e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides
  • the antibiotic e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides
  • the antibiotic may be administered systemically or locally.
  • the one or more antibiotics e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides
  • Such compositions typically include the active agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment).
  • compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • transdermal administration may be performed by iontophoresis.
  • a therapeutic agent can be formulated in a carrier system.
  • the carrier can be a colloidal system.
  • the colloidal system can be a liposome, a phospholipid bilayer vehicle.
  • the therapeutic agent is encapsulated in a liposome while maintaining the agent’s structural integrity.
  • methods to prepare liposomes See Lichtenberg, et al. , Methods Biochem. Anal., 33:337-462 (1988);
  • a therapeutically effective amount of one or more antibiotics may be defined as a concentration of inhibitor at the target tissue of 10' 32 to 10' 6 molar, e.g., approximately 10' 7 molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).
  • antibiotics e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides
  • treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
  • the mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits.
  • the mammal is a human.
  • Colonies with distinct morphotypes were collected as different strains from each patient sample and pure-cultured on PIA. For S 10 until SI 9, sputum samples were processed by the clinical microbiology laboratory at Tulane Medical Center. Pure-cultured plates were then obtained.
  • Insertional transposon mutants were confirmed via polymerase chain reaction (PCR) with gene-flanking and transposon-annealing primer sets. Primer sequences and sets used for each confirmation reaction are found in Table 1 and Table 2, respectively. Final reaction conditions were as follows: 0.4 pM forward and reverse primers, ⁇ 1 pg template DNA (genomic DNA from tested strain), IX LongAmp Taq Master Mix (Thermo Fisher Scientific), and nuclease-free FEO up to 25 pL.
  • Table 1 Sequences of PCR primers. Primers either flank gene insertion site or anneal to insertional transposon used to confirm insertional transposon mutagenesis. Optimized T m for each primer pair is reported. Table 1 shows SEQ ID NOs: 1-16 in order of appearance.
  • Genomic DNA preps from desired resistant and control lineages were whole genome sequenced using Illumina short-read sequencing (NextSeq 2000).
  • Sample libraries were prepared using Illumina DNA Prep Kit and IDT lObp UDI indices. Demultiplexing, quality control (only reads with Q>30 kept), and adapter trimming were performed using Illumina bcl-convert (v3.9.3).
  • Each sample produced a minimum of 400 Mbp high quality reads (2 x 151 bp), with an average depth of coverage of 60X of the ⁇ 6.3 Mbp genome.
  • FASTQ reads from SlO Pa 1, SP10 Pa2, SP11 Pal, S12 Pal, S12 Pa2, S12 Pa3, S7T2 Pal, S14 Pal, S15 Pal, S16 Pal, S17 Pal, S18 Pal, S18 Pa2, and S6T2 Pal were de novo assembled using unicycler ⁇ 9 with default parameters. Assembly statistics were recorded with quasfi 50 (default parameters) and samples annotated using bakta 35i (default parameters).
  • Batches’ for deduplication included: all lab evolved strains, all prospectively clinical isolates from pwCF, retrospective clinical isolates from the dataset of Lopez-Causape et al., 321 retrospective clinical isolates from the dataset of Kos et al., 352 and retrospective clinical isolates from UPMC.
  • individual spectra from all 30 replicates/treatment were compiled using tidyverse package dplyr and de facto mutational signatures from each treatment plotted with ggplot2.
  • VCF Variant call format
  • D-CONGA and D-CONGA-Q7 were synthesized using Fmoc solid-phase chemistry and purified to >95% via high performance liquid chromatography by Bio-synthesis Inc, with identity confirmed via MALDI mass spectrometry. Solutions were prepared by dissolving desired mass into 0.025% (v/v) acetic acid in water, and peptide concentration was determined by absorbance at 280 nm.
  • Table 3 Concentration of antimicrobial solutions used. All antimicrobials were dissolved in H2O, except D-CONGA and D-CONGA-Q7, which were dissolved in 0.25% acetic acid.
  • MICs were determined via microbroth dilution assay in 96-well culture plates with 2- fold serial dilutions of tested antibiotic in CAMHB, inoculated with 50 pL of 2.75 x 10 5 CFU/mL of each bacterial strain and incubated at 37°C, 200 rpm for 24 hours.
  • the IX concentration reported for each antimicrobial is the ‘starting’ concentration in Row A of the 96- well plate, with Row B being 1/2X, Row C 1/4X, Row D 1/8X, Row E 1/16X, Row F 1/32X, Row G 1/64X, and Row H no antibiotics (positive control) (Table 3).
  • MIC experiments were performed using biological triplicates (3 independent colonies/strain) plus a negative control (no inoculum) per strain/antimicrobial combination. This allowed for testing of 3 strain/antimicrobial combinations per 96-well plate (12 columns total, with 4 columns per combination).
  • Example 2 Evaluation of mutational signature analysis for prediction of MMR-status and MDR acquisition in prospective and retrospective chronic respiratory isolates of P. aeruginosa
  • Trinucleotide mutation spectra from 7 subject isolates showed similar OT enrichment in NCC and NCG and reduction in NCT and T>C enrichment in CTN and GTN that we saw with PaA/7?///A (FIG. 1A), whereas the spectra of the other 19 isolates did not display all of these characteristics (FIG. IB).
  • These 7 isolates had overall elevated levels of unique single nucleotide variants (SNVs) and indels (FIG. 2). Quantitative assessment via cosine similarity showed that spectra from these same 7 isolates were most similar to PaA/7?///A (FIG. 3).
  • mutational signature analysis can prospectively predict future rapid MDR acquisition.
  • mutational signature analysis coupled with rational combination therapy is a promising precision medicine approach that can prevent the emergence of MMR-deficiency-induced MDR in vitro.
  • Predicted MMR-deficient isolates were enriched in pwCF (31% of the total pwCF isolates), similar to previous reports, 155,272 but were also found at significant levels in RTIs (5.5% of the total RTI isolates), UTIs (2.8% of the total UTI isolates), and IAIs (2.7% of the total IAI isolates) (FIG. 7C).
  • RTIs 5.5% of the total RTI isolates
  • UTIs (2.8% of the total UTI isolates
  • IAIs 2.7% of the total IAI isolates
  • Ceftolozane-tazobactam is a new generation cephalosporin P-lactamase combination antibiotic often used to treat MDR . aeruginosa VAP, 353 and therefore infections requiring this treatment are likely enriched for MDR.
  • Example 4 Refining the MMR-deficiency-associated mutational signature in P. aeruginosa
  • a potential weakness of applying the mutational signature acquired by MMR- deficient lab strains of P. aeruginosa as a predictor of MMR-status in clinical isolates is the large difference in the genetic background of lab strains and clinical isolates. Additionally, using the signature made from only one strain may not apply well to all clinical isolates due their genetic heterogeneity. With now having a robust set of genomic data from clinical isolates from a diverse range of disease contexts and geographic locations, we next sought to employ NMF for de novo signature extraction.
  • DNA polymerases IV and V are translesion synthesis (TLS) polymerases lacking a 3’— >5’ exonuclease activity and are thus error-prone, 360 362 and they are hypothesized to not only help bacterial cells overcome antibiotic-induced DNA damage after treatment but also to promote adaptive evolution. 363 DinB favors OT transitions during synthesis, similar to SBS6 and 15. 87,162 From this, we hypothesized that the clusters of SBS6 and 15 enriched clinical isolates observed in all datasets could be a signal for previous antibiotic-induced mutagenesis. Thus, we aimed to discover and characterize mutational signatures attributable to antibiotic treatment for the first time.
  • TLS translesion synthesis
  • P aeruginosa strain MPA01 was streaked on LB and incubated overnight. From this plate, independent biological replicates (independent colonies) of MPAO1 were grown in 5 mL LB overnight. Biological replicates were serially passaged in 1/10 MIC (concentration unchanging) of each antibiotic for 10 total passages and incubated at 37°C, 200 rpm for 24 hours each passage. After 10 passages, all replicates were processed for genomic DNA isolates and whole genome sequenced using Illumina short read sequencing. Sample libraries were prepared using Illumina DNA Prep Kit and IDT lObp UDI indices.
  • VCF Variant call format
  • Ceftazidime-treated replicates showed elevation in C>G transversions in ACG and GCC, whereas cefiderocol -treated replicates did not. We suggest that these differences could be due to cytosine deamination in CpG islands or general reactive oxidative stress. [00231] Comparing these two treatment groups to the other beta-lactams, piperacillin- treatment resembles the spectra of ceftazidime-treatment but shows higher enrichment of T>Cs.
  • meropenem-treatment uniquely results in enrichment in single thymine deletions, single thymine insertions, 3bp deletions in regions with 6+ repeats, 5+bp deletions, and 5+bp deletions in regions with microhomology. Additionally, meropenem-treatment results in enrichment of C>Ain SCA contexts and overall T>G transversions. Meropenem-treatment also uniquely shows lower C>T in ACG. We suggest that this profile could be due to higher oxidative stress induced by meropenem treatment.
  • Aztreonam binds to penicillin-binding protein similar to beta-lactams but is a monobactam and is slightly different structurally. Despite having the same mechanism of action, aztreonam induced a unique mutational signature characterized by enriched C>As in VNR contexts, C>G in a GCG context, T>Gs in a ATN context, and uniquely T>As in a GTT context. Aztreonam-treatment also uniquely results in a strong signature of 3bp deletions in 6+ repeats, 5+bp deletions in 2 repeats, and 5+bp deletions in regions of 5+ repeats of microhomology. Based on this profile, we suggest that aztreonam could be inducing replicative fork stress, oxidative damage and potential bulky adducts fixed by nonhomologous end joining. The table below summarizes the combined mutational signatures described herein:
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.
  • DNA polymerase II is encoded by the DNA damage-inducible dinA gene of Escherichia coli. Proceedings of the National Academy of Sciences 87, 7663-7667 (1990).
  • Tashiro, Y. et al. Opr86 is essential for viability and is a potential candidate for a protective antigen against biofilm formation by Pseudomonas aemginosa. J Bacterial 190, 3969-3978 (2008).
  • Senkin, S. MSA reproducible mutational signature attribution with confidence based on simulations. BMC Bioinformatics 22, 540 (2021).

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Abstract

The present disclosure provides methods for predicting drug resistance in a subject with a Pseudomonas infection based on the detection of antibiotic-induced mutational signatures of Pseudomonas species.

Description

METHODS FOR PREDICTING GENOMIC ANTIBIOTIC SUSCEPTIBILITY USING ANTIBIOTIC-INDUCED MUTATIONAL SIGNATURES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/573,680 filed April 3, 2024, the entire contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present technology relates to methods for predicting drug resistance in a subject diagnosed with or suffering from a Pseudomonas infection based on the detection of antibiotic- induced mutational signatures of Pseudomonas species.
BACKGROUND
[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.
[0004] There are numerous endogenous and exogenous mutagens that could potentially be driving specific mutational signatures in bacteria, similar to human tumors.166,174 Numerous antibiotics induce mutagenesis in exposed bacterial cells,106,182,185,187 but no associated mutational signatures have yet to be characterized.
SUMMARY OF THE PRESENT TECHNOLOGY
[0005] In one aspect, the present disclosure provides a method for predicting the risk of antibiotic resistance in a subject diagnosed with or suffering from a Pseudomonas infection comprising sequencing Pseudomonas nucleic acids isolated from a biological sample obtained from the subject; generating a mutation spectrum from the sequenced Pseudomonas nucleic acids; and determining that the subject is at risk for antibiotic resistance when a Pseudomonas antibiotic-induced mutational signature is detected in the mutation spectrum.
[0006] In another aspect, the present disclosure provides a method for selecting a subject infected with a Pseudomonas species for treatment with an antibiotic therapy comprising sequencing Pseudomonas nucleic acids isolated from a biological sample obtained from the subject; generating a mutation spectrum of the Pseudomonas nucleic acid sequences; detecting a Pseudomonas antibiotic-induced mutational signature in the mutation spectrum; and selecting an antibiotic therapy based on the detected Pseudomonas antibiotic-induced mutational signature.
[0007] In some embodiments, generating the mutation spectrum comprises generating a plurality of single base substitution (SBS) contexts and a plurality of insertion/deletion (indel) profiles from the sequenced Pseudomonas nucleic acids. In some embodiments, each indel profile of the plurality of indel profiles comprise an indel size, a number of sequence unit repeats at an indel location, and the presence or absence of flanking microhomology sequences at an indel location. In some embodiments, the plurality of SBS contexts comprise a plurality of single base changes in a trinucleotide context, wherein the plurality of single base changes in the trinucleotide context comprises one or more transitions/substitutions selected from the group consisting of OA, OG, C>T, T>A, T>C, T>G, G>T, G>C, G>A, A>T, A>G, and A>C.
[0008] In any of the preceding embodiments, the Pseudomonas antibiotic-induced mutational signature comprises one or more of: an increase in OT transitions in NCG and GCN contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>A transitions in CGN and NGC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in T>C transitions in ATC, ATG, CTC, and CTG contexts relative to a reference Pseudomonas nucleic acid sample, an increase in A>G transitions in GAT, CAT, GAG, and CAG contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertions in non-repeat regions of the sequenced Pseudomonas nucleic acids relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the increase in T transitions occurs in ACG and GCG contexts and the increase in G>A transitions occurs in CGT and CGC contexts.
[0009] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in OG transversions in ACG and GCC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>C transversions in CGT and GGC contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertion in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, a carbapenem, an AMP, or an aminoglycoside. [0010] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in T>C and A>G substitutions relative to a reference Pseudomonas nucleic acid sample, an increase in OG transversions in ACG and GCC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>C transversions in CGT and GGC contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertion in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, a carbapenem, an AMP, or an aminoglycoside.
[0011] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase of 5+ base pair insertions in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample, OG transversions in ACG and GCC contexts that are comparable to a reference Pseudomonas nucleic acid sample, and G>C transversions in CGT and GGC contexts that are comparable to a reference Pseudomonas nucleic acid sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, a carbapenem, an AMP, or an aminoglycoside.
[0012] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in C>A substitutions in SCA contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>T substitutions in TGS contexts relative to a reference Pseudomonas nucleic acid sample, an increase in T>G and A>C substitutions relative to a reference Pseudomonas nucleic acid sample, a decrease in C >T substitutions in ACG contexts and G>A substitutions in CGT contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in at least one of (a) single thymine or adenine deletions, (b) single thymine or adenine insertions, (c) 5+ base pair deletions, (d) 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units, and (e) 5+ base pair deletions in regions with microhomology, relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, an AMP, or an aminoglycoside. [0013] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: a decrease in 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertions in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a monobactam, a quinolone, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, or an antimicrobial peptide (AMP).
[0014] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in single nucleotide deletions relative to a reference Pseudomonas nucleic acid sample, an increase in 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units relative to a reference Pseudomonas nucleic acid sample, an increase in OG transitions in GCT contexts and G>C transitions in AGC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in OA transitions in CCA, CCG, and GCA contexts and G>T transitions in TGG, CGG and TGC contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in T>C and/or T>G substitutions relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, a monobactam, or an antimicrobial peptide (AMP).
[0015] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in OG transitions in GCT contexts and G>C transitions in AGC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in C>A transitions in CCA, CCG, and GCA contexts and G>T transitions in TGG, CGG and TGC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in T>C and/or T>G substitutions relative to a reference Pseudomonas nucleic acid sample, an increase in at least one of : (a) C >A substitutions in VNR contexts, (b) G>T substitutions in YNB contexts, (c) C >G substitutions in a GCG contexts, (d) G>C substitutions in CGC contexts, (e) T > G substitutions in ATN contexts, (f) A>C substitutions in NAT contexts, (g) T>A substitutions in GTT contexts, and (h) A>T substitutions in AAC contexts, relative to a reference Pseudomonas nucleic acid sample, and an increase in at least one of: (a) 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units, (b) 5+ base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 2 repeating sequence units, and (c) 5+ base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 5+ repeating sequence units with microhomology, relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, a carbapenem, an AMP, or an aminoglycoside.
[0016] In some embodiments, the reference Pseudomonas nucleic acid sample is obtained from a Pseudomonas control strain that is susceptible to antibiotic therapy, is a wild type MPAO1 strain, and/or is a non-hypermutator strain.
[0017] In any of the preceding embodiments, the biological sample comprises skin tissue, throat swabs, stool, urine, blood, lung tissue, stomach tissue, or urinary tract tissue.
[0018] In any of the preceding embodiments, the Pseudomonas antibiotic-induced mutational signature is caused by prior exposure to an antibiotic. In some embodiments, the antibiotic is a P-lactamase inhibitor, a monobactam, a quinolone, an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, or an antimicrobial peptide (AMP).
[0019] In some embodiments, the antibiotic therapy comprises one or more antibiotics selected from the group consisting of a monobactam, a quinolone, an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, or an antimicrobial peptide (AMP).
[0020] In some embodiments, the monobactam is selected from among aztreonam, azactam, tigemonam, nocardicin A, BAL30072, and tabtoxin.
[0021] In some embodiments, the quinolone is selected from among Enrofloxacin (Baytril), Ciprofloxacin (i.e., Cipro and Proquin), Enoxacin (i.e., Penetrex), Gatifloxacin (i.e., Gatiflo, Tequin and Zymar), Gemifloxacin (i.e,. Factive), Levofloxacin (i.e., Levaquin), Lomefloxacin (i.e., Maxaquin), Moxifloxacin (i.e., Avelox), Norfloxacin (i.e., Noroxin), Ofloxacin (i.e., Floxin), Prulifloxacin, Sparfloxacin (i.e., Zagam), Trovafloxacin/Altrofloxacin (i.e., Trovan), Danofloxacin (i.e., Al 80), Difloxacin (i.e., Dicural), Marbofloxacin (i.e., Orbax), Orbifloxacin (i.e., Zeniquin), Cinoxacin (i.e., Cinobac), Rosoxacin, Fleroxacin, Pefloxacin, Rufloxacin, Balofloxacin, Grepafloxacin, Pazufloxacin, Temafloxacin, Tosufloxacin, Besifloxacin, Clinafloxacin, Garenoxacin, Sitafloxacin, Ibafloxacin, Pradofloxacin, and Sarafloxacin.
[0022] In some embodiments, the carbapenem is selected from among Thienamycin, doripenem, ertapenem, biapenem, tebipenem, Meropenem, Meropenem-vaborbactam, Imipenem, and Imipenem-relobactam.
[0023] In some embodiments, the cephalosporin is selected from among Cefadroxil, Cefazolin, Cephalexin, Cephradine, Cefaclor, Cefotetan, Cefoxitin, Cefprozil, Cefuroxime, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftazidime-avibactam, Ceftibuten, Ceftriaxone, Cefepime, Ceftaroline-fosamil, Ceftobiprole/medocaril, Cefiderocol, Ceftolozane-tazobactam, and moxalactam.
[0024] In some embodiments, the penicillin-based beta-lactam is selected from among amoxicillin, amoxicillin/clavulanic acid, ampicillin, benzylpenicillin, benzathine benzylpenicillin, dicloxacillin, flucioxacillin, oxacillin, cioxacillin, nafcillin, carbenicillin, ticarcillin, temocillin, mecillinam, phenoxymethylpenicillin, mezlocillin, piperacillin, and piperacillin-tazobactam.
[0025] In some embodiments, the aminoglycoside is selected from among apramycin, Tobramycin, Amikacin, Gentamicin, neomycin, streptomycin, and plazomycin.
[0026] In some embodiments, the AMP is selected from among Colistin, Dalbavancin, Daptomycin, Oritavancin, Polymyxin b, Teicoplanin, Telavancin, Vancomycin, Murepavadin, PMX-30063, Friulimicin B, PLG0206, IDR1, Omiganan, LTX-109, OP-145, DPK-060, NP101, NP108, Novexatin, Pl 13, and Ctx(Ile21)-Ha.
[0027] In some embodiments, the Pseudomonas infection is a cystic fibrosis (CF) infection, a respiratory tract infection (RTI), a urinary tract infection (UTI), a pressure sore infection, a bum infection, a wound infection, a bloodstream infection or an intra-abdominal infection (IAI).
[0028] In some embodiments, the Pseudomonas infection is caused by a Pseudomonas species selected from among P. aeruginosa, P fluorescens, P putida, P cepacia, P stutzeri, P maltophilia, and P putrefaciens.
[0029] In some embodiments, the Pseudomonas species is selected from among P. aeruginosa, P fluorescens, P putida, P cepacia, P stutzeri, P maltophilia, and P putrefaciens. [0030] In any of the preceding embodiments, the Pseudomonas nucleic acids are sequenced via whole genome sequencing (WGS).
[0031] In some embodiments, the method further comprises sequentially, simultaneously, or separately administering to the subject an effective amount of a beta-lactamase inhibitor. In some embodiments, the beta-lactamase inhibitor is selected from the group consisting of clavulanic acid, sulbactam, tazobactam, avibactam, relebactam, RG06080, and RPX7009.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIGs. 1A-1B: Trinucleotide spectra from 7 subject isolates resemble PaA/n/uA.
FIG. 1A: Trinucleotide mutation spectra of predicted MMR-deficient isolates collected from 15 subjects with bronchiectasis (CF and non-CF). FIG. IB: Trinucleotide mutation spectra of all those sample isolates predicted as WT.
[0033] FIG. 2: Mutation burden is an inaccurate determinant of MMR-status. Unique SNVs and indels per isolate following deduplication of variants across all samples, with predicted MMR-deficient isolates colored in blue/green.
[0034] FIG. 3: Seven predicted MMR-deficient subject isolates quantitatively cluster with PaAmu S'. Trinucleotide mutation spectra from patient isolates were compared to PaA/iw/A, Human AMMR, and each individual COSMIC SBS associated with MMR-deficiency and clustered based on cosine similarities.
[0035] FIG. 4: Seven predicted MMR-deficient isolates are functionally hypermutators.
Mutant frequencies of all subject isolates were measured using rifampicin reversion (rpoB mutants resistant to rifampicin per 108 viable cells). Hypermutators are defined as those having mutant frequencies above the dotted line, which represents 20-fold higher than the mutant frequency of the WT MPAO1 parent strain. Clinical isolates predicted based on mutation spectra to be MMR-deficient and hypermutant are shown in dark grey. Clinical isolates predicted to be WT are shown in light grey. Lab strains are shown in white. For each isolate, the presence (+) or absence (-) of nonsynonymous mutations in the indicated key MMR genes are denoted below.
[0036] FIGs. 5A-5B: Predicted MMR-deficient isolates acquire drug resistance in vitro.
FIGS. 5A-5B: Representative resistance acquisition shown for subject isolates. Predicted MMR- deficient subject isolates rapidly acquired resistance to (FIG. 5A) aztreonam (AZ) and (FIG. 5B) colistin (COL), while WT isolates did not. [0037] FIGs. 6A-6F: Mutational signature predicts MMR-deficient isolates and is correlated with MDR in a large independent pwCF cohort. FIG. 6A: Representative trinucleotide mutation spectra of isolates predicted as MMR-deficient from pwCF. FIG. 6B: Clustering of trinucleotide mutation spectra based on cosine similarity from patient isolates using PaA/iw/A', HumanAMMR, and each individual COSMIC SBS associated with MMR-deficiency. FIG. 6C: Percentage of predicted MMR-deficient and WT isolates containing nonsynonymous mutations in MMR genes. FIG. 6D: Comparison of the mutator status for each isolate via rifampicin reversion reported by Lopez-Causape et al. (Normal vs Hypermutator) to the MMR status predicted by mutational signature (MMR-deficient vs WT) using the same dataset. Those isolates in the upper left comer were predicted MMR-deficient and confirmed hypermutant. Two sets of isolates showed discordance between prediction and observation. Those isolates in the bottom left corner were predicted MMR-deficient but were observed not to be mutators. Those isolates in the upper right corner were predicted to be WT but observed to be hypermutators. The isolates with discordant predictions indicated by green stars (MMR-deficient, normal) or gray circles (WT, hypermutant) in FIG. 6B and discussed more in the text. FIGS. 6E-6F: Correlation of predicted MMR-deficiency using (FIG. 6E) mutational signature analysis or (FIG. 6F) hypermutation from rifampicin reversion with MDR. P-values were calculated using Fisher’s exact test and found to be (FIG. 6E) p = 0.0021 and (FIG. 6F) p=0.0009. MDR isolates were defined by clinical resistance (‘R’ via EUCAST) to >3 drugs.
[0038] FIGs. 7A-7G: Mutational signature analysis reveals occurrence of MMR- deficiency in several acute disease contexts. FIG. 7A: Trinucleotide mutation spectra of those patient isolates predicted as MMR-deficient from three different patient cohorts: cystic fibrosis (CF); respiratory tract infection (RTI); urinary tract infection (UTI); and intra-abdominal infection (IAI). FIG. 7B: Trinucleotide mutation spectra from patient isolates were clustered based on cosine similarity to Pa mutS, Human AMMR, and each individual COSMIC SBS associated with MMR-deficiency. FIGs. 7C-7G: Percentage of isolates predicted as MMR- deficient or WT across the indicated disease contexts (FIG. 7D) CF, (FIG. 7E) RTI, (FIG. 7F) UTI, and (FIG. 7G) IAI. MDR is defined by clinical resistance (‘R’ via CLSI) to >2 drugs, p = 0.0256 (D) via Fisher’s exact.
[0039] FIGs. 8A-8B: 10 trinucleotide mutation spectra in VAP isolates resemble MMR- deficiency. (FIGs. 8A, 8B) Trinucleotide mutation spectra of those patient isolates predicted as (FIG. 8A) MMR-deficient or (FIG. 8B) right below threshold. [0040] FIG. 9: Predicted MMR-deficient isolates cluster with Pa nmtS. Trinucleotide mutation spectra from patient isolates were clustered based on cosine similarity to PaXmulS, Human AMMR, and each individual COSMIC SBS associated with MMR-deficiency. Red stars represent clusters of isolates with cosine similarity to PaXmulS above 0.78, whereas orange stars represent those with cosine similarity between 0.75 and 0.77.
[0041] FIG. 10: A signature nearly identical to Pa niutS was extracted from diverse clinical isolates. NMF on 589 clinical isolates of P. aeruginosa from 4 disease contexts extracted three stable mutational signatures (VI -V3), one of which (V3) has a 0.98 cosine similarity to PaXmulS (“MMR-”, bottom).
[0042] FIGs. 11A-11B: Aztreonam- and ciprofloxacin-treatment induces mutagenesis in P. aeruginosa. FIG. 11 A: Viable cells recovered on nonselective CAMHB agar in untreated (WT), ciprofloxacin-treated (+CIPRO), or aztreonam-treated (+AZ) groups. FIG. 11B: Mutant frequencies of five replicates per treatment group were measured using rifampicin reversion (rpoB mutants resistant to rifampicin per 108 viable cells). Data is shown as means ± SD, n=3 (WT) or n=5 (+CIPRO or +AZ). ****, p<0.0001, one-way ANOVA with multiple comparisons.
[0043] FIG. 12: Aztreonam- and ciprofloxacin-treatment induce specific mutational signatures that resemble human SBS6. Compiled trinucleotide spectra from lab-construction (“CIP” and “AZ”), de novo extracted signatures (“VI” and “V2”), and human SBS6 are shown.
[0044] FIG. 13: Clinical isolates of P. aeruginosa show enrichment in aztreonam- and ciprofloxacin-induced signatures. Red stars represent clusters of predicted MMR-deficient isolates. Orange star represents cluster with SBS6 and antibiotic-induced signature enrichment.
[0045] FIG. 14: Trinucleotide mutation spectra of all sample collapsed by treatment group. Shown are n = 19 replicated treated with piperacillin, n = 13 treated with ceftazidime, n = 15 treated with cefiderocol, n = 16 with meropenem, n = 87 with aztreonam, n = 20 with ciprofloxacin, and n = 17 with tobramycin. Treatment groups are clustered and annotated on figure by drug class, structural similarities, or mechanism of action similarities to illustrate differences in mutation spectra despite these characteristics.
[0046] FIG. 15: Indel mutation spectra of all sample collapsed by treatment group.
Shown are n = 19 replicated treated with piperacillin, n = 13 treated with ceftazidime, n = 15 treated with cefiderocol, n = 16 with meropenem, n = 87 with aztreonam, n = 20 with ciprofloxacin, and n = 17 with tobramycin. Treatment groups are clustered and annotated on figure by drug class, structural similarities, or mechanism of action similarities to illustrate differences in mutation spectra despite these characteristics.
[0047] FIG. 16: Classification of small deletions and insertions for mutational signature analysis as represented by exemplary SEQ ID NOs: 19-109.
DETAILED DESCRIPTION
[0048] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.
[0049] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson el al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg el al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).
[0050] Identification of antibiotic-induced mutational signatures could indicate prior antibiotic exposure and/or pre-existing antibiotic resistance of a clinical Pseudomonas signature, serving as a proxy for antibiotic susceptibility (AST). The Examples described herein demonstrate the application of mutational signature analysis as a method of genomics-based AST by discovering and validating antibiotic-induced signatures for the first time and demonstrating their presence in drug-resistant Pseudomonas strains.
Definitions
[0051] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in the present disclosure. Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.
[0052] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
[0053] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.
[0054] As used herein, the terms “amplify” or “amplification” with respect to nucleic acid sequences, refer to methods that increase the representation of a population of nucleic acid sequences in a sample. Nucleic acid amplification methods, such as PCR, isothermal methods, rolling circle methods, etc., are well known to the skilled artisan. Copies of a particular nucleic acid sequence generated in vitro in an amplification reaction are called “amplicons” or “amplification products”. [0055] The terms “complementary” or “complementarity” as used herein with reference to polynucleotides (z.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) refer to the base-pairing rules. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3’ end of the other, is in “antiparallel association.” For example, the sequence “5'-A-G-T-3”’ is complementary to the sequence “3’-T-C-A-5.” Certain bases not commonly found in naturally-occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7-deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA). Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. A complement sequence can also be an RNA sequence complementary to the DNA sequence or its complement sequence, and can also be a cDNA.
[0056] The IUPAC nucleotide code is summarized below:
[0057] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." A “control nucleic acid sample” or “reference nucleic acid sample” as used herein, refers to nucleic acid molecules from a control or reference sample. In certain embodiments, the reference or control nucleic acid sample is a wild type or a non-mutated DNA or RNA sequence. In certain embodiments, the reference nucleic acid sample is purified or isolated (e.g., it is removed from its natural state).
[0058] “Detecting” as used herein refers to determining the presence of a mutational signature in a sample comprising a Pseudomonas species. Detection does not require the method to provide 100% sensitivity. Analysis of nucleic acid markers can be performed using techniques known in the art including, but not limited to, sequence analysis, and electrophoretic analysis. Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears etaL, Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al. , Methods Mol. Cell Biol, 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al., Nat. Biotechnol, 16:381-384 (1998)), and sequencing by hybridization. Chee et al., Science, 274:610-614 (1996); Drmanac et al., Science, 260: 1649- 1652 (1993); Drmanac et al., Nat. Biotechnol, 16:54-58 (1998). Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Additionally, next generation sequencing methods can be performed using commercially available kits and instruments from companies such as the Life Technologies/Ion Torrent PGM or Proton, the Illumina HiSEQ or MiSEQ, and the Roche/454 next generation sequencing system.
[0059] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.
[0060] “ Gene” as used herein refers to a DNA sequence that comprises regulatory and coding sequences necessary for the production of an RNA, which may have a non-coding function (e.g., a ribosomal or transfer RNA) or which may include a polypeptide or a polypeptide precursor. The RNA or polypeptide may be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. Although a sequence of the nucleic acids may be shown in the form of DNA, a person of ordinary skill in the art recognizes that the corresponding RNA sequence will have a similar sequence with the thymine being replaced by uracil, z.e., "T" is replaced with "U."
[0061] As used herein, “hypermutator” refers to a phenotype that exhibits rapid adaptive evolution and antibiotic resistance acquisition.
[0062] As used herein, the terms “individual”, “patient”, or “subject” are used interchangeably and refer to an individual organism, a vertebrate, a mammal, or a human. In a preferred embodiment, the individual, patient or subject is a human.
[0063] As used herein, a “mutation” of a gene refers to the presence of a variation within the gene or gene product that affects the expression and/or activity of the gene or gene product as compared to the normal or wild-type gene or gene product. The genetic mutation can result in changes in the quantity, structure, and/or activity of the gene or gene product in a mutant bacterial cell, as compared to its quantity, structure, and/or activity, in a control bacterial cell.
[0064] For example, a mutation can have an altered nucleotide sequence (e.g., a mutation), amino acid sequence, expression level, protein level, protein activity, in an infected tissue or infected cell, as compared to a normal, healthy tissue or cell. Exemplary mutations include, but are not limited to, point mutations (e.g., silent, missense, or nonsense), deletions, insertions, inversions, linking mutations, duplications, translocations, inter- and intra-chromosomal rearrangements. Mutations can be present in the coding or non-coding region of the gene. In certain embodiments, the mutations are associated with a phenotype, e.g., an antibiotic-resistant or antibiotic-susceptibility phenotype.
[0065] As used herein, “microhomology” is defined as one or more base pairs (bp) of perfectly matching sequence shared between the proximal and distal reference sequences surrounding a breakpoint (e.g., an indel location).
[0066] As used herein, a “mutational signature” is a discreet probability distribution of genetic mutations made by a specific mutagenic process. The specific mutagenic process may be one that occurs in nature, such as a mutagenic process in a bacterium that responds to a naturally occurring stressor in a native bacterial environment. In other embodiments, the specific mutagenic process may be intentionally induced, such as induction of a mutagenic process in a bacterium by treating the bacterium with an antibiotic. In some embodiments, the mutational signature is based on the trinucleotide mutation (SBS) profiles and indel spectra of nucleic acids present in a biological sample.
[0067] “Next-generation sequencing or NGS” as used herein, refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a high throughput parallel fashion (e.g., greater than 103, 104, 105 or more molecules are sequenced simultaneously). In one embodiment, the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment. Next generation sequencing methods are known in the art, and are described, e.g. in Metzker, M. Nature Biotechnology Reviews 11 :31-46 (2010).
[0068] As used herein, “oligonucleotide” refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals. The nucleic acid bases are arranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide. The most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position. Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group. Oligonucleotides of the method which function as primers or probes are generally at least about 10-15 nucleotides long and more preferably at least about 15 to 25 nucleotides long, although shorter or longer oligonucleotides may be used in the method. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including, for example, chemical synthesis, DNA replication, restriction endonuclease digestion of plasmids or phage DNA, reverse transcription, PCR, or a combination thereof. The oligonucleotide may be modified e.g., by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.
[0069] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
[0070] As used herein, a “repeat region” refers to a region including a repeating sequence unit that is being inserted or deleted in the context of a directly neighboring/adjacent Pseudomonas nucleic acid sequence. Exemplary repeat regions including a repeating sequence unit [X] occurring at an indel location in a Pseudomonas nucleic acid sequence (e.g., ACCCC[X]nGCGGC (SEQ ID NO: 17) and ACCAA[X]nGCGGC (SEQ ID NO: 18), wherein n= 0, 1, 2, 3, 4, 5, 6 or more)) are described in FIG. 16. In some embodiments, a single repeating sequence unit [X] may comprise from about 1 to about 8 individual nucleotides. Additionally or alternatively, in some embodiments, the individual nucleotides present in a single repeating sequence unit [X] may be the same or distinct. As used herein, a “non-repeat region” refers to a region that lacks a repeating sequence unit in the context of a directly adjacent Pseudomonas nucleic acid sequence. [0071] As used herein, the term “sample” refers to clinical samples obtained from a patient or isolated microorganisms. In preferred embodiments, a sample is obtained from a biological source (z.e., a "biological sample"), such as tissue, bodily fluid, or microorganisms collected from a subject. Sample sources include, but are not limited to, mucus, sputum (processed or unprocessed), bronchial alveolar lavage (BAL), bronchial wash (BW), blood, bodily fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsy material).
[0072] The term “sensitivity,” as used herein in reference to the methods of the present technology, is a measure of the ability of a method to detect a preselected sequence variant in a heterogeneous population of sequences. A method has a sensitivity of S % for variants of F % if, given a sample in which the preselected sequence variant is present as at least F % of the sequences in the sample, the method can detect the preselected sequence at a preselected confidence of C %, S % of the time. By way of example, a method has a sensitivity of 90% for variants of 5% if, given a sample in which the preselected variant sequence is present as at least 5% of the sequences in the sample, the method can detect the preselected sequence at a preselected confidence of 99%, 9 out of 10 times (F=5%; C=99%; S=90%). Exemplary sensitivities include at least 50, 60, 70, 80, 90, 95, 98, and 99%.
[0073] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
[0074] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
[0075] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.
[0076] “Single base substitutions” or “SBS” are defined as a replacement of a single nucleotide base with another single nucleotide base. Exemplary possible substitutions (e.g., labels): OA, OG, OT, T>A, T>C, and T>G. These SBS classes can be further expanded considering the nucleotide context, e.g., considering not only the mutated base, but also the bases immediately 5’ and 3’. In some embodiments, a point mutation profile of a patient may be determined using the conventional 96 SBS mutation type classification or matrices.
[0077] As used herein, “SNVs” or “single nucleotide variants” are general terms for germline or somatic single nucleotide changes in a nucleic acid e.g., DNA, RNA) sequence.
[0078] Specificity,” as used herein, is a measure of the ability of a method to distinguish a truly occurring preselected sequence variant from sequencing artifacts or other closely related sequences. It is the ability to avoid false positive detections. False positive detections can arise from errors introduced into the sequence of interest during sample preparation, sequencing error, or inadvertent sequencing of closely related sequences like pseudo-genes or members of a gene family. A method has a specificity of X % if, when applied to a sample set of Niotai sequences, in which Xmie sequences are truly variant and X\<>t true are not truly variant, the method selects at least X % of the not truly variant as not variant. E.g., a method has a specificity of 90% if, when applied to a sample set of 1,000 sequences, in which 500 sequences are truly variant and 500 are not truly variant, the method selects 90% of the 500 not truly variant sequences as not variant. Exemplary specificities include at least 50, 60, 70, 80, 90, 95, 98, and 99%.
[0079] “Treating” or “treatment” as used herein covers the treatment of a disease or condition described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or condition, i.e., arresting its development; (ii) relieving a disease or condition, i.e., causing regression of the condition; (iii) slowing progression of the condition; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or condition. Therapeutic effects of treatment include, without limitation, inhibiting recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
[0080] It is also to be appreciated that the various modes of treatment of diseases as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition. Antibiotic compositions including the fluoroquinolones., carbapenems, cephalosporins., penicillin-based beta-lactams, antimicrobial peptides, and/or aminoglycosides
Quinolones
[0081] Quinolones are a class of antibiotics that all share a 4-quinolone bactericidal core structure. Fluoroquinolones are a sub-class of quinolone antibiotics that are known to interfere with bacterial DNA replication. Fluoroquinolones further incorporate fluorine into the structure, and are broad spectrum antibiotics. Quinolones include, but are not limited to, Enrofloxacin (Baytril), Ciprofloxacin (i.e., Cipro and Proquin), Enoxacin (i.e., Penetrex), Gatifloxacin (i.e., Gatiflo, Tequin and Zymar), Gemifloxacin (i.e,. Factive), Levofloxacin (i.e., Levaquin), Lomefloxacin (i.e., Maxaquin), Moxifloxacin (i.e., Avelox), Norfloxacin (i.e., Noroxin), Ofloxacin (i.e., Floxin), Prulifloxacin, Sparfloxacin (i.e., Zagam), Trovafloxacin/Altrofloxacin (i.e., Trovan), Danofloxacin (i.e., A180), Difloxacin (i.e., Dicural), Marbofloxacin (i.e., Orbax), Orbifloxacin (i.e., Zeniquin), Cinoxacin (i.e., Cinobac), Rosoxacin, Fleroxacin, Pefloxacin, Rufloxacin, Balofloxacin, Grepafloxacin, Pazufloxacin, Temafloxacin, Tosufloxacin, Besifloxacin, Clinafloxacin, Garenoxacin, Sitafloxacin, Ibafloxacin, Pradofloxacin, and Sarafloxacin, and conjugates thereof. In some embodiments, the methods of the present technology comprise selecting for administration or administering a fluroquinolone. In some embodiments, the methods of the present technology comprise selecting for administration or administering one or more of Enrofloxacin (Baytril), Ciprofloxacin (i.e., Cipro and Proquin), Enoxacin (i.e., Penetrex), Gatifloxacin (i.e., Gatiflo, Tequin and Zymar), Gemifloxacin (i.e,. Factive), Levofloxacin (i.e., Levaquin), Lomefloxacin (i.e., Maxaquin), Moxifloxacin (i.e., Avelox), Norfloxacin (i.e., Noroxin), Ofloxacin (i.e., Floxin), Prulifloxacin, Sparfloxacin (i.e., Zagam), Trovafloxacin/Altrofloxacin (i.e., Trovan), Danofloxacin (i.e., A180), Difloxacin (i.e., Dicural), Marbofloxacin (i.e., Orbax), Orbifloxacin (i.e., Zeniquin), Cinoxacin (i.e., Cinobac), Rosoxacin, Fleroxacin, Pefloxacin, Rufloxacin, Balofloxacin, Grepafloxacin, Pazufloxacin, Temafloxacin, Tosufloxacin, Besifloxacin, Clinafloxacin, Garenoxacin, Sitafloxacin, Ibafloxacin, Pradofloxacin, and Sarafloxacin, and conjugates thereof.
Carbapenems
[0082] Carbapenems are a class of antibiotics in the P-lactam family that inhibit bacterial cell-wall biosynthesis. Carbapenems are broad spectrum, acting against Gram-negative and Gram-positive bacteria. Carbapenems include, but are not limited to, Thienamycin, imipenem, meropenem, doripenem, ertapenem, biapenem, tebipenem, Meropenem-vaborbactam, Imipenem- relobactam, and conjugates thereof. In some embodiments, the methods of the present technology comprise selecting for administration or administering a carbapenem. In some embodiments, the methods of the present technology comprise selecting for administration or administering one or more of Thienamycin, imipenem, meropenem, doripenem, ertapenem, biapenem, tebipenem, Meropenem-vaborbactam, Imipenem-relobactam, and conjugates thereof.
Cephalosporins
[0083] Cephalosporins are bactericidal P-lactam antibiotics that inhibit enzymes in the cell wall of susceptible bacteria, disrupting cell wall synthesis. Cephalosporins are broad spectrum, acting against Gram-negative and Gram-positive bacteria. Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin, Cephalexin, Cephradine, Cefaclor, Cefotetan, Cefoxitin, Cefprozil, Cefuroxime, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftazidime-avibactam, Ceftibuten, Ceftriaxone, Cefepime, Ceftaroline-fosamil, Ceftobiprole/medocaril, Cefiderocol, Ceftolozane-tazobactam, moxalactam, and conjugates thereof. In some embodiments, the methods of the present technology comprise selecting for administration or administering a cephalosporin. In some embodiments, the methods of the present technology comprise selecting for administration or administering one or more of Cefadroxil, Cefazolin, Cephalexin, Cephradine, Cefaclor, Cefotetan, Cefoxitin, Cefprozil, Cefuroxime, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefoperazone/sulbactam, Cefotaxime, Cefpodoxime, Ceftazidime/avibactam, Ceftibuten, Ceftriaxone, Cefepime, Ceftaroline/fosamil, Ceftobiprole/medocaril, Cefiderocol Ceftolozane/tazobactam, moxalactam, and conjugates thereof.
Penicillin-based beta-lactams
[0084] Penicillin antibiotics are a broad-spectrum class of bactericidal P-lactam antibiotics. Penicillin antibiotics interrupt bacterial cell-wall formation by binding to essential penicillin- binding proteins (PBPs). Penicillin antibiotics include, but are not limited to, amoxicillin, amoxicillin/clavulanic acid, ampicillin, benzylpenicillin, benzathine benzylpenicillin, dicloxacillin, flucloxacillin, oxacillin, cioxacillin, nafcillin, carbenicillin, ticarcillin, temocillin, mecillinam, phenoxymethylpenicillin, mezlocillin, piperacillin, piperacillin-tazobactam, and conjugates thereof. In some embodiments, the methods of the present technology comprise selecting for administration or administering a penicillin antibiotic. In some embodiments, the methods of the present technology comprise selecting for administration or administering one or more of amoxicillin, amoxicillin/clavulanic acid, ampicillin, benzylpenicillin, benzathine benzylpenicillin, dicloxacillin, flucioxacillin, oxacillin, cioxacillin, nafcillin, carbenicillin, ticarcillin, temocillin, mecillinam, phenoxymethylpenicillin, piperacillin, piperacillin- tazobactam, and conjugates thereof.
Monobactams
[0085] Monobactams are a class of bactericidal P-lactam antibiotics that are monocyclic. Monobactams are typically used to treat aerobic Gram-negative bacterial infections. Monobactams include, but are not limited to, aztreonam, tigemonam, nocardicin A, carumonam, tabtoxin, BAL30072, and conjugates thereof. In some embodiments, the methods of the present technology comprise selecting for administration or administering a monobactam. In some embodiments, the methods of the present technology comprise selecting for administration or administering one or more of aztreonam, tigemonam, nocardicin A, carumonam, tabtoxin, BAL30072, and conjugates thereof.
Antimicrobial Peptides
[0086] Antimicrobial Peptides (AMPs), are a class of small peptides with antimicrobial activity, including against bacteria, viruses, fungi, and protozoa. Antimicrobial peptides include aniconic peptides, linear cationic a-helical peptides, cationic peptides enriched for specific amino acids, and aniconic/cationic peptides that form disulfide bonds. AMPs are numerous, and can include those described in Malak Pirtskhalava, et al., DBAASP v3: database of antimicrobial/cytotoxic activity and structure of peptides as a resource for development of new therapeutics, Nucleic Acids Research, Volume 49, Issue DI, 8 January 2021, Pages D288-D297. AMPs having anti-bacterial activity include, but are not limited to, Colistin, Dalbavancin, Daptomycin, Oritavancin, Polymyxin b, Teicoplanin, Telavancin, Vancomycin, Murepavadin, PMX-30063, Friulimicin B, PLG0206, IDR1, Omiganan, LTX-109, OP-145, DPK-060, NP101, NP108, Novexatin, Pl 13, Ctx(Ile21)-Ha, and conjugates thereof. In some embodiments, the methods of the present technology comprise selecting for administration or administering an antimicrobial peptide. In some embodiments, the methods of the present technology comprise selecting for administration or administering one or more of Colistin, Dalbavancin, Daptomycin, Oritavancin, Polymyxin b, Teicoplanin, Telavancin, Vancomycin, Murepavadin, PMX-30063, Friulimicin B, PLG0206, IDR1, Omiganan, LTX-109, OP-145, DPK-060, NP101, NP108, Novexatin, Pl 13, Ctx(Ile21)-Ha, and conjugates thereof. Aminoglycosides
[0087] Aminoglycosides are a class of broad-spectrum antibiotics for Gram-negative organisms that share an aminocyclitol ring. Aminoglycosides typically act by interfering with bacterial protein synthesis. Aminoglycosides include, but are not limited to, apramycin, Tobramycin, Amikacin, Gentamicin, neomycin, streptomycin, plazomycin, and conjugates thereof. In some embodiments, the methods of the present technology comprise selecting for administration or administering an aminoglycoside. In some embodiments, the methods of the present technology comprise selecting for administration or administering one or more of apramycin, Tobramycin, Amikacin, Gentamicin, neomycin, streptomycin, plazomycin, and conjugates thereof.
P-Lactamase inhibitors
[0088] P-lactamase enzymes confer resistance to P-lactam antibiotics, including penicillin antibiotics, by digesting the P-lactams. P-lactamase inhibitors are a class of compounds that inhibit P-lactamase enzymes, and can be used synergistically with P-lactam antibiotics. P- lactamase inhibitors include, but are not limited to, clavulanic acid, sulbactam, tazobactam, avibactam, relebactam, RG06080, RPX7009, and conjugates thereof. In some embodiments, a penicillin antibiotic and a P-lactamase inhibitor are administered simultaneously, sequentially, or separately, including as part of the same or different compositions. In some embodiments, the methods of the present technology further comprise sequentially, simultaneously, or separately administering a therapeutically effective amount of a P-lactamase inhibitor. In some embodiments, the P-lactamase inhibitor is selected from the group consisting of clavulanic acid, sulbactam, tazobactam, avibactam, relebactam, RG06080, RPX7009, and conjugates thereof.
Antibiotic Compositions
[0089] The pharmaceutical compositions of the present technology can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others. Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. In certain embodiments, the compositions disclosed herein are formulated for administration to a mammal, such as a human.
[0090] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
[0091] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Compositions formulated for parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion.
[0092] In order to prolong the effect of a compound of the present disclosure, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactidepolyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.
[0093] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents such as phosphates or carbonates.
[0094] Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. [0095] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
NGS platforms
[0096] In some embodiments, high throughput, massively parallel sequencing employs sequencing-by-synthesis with reversible dye terminators. In other embodiments, sequencing is performed via sequencing-by-ligation. In yet other embodiments, sequencing is single molecule sequencing. Examples of Next Generation Sequencing techniques include, but are not limited to pyrosequencing, Reversible dye-terminator sequencing, SOLiD sequencing, Ion semiconductor sequencing, Helioscope single molecule sequencing etc.
[0097] The Ion Torrent™ (Life Technologies, Carlsbad, CA) amplicon sequencing system employs a flow-based approach that detects pH changes caused by the release of hydrogen ions during incorporation of unmodified nucleotides in DNA replication. For use with this system, a sequencing library is initially produced by generating DNA fragments flanked by sequencing adapters. In some embodiments, these fragments can be clonally amplified on particles by emulsion PCR. The particles with the amplified template are then placed in a silicon semiconductor sequencing chip. During replication, the chip is flooded with one nucleotide after another, and if a nucleotide complements the DNA molecule in a particular microwell of the chip, then it will be incorporated. A proton is naturally released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH. The pH of the solution then changes in that well and is detected by the ion sensor. If homopolymer repeats are present in the template sequence, multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal. [0098] The 454TM GS FLX ™ sequencing system (Roche, Germany), employs a light-based detection methodology in a large-scale parallel pyrosequencing system. Pyrosequencing uses DNA polymerization, adding one nucleotide species at a time and detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates. For use with the 454™ system, adapter-ligated DNA fragments are fixed to small DNA-capture beads in a water-in-oil emulsion and amplified by PCR (emulsion PCR). Each DNA-bound bead is placed into a well on a picotiter plate and sequencing reagents are delivered across the wells of the plate. The four DNA nucleotides are added sequentially in a fixed order across the picotiter plate device during a sequencing run. During the nucleotide flow, millions of copies of DNA bound to each of the beads are sequenced in parallel. When a nucleotide complementary to the template strand is added to a well, the nucleotide is incorporated onto the existing DNA strand, generating a light signal that is recorded by a CCD camera in the instrument.
[0099] Sequencing technology based on reversible dye-terminators: DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed. Four types of reversible terminator bases (RT -bases) are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle.
[00100] Helicos's single-molecule sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface. At each cycle, DNA polymerase and a single species of fluorescently labeled nucleotide are added, resulting in template-dependent extension of the surface-immobilized primer-template duplexes. The reads are performed by the Helioscope sequencer. After acquisition of images tiling the full array, chemical cleavage and release of the fluorescent label permits the subsequent cycle of extension and imaging.
[00101] Sequencing by synthesis (SBS), like the "old style" dye-termination electrophoretic sequencing, relies on incorporation of nucleotides by a DNA polymerase to determine the base sequence. A DNA library with affixed adapters is denatured into single strands and grafted to a flow cell, followed by bridge amplification to form a high-density array of spots onto a glass chip. Reversible terminator methods use reversible versions of dye-terminators, adding one nucleotide at a time, detecting fluorescence at each position by repeated removal of the blocking group to allow polymerization of another nucleotide. The signal of nucleotide incorporation can vary with fluorescently labeled nucleotides, phosphate-driven light reactions and hydrogen ion sensing having all been used. Examples of SBS platforms include Illumina GA and HiSeq 2000. The MiSeq® personal sequencing system (Illumina, Inc.) also employs sequencing by synthesis with reversible terminator chemistry.
[00102] In contrast to the sequencing by synthesis method, the sequencing by ligation method uses a DNA ligase to determine the target sequence. This sequencing method relies on enzymatic ligation of oligonucleotides that are adjacent through local complementarity on a template DNA strand. This technology employs a partition of all possible oligonucleotides of a fixed length, labeled according to the sequenced position. Oligonucleotides are annealed and ligated and the preferential ligation by DNA ligase for matching sequences results in a dinucleotide encoded color space signal at that position (through the release of a fluorescently labeled probe that corresponds to a known nucleotide at a known position along the oligo). This method is primarily used by Life Technologies’ SOLiD™ sequencers. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing only copies of the same DNA molecule, are deposited on a solid planar substrate.
[00103] SMRT™ sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero-mode wave-guides (ZMWs)-small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labeled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring at the bottom of the well is detected. The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.
Prognostic and Theranostic Methods of the Present Technology
[00104] The methods of the present technology involve the use of trinucleotide mutation (SBS) profiles and indel spectra to predict antibiotic susceptibility in a patient diagnosed with or suffering from a Pseudomonas infection. SBS signatures result from recurring trinucleotide patterns of the transit! on/transversi on types of somatic single nucleotide variants (SNVs) and their flanking nucleotides, whereas indel signatures are defined according to size, nucleotides affected, and the presence of repetitive/microhomology regions.
[00105] In one aspect, the present disclosure provides a method for predicting the risk of antibiotic resistance in a subject diagnosed with or suffering from a Pseudomonas infection comprising sequencing Pseudomonas nucleic acids isolated from a biological sample obtained from the subject; generating a mutation spectrum from the sequenced Pseudomonas nucleic acids; and determining that the subject is at risk for antibiotic resistance when a Pseudomonas antibiotic-induced mutational signature is detected in the mutation spectrum. In some embodiments, the Pseudomonas infection is caused by a Pseudomonas species selected from among P. aeruginosa, P fluorescens, P putida, P cepacia, P stutzeri, P maltophilia, and P putrefaciens.
[00106] In another aspect, the present disclosure provides a method for selecting a subject infected with a Pseudomonas species for treatment with an antibiotic therapy comprising sequencing Pseudomonas nucleic acids isolated from a biological sample obtained from the subject; generating a mutation spectrum of the Pseudomonas nucleic acid sequences; detecting a Pseudomonas antibiotic-induced mutational signature in the mutation spectrum; and selecting an antibiotic therapy based on the detected Pseudomonas antibiotic-induced mutational signature. In some embodiments, the Pseudomonas species is selected from among P. aeruginosa, P fluorescens, P putida, P cepacia, P stutzeri, P maltophilia, and P putrefaciens. In some embodiments, the Pseudomonas species has been previously exposed to an antibiotic, whereas in other embodiments the Pseudomonas species has not been previously exposed to an antibiotic.
[00107] In some embodiments, generating the mutation spectrum comprises generating a plurality of single base substitution (SBS) contexts and a plurality of insertion/deletion (indel) profiles from the sequenced Pseudomonas nucleic acids. Combinatorial analysis using both SBS and indel profiles allows for the use of more complex mutational signatures. In some embodiments, each indel profile of the plurality of indel profiles comprise an indel size, a number of sequence unit repeats at an indel location, and the presence or absence of flanking microhomology sequences at an indel location.
[00108] In some embodiments, the indel size is about 1 base pair to about 10 base pairs. In some embodiments, the indel size is 1 base pair. In some embodiments, the indel size is 2 base pairs. In some embodiments, the indel size is 3 base pairs. In some embodiments, the indel size is 4 base pairs. In some embodiments, the indel size is 5 base pairs. In some embodiments, the indel size is 6 base pairs. In some embodiments, the indel size is 7 base pairs. In some embodiments, the indel size is 8 base pairs. In some embodiments, the indel size is 9 base pairs. In some embodiments, the indel size is 10 base pairs. [00109] In some embodiments, the number of sequence unit repeats at an indel location is about 0 to about 10. In some embodiments, the number of sequence unit repeats at an indel location is 0. In some embodiments, the number of sequence unit repeats at an indel location is 1. In some embodiments, the number of sequence unit repeats at an indel location is 2. In some embodiments, the number of sequence unit repeats at an indel location is 3. In some embodiments, the number of sequence unit repeats at an indel location is 4. In some embodiments, the number of sequence unit repeats at an indel location is 5. In some embodiments, the number of sequence unit repeats at an indel location is 6. In some embodiments, the number of sequence unit repeats at an indel location is 7. In some embodiments, the number of sequence unit repeats at an indel location is 8. In some embodiments, the number of sequence unit repeats at an indel location is 9. In some embodiments, the number of sequence unit repeats at an indel location is 10.
[00110] In some embodiments, the length of the flanking microhomology sequences at an indel location is about 1 base pair to about 10 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 1 base pair. In some embodiments, the length of the flanking microhomology sequences at an indel location is 2 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 3 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 4 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 5 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 6 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 7 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 8 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 9 base pairs. In some embodiments, the length of the flanking microhomology sequences at an indel location is 10 base pairs.
[00111] In some embodiments, the plurality of SBS contexts comprise a plurality of single base changes in a trinucleotide context, wherein the plurality of single base changes in the trinucleotide context comprises one or more transitions/transversions/substitutions selected from the group consisting of OA, OG, OT, T>A, T>C, T>G, G>T, G>C, G>A, A>T, A>G, and A>C. [00112] In any of the preceding embodiments, the Pseudomonas antibiotic-induced mutational signature comprises one or more of: an increase in OT transitions in NCG and GCN contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>A transitions in CGN and NGC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in T>C transitions in ATC, ATG, CTC, and CTG contexts relative to a reference Pseudomonas nucleic acid sample, an increase in A>G transitions in GAT, CAT, GAG, and CAG contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertions in non-repeat regions of the sequenced Pseudomonas nucleic acids relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the increase in C>T transitions in NCG and GCN contexts relative to a reference Pseudomonas nucleic acid sample, the increase in G>A transitions in CGN and NGC contexts relative to a reference Pseudomonas nucleic acid sample, the increase in T>C transitions in ATC, ATG, CTC, and CTG contexts relative to a reference Pseudomonas nucleic acid sample, the increase in A>G transitions in GAT, CAT, GAG, and CAG contexts relative to a reference Pseudomonas nucleic acid sample, and the increase in cytosine or guanine insertions in non-repeat regions of the sequenced Pseudomonas nucleic acids relative to a reference Pseudomonas nucleic acid sample are increases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample. In some embodiments, the increase in C>T transitions occurs in ACG and GCG contexts and the increase in G>A transitions occurs in CGT and CGC contexts.
[00113] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in OG transversions in ACG and GCC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>C transversions in CGT and GGC contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertion in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the increase in C>G transversions in ACG and GCC contexts relative to a reference Pseudomonas nucleic acid sample, the increase in G>C transversions in CGT and GGC contexts relative to a reference Pseudomonas nucleic acid sample, and the increase in cytosine or guanine insertion in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample are increases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, a carbapenem, an AMP, or an aminoglycoside.
[00114] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in T>C and A>G substitutions relative to a reference Pseudomonas nucleic acid sample, an increase in OG transversions in ACG and GCC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>C transversions in CGT and GGC contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertion in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the increase in T>C and A>G substitutions relative to a reference Pseudomonas nucleic acid sample, the increase in OG transversions in ACG and GCC contexts relative to a reference Pseudomonas nucleic acid sample, the increase in G>C transversions in CGT and GGC contexts relative to a reference Pseudomonas nucleic acid sample, and the increase in cytosine or guanine insertion in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample are increases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, a carbapenem, an AMP, or an aminoglycoside.
[00115] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase of 5+ base pair insertions in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample, OG transversions in ACG and GCC contexts that are comparable to a reference Pseudomonas nucleic acid sample, and G>C transversions in CGT and GGC contexts that are comparable to a reference Pseudomonas nucleic acid sample. In some embodiments, the increase of 5+ base pair insertions in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample is an increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, a carbapenem, an AMP, or an aminoglycoside.
[00116] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in OA substitutions in SCA contexts relative to a reference Pseudomonas nucleic acid sample, an increase in G>T substitutions in TGS contexts relative to a reference Pseudomonas nucleic acid sample, an increase in T>G and A>C substitutions relative to a reference Pseudomonas nucleic acid sample, a decrease in C >T substitutions in ACG contexts and G>A substitutions in CGT contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in at least one of (a) single thymine or adenine deletions, (b) single thymine or adenine insertions, (c) 5+ base pair deletions, (d) 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units, and (e) 5+ base pair deletions in regions with microhomology, relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the increase in OA substitutions in SCA contexts relative to a reference Pseudomonas nucleic acid sample, the increase in G>T substitutions in TGS contexts relative to a reference Pseudomonas nucleic acid sample, the increase in T>G and A>C substitutions relative to a reference Pseudomonas nucleic acid sample, and the increase in at least one of (a) single thymine or adenine deletions, (b) single thymine or adenine insertions, (c) 5+ base pair deletions, (d) 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units, and (e) 5+ base pair deletions in regions with microhomology, relative to a reference Pseudomonas nucleic acid sample are increases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample. In some embodiments, the decrease in C >T substitutions in ACG contexts and G>A substitutions in CGT contexts relative to a reference Pseudomonas nucleic acid sample is a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, an AMP, or an aminoglycoside.
[00117] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: a decrease in 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units relative to a reference Pseudomonas nucleic acid sample, and an increase in cytosine or guanine insertions in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the decrease in 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units relative to a reference Pseudomonas nucleic acid sample, is a decrease of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample. In some embodiments, the increase in cytosine or guanine insertions in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample is an increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a monobactam, a quinolone, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, or an antimicrobial peptide (AMP).
[00118] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in single nucleotide deletions relative to a reference Pseudomonas nucleic acid sample, an increase in 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units relative to a reference Pseudomonas nucleic acid sample, an increase in G transitions in GCT contexts and G>C transitions in AGC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in OA transitions in CCA, CCG, and GCA contexts and G>T transitions in TGG, CGG and TGC contexts relative to a reference Pseudomonas nucleic acid sample, and an increase in T>C and/or T>G substitutions relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the increase in single nucleotide deletions relative to a reference Pseudomonas nucleic acid sample, the increase in 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units relative to a reference Pseudomonas nucleic acid sample, the increase in OG transitions in GCT contexts and G>C transitions in AGC contexts relative to a reference Pseudomonas nucleic acid sample, the increase in C>A transitions in CCA, CCG, and GCA contexts and G>T transitions in TGG, CGG and TGC contexts relative to a reference Pseudomonas nucleic acid sample, and the increase in T>C and/or T>G substitutions relative to a reference Pseudomonas nucleic acid sample are increases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, a monobactam, or an antimicrobial peptide (AMP).
[00119] In some embodiments, the Pseudomonas antibiotic-induced mutational signature further comprises one or more of: an increase in OG transitions in GCT contexts and G>C transitions in AGC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in OA transitions in CCA, CCG, and GCA contexts and G>T transitions in TGG, CGG and TGC contexts relative to a reference Pseudomonas nucleic acid sample, an increase in T>C and/or T>G substitutions relative to a reference Pseudomonas nucleic acid sample, an increase in at least one of : (a) C >A substitutions in VNR contexts, (b) G>T substitutions in YNB contexts, (c) C >G substitutions in a GCG contexts, (d) G>C substitutions in CGC contexts, (e) T > G substitutions in ATN contexts, (f) A>C substitutions in NAT contexts, (g) T>A substitutions in GTT contexts, and (h) A>T substitutions in AAC contexts, relative to a reference Pseudomonas nucleic acid sample, and an increase in at least one of: (a) 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units, (b) 5+ base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 2 repeating sequence units, and (c) 5+ base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 5+ repeating sequence units with microhomology, relative to a reference Pseudomonas nucleic acid sample. In some embodiments, the increase in OG transitions in GCT contexts and G>C transitions in AGC contexts relative to a reference Pseudomonas nucleic acid sample, the increase in C>A transitions in CCA, CCG, and GCA contexts and G>T transitions in TGG, CGG and TGC contexts relative to a reference Pseudomonas nucleic acid sample, the increase in T>C and/or T>G substitutions relative to a reference Pseudomonas nucleic acid sample, the increase in at least one of : (a) C >A substitutions in VNR contexts, (b) G>T substitutions in YNB contexts, (c) C >G substitutions in a GCG contexts, (d) G>C substitutions in CGC contexts, (e) T > G substitutions in ATN contexts, (f) A>C substitutions in NAT contexts, (g) T>A substitutions in GTT contexts, and (h) A>T substitutions in AAC contexts, relative to a reference Pseudomonas nucleic acid sample, and the increase in at least one of: (a) 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units, (b) 5+ base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 2 repeating sequence units, and (c) 5+ base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 5+ repeating sequence units with microhomology, relative to a reference Pseudomonas nucleic acid sample are increases of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% relative to the reference sample. In some embodiments, the method further comprises administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, a carbapenem, an AMP, or an aminoglycoside.
[00120] In some embodiments, the reference Pseudomonas nucleic acid sample is obtained from a Pseudomonas control strain that is susceptible to antibiotic therapy, is a wild type MPA01 strain, and/or is a non-hypermutator strain. In some embodiments, the Pseudomonas control strain is susceptible to antibiotic therapy. In some embodiments, the Pseudomonas control strain is a wild type MPAO1 strain. In some embodiments, the Pseudomonas control strain is a non-hypermutator strain. In some embodiments, the reference Pseudomonas nucleic acid sample is obtained from a Pseudomonas control strain that is MMR proficient (pMMR). In other embodiments, the reference Pseudomonas nucleic acid sample is obtained from a Pseudomonas control strain that is MMR deficient (dMMR).
[00121] In any of the preceding embodiments, the biological sample comprises skin tissue, throat swabs, stool, urine, blood, lung tissue, stomach tissue, or urinary tract tissue. Samples can be collected using any appropriate means to ensure nucleic acid stability for further processing and analysis.
[00122] In any of the preceding embodiments, the Pseudomonas antibiotic-induced mutational signature is caused by prior exposure to an antibiotic. In some embodiments, the antibiotic is a P-lactamase inhibitor, a monobactam, a quinolone, an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, or an antimicrobial peptide (AMP).
[00123] In some embodiments, the antibiotic therapy comprises one or more antibiotics selected from the group consisting of a monobactam, a quinolone, an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, or an antimicrobial peptide (AMP). [00124] In some embodiments, the monobactam is selected from among aztreonam, azactam, tigemonam, nocardicin A, BAL30072, and tabtoxin.
[00125] In some embodiments, the quinolone is selected from among Enrofloxacin (Baytril), Ciprofloxacin (i.e., Cipro and Proquin), Enoxacin (i.e., Penetrex), Gatifloxacin (i.e., Gatiflo, Tequin and Zymar), Gemifloxacin (i.e,. Factive), Levofloxacin (i.e., Levaquin), Lomefloxacin (i.e., Maxaquin), Moxifloxacin (i.e., Avelox), Norfloxacin (i.e., Noroxin), Ofloxacin (i.e., Floxin), Prulifloxacin, Sparfloxacin (i.e., Zagam), Trovafloxacin/Altrofloxacin (i.e., Trovan), Danofloxacin (i.e., Al 80), Difloxacin (i.e., Dicural), Marbofloxacin (i.e., Orbax), Orbifloxacin (i.e., Zeniquin), Cinoxacin (i.e., Cinobac), Rosoxacin, Fleroxacin, Pefloxacin, Rufloxacin, Balofloxacin, Grepafloxacin, Pazufloxacin, Temafloxacin, Tosufloxacin, Besifloxacin, Clinafloxacin, Garenoxacin, Sitafloxacin, Ibafloxacin, Pradofloxacin, and Sarafloxacin.
[00126] In some embodiments, the carbapenem is selected from among Thienamycin, doripenem, ertapenem, biapenem, tebipenem, Meropenem, Meropenem-vaborbactam, Imipenem, and Imipenem-relobactam.
[00127] In some embodiments, the cephalosporin is selected from among Cefadroxil, Cefazolin, Cephalexin, Cephradine, Cefaclor, Cefotetan, Cefoxitin, Cefprozil, Cefuroxime, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftazidime-avibactam, Ceftibuten, Ceftriaxone, Cefepime, Ceftaroline-fosamil, Ceftobiprole/medocaril, Cefiderocol, Ceftolozane-tazobactam, and moxalactam.
[00128] In some embodiments, the penicillin-based beta-lactam is selected from among amoxicillin, amoxicillin/clavulanic acid, ampicillin, benzylpenicillin, benzathine benzylpenicillin, dicloxacillin, flucioxacillin, oxacillin, cioxacillin, nafcillin, carbenicillin, ticarcillin, temocillin, mecillinam, phenoxymethylpenicillin, mezlocillin, piperacillin, and piperacillin-tazobactam.
[00129] In some embodiments, the aminoglycoside is selected from among apramycin, Tobramycin, Amikacin, Gentamicin, neomycin, streptomycin, and plazomycin.
[00130] In some embodiments, the AMP is selected from among Colistin, Dalbavancin, Daptomycin, Oritavancin, Polymyxin b, Teicoplanin, Telavancin, Vancomycin, Murepavadin, PMX-30063, Friulimicin B, PLG0206, IDR1, Omiganan, LTX-109, OP-145, DPK-060, NP101, NP108, Novexatin, Pl 13, and Ctx(Ile21)-Ha. [00131] In some embodiments, the Pseudomonas infection is a cystic fibrosis (CF) infection, a respiratory tract infection (RTI), a urinary tract infection (UTI), a pressure sore infection, a bum infection, a wound infection, a bloodstream infection or an intra-abdominal infection (IAI).
[00132] In any of the preceding embodiments, the Pseudomonas nucleic acids are sequenced via whole genome sequencing (WGS).
[00133] In some embodiments, the method further comprises sequentially, simultaneously, or separately administering to the subject an effective amount of a beta-lactamase inhibitor. In some embodiments, the beta-lactamase inhibitor is selected from the group consisting of clavulanic acid, sulbactam, tazobactam, avibactam, relebactam, RG06080, and RPX7009.
Modes of Administration and Effective Dosages
[00134] Any method known to those in the art for contacting a cell, organ or tissue with one or more antibiotics (e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based betalactams, antimicrobial peptides, or aminoglycosides) disclosed herein may be employed.
Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of one or more antibiotics (e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides) to a mammal, suitably a human. When used in vivo for therapy, the one or more antibiotics (e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides) described herein are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the disease state of the subject, the characteristics of the particular antibiotic (e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides) used, e.g., its therapeutic index, and the subject’s history.
[00135] The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of one or more antibiotics (e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides) useful in the methods may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The antibiotic (e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides) may be administered systemically or locally.
[00136] The one or more antibiotics (e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides) described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment or prevention of a disease or condition described herein. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
[00137] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment).
[00138] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
[00139] The pharmaceutical compositions having one or more antibiotics (e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides) disclosed herein can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
[00140] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[00141] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
[00142] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.
[00143] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed by iontophoresis.
[00144] A therapeutic agent can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic agent is encapsulated in a liposome while maintaining the agent’s structural integrity. One skilled in the art would appreciate that there are a variety of methods to prepare liposomes. (See Lichtenberg, et al. , Methods Biochem. Anal., 33:337-462 (1988);
Anselem, et al., Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.
[00145] The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic agent can be embedded in the polymer matrix, while maintaining the agent’s structural integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly a-hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother ., 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).
[00146] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99/15154 (Tracy, et al.), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale, et al.), PCT publication WO 96/40073 (Zale, et al.), and PCT publication WO 00/38651 (Shah, et al.). U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.
[00147] In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
[00148] The therapeutic compounds can also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, e.g., Chonn and Cullis, “Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in Biotechnology 6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods, 4(3):201-9 (1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery: Progress and Problems,” Trends BiotechnoL, 13(12):527-37 (1995). Mizguchi, et al., Cancer Lett., 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro.
[00149] Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
[00150] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine useful doses in humans accurately. Levels in plasma may be measured, for example, by high performance liquid chromatography.
[00151] Typically, an effective amount of the one or more antibiotics (e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides) disclosed herein sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of the therapeutic compound ranges from 0.001- 10,000 micrograms per kg body weight. In one embodiment, one or more antibiotic (e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides) concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
[00152] In some embodiments, a therapeutically effective amount of one or more antibiotics (e.g., fluoroquinolones, carbapenems, cephalosporins, penicillin-based beta-lactams, antimicrobial peptides, or aminoglycosides) may be defined as a concentration of inhibitor at the target tissue of 10'32 to 10'6 molar, e.g., approximately 10'7 molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).
[00153] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
[00154] The mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human.
EXAMPLES
[00155] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this present application. The present application will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the present application but, of course, should not be construed as in any way limiting its scope. Example 1: Materials and methods for Examples 1-5 [00156] Human subject isolate collection
[00157] Adult subjects with CF or non-CF bronchiectasis (8 male, 9 female) and known history of P. aeruginosa chronic respiratory colonization were recruited from Tulane Medical Center (Tulane IRB 2019-1840) and informed consent was obtained. For SI until S9: spontaneous sputum samples were collected in sterile screw-cap cups for processing. Samples were processed within 24 hours of collection, kept at 4° C overnight if needed or processed directly from room temperature if within 4 hours of collection. Sputum samples were processed with equal volume 6.5mM DTT, vortexed for 45 seconds and rotated for 30 minutes. Processed sputum was streaked onto PIA for P. aeruginosa isolation. Colonies with distinct morphotypes were collected as different strains from each patient sample and pure-cultured on PIA. For S 10 until SI 9, sputum samples were processed by the clinical microbiology laboratory at Tulane Medical Center. Pure-cultured plates were then obtained.
[00158] Bacterial strains and growth conditions
[00159] For this work, MPA01 and insertional transposon knockouts, MP AO1 - IkmutS and MP AO1 - HmutL, were used. Transposon knockouts were genotypically verified by PCR and phenotypically validated as hypermutators via rifampicin reversion before experimentation.
[00160] WT parent strain MPA01 and transposon mutants MPA01 -AmutS (strain PW7149), MP AO1 -AmutL (strain PW5709), MPAOl- Z 4 (strain PW8371), MPAO I - /'c/k (strain PW9164), MPAO1 -ApilD (strain PW8672), MPAOl- rA/C (strain PW3043), and MPAO1- ApmrB (strain PW9023) were purchased from the Pseudomonas aeruginosa two allele transposon library of Colin Manoil, PhD at the University of Washington (funded by grant no. NIH P30 DK089507).284,285 P. aeruginosa strains were initially streaked on Pseudomonas Isolation Agar (PIA) (BD Difco). All strains were cultured in Luria Bertani (LB) broth (Miller) (VWR Life Sciences) at 37°C at 220 rpm for 18 hours to make glycerol (40% v/v) frozen stocks stored at -80°C prior to experimentation. An initial set of 3 glycerol stocks were made for all strains, and the working glycerol stock was replaced when colony density was significantly decreased upon streaking on LB.
[00161] Confirmation of insertional transposon knockout mutants
[00162] Insertional transposon mutants were confirmed via polymerase chain reaction (PCR) with gene-flanking and transposon-annealing primer sets. Primer sequences and sets used for each confirmation reaction are found in Table 1 and Table 2, respectively. Final reaction conditions were as follows: 0.4 pM forward and reverse primers, < 1 pg template DNA (genomic DNA from tested strain), IX LongAmp Taq Master Mix (Thermo Fisher Scientific), and nuclease-free FEO up to 25 pL. Thermocycling conditions were as follows: Denaturation at 94 °C for 5 minutes, 30 cycles of amplification (94 °C for 30 seconds, optimal Tm for 60 seconds (Table 1), 65 °C for 60 seconds), and final extension at 65 °C for 10 minutes. Primer sets were first optimized on MPA01. PCR products were visualized on 1% agarose gels via electrophoresis and mutant expected sizes were confirmed (Table 2).
[00163] Table 1: Sequences of PCR primers. Primers either flank gene insertion site or anneal to insertional transposon used to confirm insertional transposon mutagenesis. Optimized Tm for each primer pair is reported. Table 1 shows SEQ ID NOs: 1-16 in order of appearance.
[00164] Table 2 PCR reactions and expected sizes. PCR reactions to confirm each insertional transposon mutant and corresponding expected sizes upon amplification of genomic DNA from the respective knockout strain are reported.
[00165] Rifampicin reversion mutant frequency assays
[00166] Bacteria were streaked from glycerol stocks on LB agar, and independent triplicate colonies were inoculated into 10 mL Cation- Adjusted Mueller-Hinton broth (CAMHB) (BD BBL) and incubated for 18 hours overnight. 1 mL of each culture was pelleted at 4,000 x g for 10 minutes, and the pellet was washed with sterile IX phosphate buffered saline (PBS) 3 times. The pellet was resuspended in 1 mL IX PBS and serially diluted 10-fold in IX PBS. Dilutions were spotted (10 pL) and spread (100 pL) on CAMHB agar and CAMHB agar containing 100 pg/mL rifampicin and incubated at 37°C for 24 hours. Mutant frequency was determined by the following:14 colonies grown on CAMHB + rifampicin Mutant frequency = - - - x 100 colonies grown on CAMHB
[00167] All mutant frequences were normalized to that of WT MPA01 (1 O’8) and are therefore expressed as Mutant frequency x 10'8 viable cells.
[00168] DNA purification
[00169] For subject isolates, isolates were streaked onto LB and statically grown for 24-48 hours (depending on normal/slow-growth phenotype) at 37 °C. One colony/strain was then inoculated into 5 mL LB and grown at 37 °C shaking at 220 rpm for 18-48 hours (depending on normal/slow-growth phenotype). Liquid culture was then used for downstream DNA purification.
[00170] Aliquoted cultures of evolved clones from each lineage were stored at -80°C after time of emergence of resistance and experimental endpoint. Frozen aliquoted cultures were thawed and pelleted (500 pL) at 4,000 x g for 10 minutes, and the supernatant was discarded. Genomic DNA was isolated using a Qiagen DNeasy Blood and Tissue Kit per manufacturer’s instructions. DNA quality and concentration was determined via absorbance at 260 nm, 280 nm, and 230 nm.
[00171] Whole genome sequencing and variant calling
[00172] Genomic DNA preps from desired resistant and control lineages were whole genome sequenced using Illumina short-read sequencing (NextSeq 2000). Sample libraries were prepared using Illumina DNA Prep Kit and IDT lObp UDI indices. Demultiplexing, quality control (only reads with Q>30 kept), and adapter trimming were performed using Illumina bcl-convert (v3.9.3). Each sample produced a minimum of 400 Mbp high quality reads (2 x 151 bp), with an average depth of coverage of 60X of the ~6.3 Mbp genome.
[00173] Paired end reads were aligned to the PAO1 reference genome (NCBI accession #NC_002516.2) and variants were called to reference using breseq (vO.36.1).286 All variants called were at 100% frequency unless otherwise specified.
[00174] For all analyzed clinical isolate, nonsynonymous mutations called to PAO1 using breseq were notated in the following genes: mutS, mull., uvrl). mexR, mexA, mexB, oprM, oprD, mexY, mexX, mexZ, mexS, mexT, mexE, mexF, oprN, nalD, nalC, armR, oprJ, mexD, mexC, njxB, pmrA, pmrB, opr86.
[00175] For in vitro induction of mutational signatures associated with aztreonam or ciprofloxacin and MPAO1 -EmutS lab evolved clones, breseq (polymorphism mode) was used and variants down to 5% frequency were called.
[00176] De novo genome assembly of subject isolates
[00177] FASTQ reads from SlO Pa 1, SP10 Pa2, SP11 Pal, S12 Pal, S12 Pa2, S12 Pa3, S7T2 Pal, S14 Pal, S15 Pal, S16 Pal, S17 Pal, S18 Pal, S18 Pa2, and S6T2 Pal were de novo assembled using unicycler^9 with default parameters. Assembly statistics were recorded with quasfi50 (default parameters) and samples annotated using bakta35i (default parameters).
[00178] Mutational signature analysis
[00179] ‘ Batches’ for deduplication included: all lab evolved strains, all prospectively clinical isolates from pwCF, retrospective clinical isolates from the dataset of Lopez-Causape et al.,321 retrospective clinical isolates from the dataset of Kos et al.,352 and retrospective clinical isolates from UPMC. For antibiotic-treated lab evolved strains, individual spectra from all 30 replicates/treatment were compiled using tidyverse package dplyr and de facto mutational signatures from each treatment plotted with ggplot2.
[00180] Variant call format (VCF) files containing all variants called to PAO1 for each sample were deduplicated using bcftools2 1, removing any variant shared with at least one other sample from all samples, to create unique VCFs for each sample. For the construction of PaA/7w/,S', only the final timepoints for each independent lineage were included, excluding longitudinal sequences from the same lineage at different timepoints. This was done to avoid inappropriate deduplication of potential de novo mutations that would be shared among timepoints from the same lineage.
[00181] Unique VCFs were loaded into and parsed in R using the tidyverse suite.288 For each mutation, the reference base was retrieved from the PAO1 reference genome sequence along with flanking reference bases on both the 3’ and 5’ ends to produce the trinucleotide mutation context. Where necessary, trinucleotide contexts were converted to their reverse complement to reflect one of the canonical 6 types of bases changes (C>{A,G,Tj or T>{A,C,G}). Individual mutation spectra were plotted in their 96-trinucleotide context.
[00182] Due to few total detected de novo mutations, all mutations from MP AO1 - ^nmlS samples were also compiled into one signature using tidyverse2** package dplyr and de facto mutational signature plotted with ggplot2. Established human (hg38) MMR-deficiency- associated mutational signatures were retrieved from the Catalogue of Somatic Mutations in Cancer (COSMIC; SBS6/15/21/26/44). Human MMR signatures were also additively combined into a compiled signature and proportions were normalized by the number of combined signatures (n=5). Cosine similarities between the mutation distributions of relevant samples and the COSMIC MMR-associated signatures were calculated using via the R package MutationalP atter ns .289
[00183] In vitro adaptive evolution
[00184] A selection of prospectively collected P. aeruginosa isolated from subjects with bronchiectasis were passaged in aztreonam and colistin to observe resistance acquisition. See
[00185] Antimicrobial preparation [00186] 20X solutions (Table 3) for each tested antimicrobial were prepared (Table 3). For all antibiotics, the solvent was nuclease-free, sterile-filtered (0.2 pM filter) ddFLO. Solutions were aliquoted (1 mL) and stored at -20 °C.
[00187] D-CONGA and D-CONGA-Q7 were synthesized using Fmoc solid-phase chemistry and purified to >95% via high performance liquid chromatography by Bio-synthesis Inc, with identity confirmed via MALDI mass spectrometry. Solutions were prepared by dissolving desired mass into 0.025% (v/v) acetic acid in water, and peptide concentration was determined by absorbance at 280 nm.
[00188] Table 3: Concentration of antimicrobial solutions used. All antimicrobials were dissolved in H2O, except D-CONGA and D-CONGA-Q7, which were dissolved in 0.25% acetic acid.
Antimicrobial 20X 2X IX
Chloramphenicol 8 mg/mL 800 pg/mL 400 pg/mL
Streptomycin 1.28 mg/mL 128 pg/mL 64 pg/mL
Ciprofloxacin 320 pg/mL 32 pg/mL 16 pg/mL
Aztreonam 2.56 mg/mL 256 pg/mL 128 pg/mL
Polymyxin B 640 pg/mL 64 pg/mL 32 pg/mL
Colistin 320 pg/mL 32 pg/mL 16 pg/mL
D-CONGA 1280 pM 128 pM 64 pM
D-CONGA-Q7 1280 pM 128 pM 64 pM
[00189] Antimicrobial susceptibility testing
[00190] MICs were determined via microbroth dilution assay in 96-well culture plates with 2- fold serial dilutions of tested antibiotic in CAMHB, inoculated with 50 pL of 2.75 x 105 CFU/mL of each bacterial strain and incubated at 37°C, 200 rpm for 24 hours. The IX concentration reported for each antimicrobial is the ‘starting’ concentration in Row A of the 96- well plate, with Row B being 1/2X, Row C 1/4X, Row D 1/8X, Row E 1/16X, Row F 1/32X, Row G 1/64X, and Row H no antibiotics (positive control) (Table 3). MIC experiments were performed using biological triplicates (3 independent colonies/strain) plus a negative control (no inoculum) per strain/antimicrobial combination. This allowed for testing of 3 strain/antimicrobial combinations per 96-well plate (12 columns total, with 4 columns per combination).
[00191] In vitro adaptive evolution
[00192] Serial passaging of bacterial strains was performed in 96-well culture plates with 2- fold serial dilutions of antibiotic in CAMHB, inoculated with 50 pL 2.75 x 105 CFU/mL of each strain and incubated at 37°C, 200 rpm for 24 hours. See Antimicrobial susceptibility testing for more details. Next, bacteria taken from the well with the highest concentration of antibiotic that still exhibited growth (compared to negative control with no inoculum) was diluted 1 : 100 into 5 mL of fresh LB containing no antibiotics and grown overnight. The bacteria were then serially passaged in the same antibiotic. The bacteria were serially passaged a total of 10 times.
[00193] MICs were determined for all antibiotics (aztreonam, ciprofloxacin, D-CONGA, D- CONGA-Q7, polymyxin B) after 1, 4, 7, and 10 complete passages in treatment (see Antimicrobial susceptibility testing). 2.75 x 105 CFU/mL from the overnight LB culture following the denoted passage number (e.g. the overnight LB culture grown after 4 passages in AZ for ‘Passage 4’ AZ-induced cross-resistance data) were used as inoculum.
[00194] Public dataset analyses
[00195] FASTQ files for all samples from datasets of Lopez-Causape et al.321 and Kos et al.352 were retrieved from NCBI Sequence Read Archive (SRA) Run Selector using sra-tools. From the dataset of Lopez-Causape et al., the following samples were excluded from this study due to missing or insufficient quality reads: 004-526, 006-7204, 021-9884. 021-2955, 021-4234, 022-5179, 022-5546, 023-2344, 023-6966, 023-9557, 024-1092, 024-7416, 025-6546, 025-9260, 025-7986, OB2 38, OB2 50, OB2 23. From the dataset of Kos et al., the following samples were excluded from this study due to missing or insufficient quality reads: AZPAE13756, AZPAE13757, AZPAE13848, AZPAE13850, AZPAE13853, AZPAE13856, AZPAE13858, AZPAE13860, AZPAE13864, AZPAE13866, AZPAE13872, AZPAE13876, AZPAE13877, AZPAE13879, AZPAE13880, AZPAE14352, AZPAE14353, AZPAE14359, AZPAE14372, AZPAE14373, AZPAE14379, AZPAE14381, AZPAE14390, AZPAE14393, AZPAE14394, AZPAE14395, AZPAE14398, AZPAE14402, AZPAE14403, AZPAE14404, AZPAE14410, AZPAE14415, AZPAE14422, AZPAE14437, AZPAE14441, AZPAE14442, AZPAE14443, AZPAE14453, AZPAE14463, AZPAE14499, AZPAE14505, AZPAE14509, AZPAE14526, AZPAE14533, AZPAE14535, AZPAE14538, AZPAE14550, AZPAE14554, AZPAE14557, AZPAE14566, AZPAE14570, AZPAE14687, AZPAE14689, AZPAE14690, AZPAE14691, AZPAE14692, AZPAE14693, AZPAE14835, AZPAE14886, AZPAE14887, AZPAE14941, AZPAE14949, AZP AE15042.
[00196] In vitro induction of antibiotic-associated mutational signatures
[00197] 30 biological replicates (independent colonies) of MPAO1 were grown in 5 mL LB overnight (see Bacterial strains and growth conditions'). In separate 2 mL LB cultures, each replicate was treated with 1/10 MIC of either aztreonam (200 ng/mL) or ciprofloxacin (50 ng/mL) and inoculated with 103 CFU. All cultures were incubated overnight. Induced mutagenesis was confirmed using rifampicin reversion (see Rifampicin reversion mutant frequency assay) and then cultures were used for downstream DNA isolation, WGS and mutational signature analysis (See DNA purification, Whole genome sequencing and variant calling, and Mutational signature analysis).
[00198] De novo signature extraction
[00199] De novo mutational signature extraction was performed via non-negative matrix factorization (NMF)166 using the R Package MutationalP atterns 2 9 Batches for de novo extraction are as follows: all clinical isolates (prospective collection, samples from Lopez- Causape et a., samples from Kos et al., retrospective samples from UPMC) and all lab clones treated with aztreonam or ciprofloxacin.
[00200] Statistical analyses
[00201] Fisher’s exact test was used to assess correlation of predicted MMR-status with MDR. Mutational signatures were correlated to resistance patterns using two-sample Kolmogorov- Smirnov tests (one for each factor-candidate signature pairing) followed by Benjamini -Hochberg procedure for controlling false discovery rate to control for multiple comparisons.166 All figures were created and statistical tests were done using GraphPad Prism
9.3.1. Example 2: Evaluation of mutational signature analysis for prediction of MMR-status and MDR acquisition in prospective and retrospective chronic respiratory isolates of P. aeruginosa
[00202] Our results thus far suggest that MMR-deficiency produces a distinct mutational signature and drives rapid MDR acquisition, but that this can be targeted with rational combinations of treatments. We speculated that identification of the MMR-deficiency-associated mutational signature itself could be a predictor of rapid drug resistance acquisition and potentially guide targeted treatment with combination therapy. To test this, we analyzed WGS from the 26 prospectively collected P. aeruginosa from CF or non-CF bronchiectasis (data not shown). Following variant calling to PAO1, we plotted trinucleotide mutation spectra of only mutations unique to each sample.
[00203] Trinucleotide mutation spectra from 7 subject isolates showed similar OT enrichment in NCC and NCG and reduction in NCT and T>C enrichment in CTN and GTN that we saw with PaA/7?///A (FIG. 1A), whereas the spectra of the other 19 isolates did not display all of these characteristics (FIG. IB). These 7 isolates had overall elevated levels of unique single nucleotide variants (SNVs) and indels (FIG. 2). Quantitative assessment via cosine similarity showed that spectra from these same 7 isolates were most similar to PaA/7?///A (FIG. 3). From cosine similarity analysis and clustering results, all isolates with a cosine similarity with PaA/7?///A above 0.78 were predicted to be MMR-deficient, and all below 0.78 as WT, resulting in 7 predicted MMR-deficient isolates and 19 WT (27% isolates MMR-deficient).
[00204] Interestingly, different subject isolates clustered based on decreasing degrees of similarity to the P. aeruginosa MMR-deficient mutational signature, with some being more similar to HumanAMMR, SBS6, or SBS15 than PatsmutS (FIG. 3). This same cluster (S18 Pal to S16 Pal) also showed elevated similarity to SBS44 compared to PaAmz/tA These six isolates show OT enrichment but were lacking the dramatic OT diminishment in NCT contexts and T>C enrichment, suggesting these are key distinguishing factors for prediction of MMR-status.
[00205] Another notable finding was that the mutation spectrum of S6T1 Pa2 resembled PaAmwAS', while the spectrum of S6T1 Pal did not (FIGs. 1A and IB). Both isolates were collected from the same patient at the same time indicating infections from distinct isolates. A longitudinal isolate from the same subject, S6T2 Pal, did not resemble Pa mutS either (FIG. IB) Our results predicted that S6T1 Pa2 would be MMR-deficient and more rapidly acquire MDR during in vitro experiments than the predicted MMR-proficient S6T1 Pal or S6T2 Pal. [00206] Predictions of MMR-status were functionally validated by assessing for hypermutator phenotype via rifampicin reversion frequency (FIG. 4). Additionally, all predicted MMR- deficient isolates had a nonsynonymous mutation in mutS, mull.. or uvrD. However, many validated WT isolates also had mutations in an MMR gene, indicating that assessing for genotype is an inaccurate determinant of MMR-status (FIG. 4).
[00207] We next asked how well the PaA/izzz/A mutational signature can predict rapid drug resistance in clinical isolates. We measured resistance acquisition and efficacy of combination therapy in a set of predicted MMR-deficient and WT patient samples in vitro. We then evolved strains in AZ alone, COL alone, and combined AZ + COL and measured resistance acquisition (data not shown). All three isolates demonstrating starting clinical resistance to AZ (S6T1 Pa2, S8 Pal, and S9 Pal) acquired further resistance to AZ alone (FIG. 5A, gray lines) and rapidly acquired resistance to COL (FIG. 5B, gray lines). S9 Pa2 was heterogeneously susceptible to both monotherapies, but all clones rapidly acquired resistance similar to MMR-deficient P. aeruginosa laboratory strains (FIGs. 5A and 5B). Together, these results indicate that mutational signature analysis can prospectively predict future rapid MDR acquisition. Without wishing to be bound by theory, it is believed that mutational signature analysis coupled with rational combination therapy is a promising precision medicine approach that can prevent the emergence of MMR-deficiency-induced MDR in vitro.
[00208] Although these results strongly suggest the diagnostic promise of mutational signature analysis, our test population was limited to subjects from one geographic area (LA, USA). To expand the breadth of our work’s translation, we applied our pipeline on publicly available WGS reads of 131 P. aeruginosa isolates from 50 pwCF in Spain.321 Trinucleotide mutation spectra from 17 isolates strongly resembled PaA/izzz/A and showed enrichment and diminishment in key discussed C>T and T>C contexts (FIG. 6A). Cosine similarity analysis predicted these 17 isolates as MMR-deficient and the remaining 114 as WT (13% MMR- deficient). A small cluster of 6 isolates showed higher similarity to HumanAMMR, SBS6, and SBS15 than PaA/izzz/A (FIG. 6B). Similar to our collected isolates, predicted MMR-deficient and WT isolates were indistinguishable by MMR genotype (FIG. 6C).
[00209] These isolates had accompanying data on clinical resistance and hypermutator phenotype, allowing us the opportunity to validate our predictions. Of the 17 predicted MMR- deficient isolates, 11 were reported as hypermutators via rifampicin reversion (FIG. 6D, upper left). All of the 6 remaining predicted MMR-deficient that were not hypermutators (FIG. 6D, bottom left), however, clustered with the PaDmulS samples (FIG. 6B, stars). This suggests that these samples were at some point MMR-deficient. The remaining 7 predicted WT isolates that were reported to be hypermutators (FIG. 6D, upper right) did not cluster with the VaDmutS samples (FIG. 6B, circles). The hypermutator phenotypes in these samples are candidates for alternative causes. Despite incomplete agreement with hypermutator phenotype, predicted MMR-deficient isolates show strong correlation (p = 0.0021 via Fisher’s exact) with MDR (defined by ‘R’ via EUCAST to >3 drugs) (FIG. 6E), as does the hypermutator phenotype like previously documented (FIG. 6F).194 Our application of mutational signature analysis in clinical isolates of P. aeruginosa shows that it can rather robustly, easily, and accurately screen for MMR-deficiency and identify isolates prone to rapid MDR acquisition or enriched for MDR.
Example 3: Applying mutational signature analysis in acute disease contexts
[00210] Previous studies linking MMR-deficiency and MDR in P. aeruginosa were largely focused on isolates from pwCF or other chronic lung infection contexts.155,194’272’277 An early study screening isolates from intensive care units attributed the contribution of hypermutation in acute P. aeruginosa infections to <1%.269 However, a few recent clinical case studies have suggested that MMR-deficient P. aeruginosa may be playing a more prominent role in acute disease contexts.195,203
[00211] We used mutational signature analysis to investigate the frequency of MMR- deficiency using publicly available datasets of WGS reads from 325 P. aeruginosa clinical isolates in four important human disease contexts: respiratory tract infections (RTIs), urinary tract infections (UTIs), intraabdominal infections (IAIS), and pwCF. Trinucleotide spectra from 22 isolates were predicted to be MMR-deficient via qualitative similarity with PaA/iw/A' (FIG. 7 A) and cosine similarity analysis (FIG. 7B). Predicted MMR-deficient isolates were enriched in pwCF (31% of the total pwCF isolates), similar to previous reports,155,272 but were also found at significant levels in RTIs (5.5% of the total RTI isolates), UTIs (2.8% of the total UTI isolates), and IAIs (2.7% of the total IAI isolates) (FIG. 7C). We again observed a cluster of isolates with similarity to SBS6 that appeared across all four disease contexts (FIG. 7B). MMR-deficient isolates from pwCF were significantly correlated (p = 0.0256) with MDR (FIG. 7D) as seen previously.194 Predicted MMR-deficient isolates trend towards higher rates of MDR in RTIs (FIG. 7E) and UTIs (FIG. 7F), but not IAIs (Figure 7G). These results illuminate a potential larger and more appreciable role of MMR-deficiency in acute infections of P. aeruginosa than previously accepted. [00212] P. aeruginosa is one of the primary causes of VAP and results in a 13.5% mortality rate in this context.240 In MDR strains, however, attributable mortality is over 30%.239 Previous studies have documented MMR-deficient strains in VAP cases.203 VAP-causing strains tend to be enriched for MDR due to extensive previous drug exposure and hospital circulation.6 Therefore, we reasoned that MMR-deficiency could be enriched in MDR P. aeruginosa causing VAP.
[00213] To test this, we investigated a retrospective cohort of 102 subjects with P. aeruginosa VAP collected at University of Pittsburgh Medical Center. All included subjects were initially treated with ceftolozane-tazobactam upon first positive P. aeruginosa culture. Ceftolozane- tazobactam is a new generation cephalosporin P-lactamase combination antibiotic often used to treat MDR . aeruginosa VAP,353 and therefore infections requiring this treatment are likely enriched for MDR.
[00214] Whole genomes of baseline isolates (i.e. the first isolate recorded for each subject) were aligned and variants called to PAO1, and trinucleotide spectra of unique variants were plotted. Five isolates qualitatively resembled (FIG. 8A) and clustered strongly with the PaA/iw/A' signature (FIG. 9, dark grey stars), and thus were predicted as MMR-deficient. Again, we see a cluster with strong enrichment to SBS6 and 15 (FIG. 9).
[00215] Spectra from an additional five isolates closely resembled PaA/iw/A' (FIG. 8B) and had cosine similarities closely approaching (0.75-0.77) our quantitative threshold (0.78) for distinguishing MMR-deficient and WT isolates. Compared to the former five isolates, these isolates cluster further away from PaA/iw/A' and show elevated similarity to SBS6, SBS15, and SBS44 (FIG. 9, light gray stars). They additionally show notably more mutations in C>A, C>G, T>A, and T>G (FIGs. 8A and 8B). Three out of five isolates show enrichment in GTA>GTG and all show enrichment in CCG>CAG and GCG>GAG (FIG. 8B). Higher mutation burdens in other contexts, which can be due to other mutagenic processes, could be artificially decreasing the cosine similarity to PaA/iw/A' and altering clustering patterns. Consequently, these results suggest that the proportion of MMR-deficiency in VAP contexts could be as high as 10%.
Example 4: Refining the MMR-deficiency-associated mutational signature in P. aeruginosa [00216] A potential weakness of applying the mutational signature acquired by MMR- deficient lab strains of P. aeruginosa as a predictor of MMR-status in clinical isolates is the large difference in the genetic background of lab strains and clinical isolates. Additionally, using the signature made from only one strain may not apply well to all clinical isolates due their genetic heterogeneity. With now having a robust set of genomic data from clinical isolates from a diverse range of disease contexts and geographic locations, we next sought to employ NMF for de novo signature extraction.
[00217] After non-negative matrix factorization (NMF) iterations, we stably extracted three distinct signatures from the cumulative 589 clinical isolates (FIG. 10, V1-V3). V3 remarkably resembles PaA/iw/A', with a 0.98 cosine similarity (FIG. 10, V3 and MMR-). We speculate that VI and V2 appear to be cumulations of, rather than distinct, signatures and could be clarified with further rounds of NMF on larger datasets. These results promisingly show that a lab- constructed signature can highly resemble a computationally extracted one of the same mutagenic processes, and additionally validate our previous findings using PaA/iw/A' to predict MMR-status in clinical isolates.
Example 5: Discovery and application of antibiotic-induced mutational signatures to predict drug resistance
[00218] Interestingly, in all four clinical isolate datasets analyzed, we observed clustering of isolates with high cosine similarity to human SBS6 and sometimes human SBS15 but with less similarity to PaA/iw/A'. Along with selecting for hypermutators, antibiotic treatment is also known to transiently increase mutation rates via /'/ A-mediated suppression of mutS and upregulation of DNA polymerases IV (DinB) and V (UmuD’2C).354 359 DNA polymerases IV and V are translesion synthesis (TLS) polymerases lacking a 3’— >5’ exonuclease activity and are thus error-prone,360 362 and they are hypothesized to not only help bacterial cells overcome antibiotic-induced DNA damage after treatment but also to promote adaptive evolution.363 DinB favors OT transitions during synthesis, similar to SBS6 and 15.87,162 From this, we hypothesized that the clusters of SBS6 and 15 enriched clinical isolates observed in all datasets could be a signal for previous antibiotic-induced mutagenesis. Thus, we aimed to discover and characterize mutational signatures attributable to antibiotic treatment for the first time.
[00219] We chose to start with ciprofloxacin and aztreonam, as both are from drug classes previously shown to induce mutagenesis in bacteria,106,182 such as P. aeruginosa and E. coh. and are commonly used to treat pwCF and are used consistently throughout this work. Following the approach of prior work investigating antibiotic-induced mutagenesis,106,182,185 we treated 30 biological replicates, to maximize total numbers of mutations, of WT P. aeruginosa overnight in either 1/10 MIC of ciprofloxacin or aztreonam. 10 replicates from each treatment group were used to confirm elevated mutagenesis via rifampicin reversion frequency. Growth in even just 1/10 MIC of either aztreonam or ciprofloxacin reduced the number of recovered viable cells by approximately one log-fold (FIG. 11 A), although this could be due to low seed number of cells in the inoculum. Treatment with aztreonam or ciprofloxacin induced mutagenesis about 40-fold higher than baseline (FIG. 11B), which is about 2-fold more than previously reported. However, mutant frequencies from treated clones were highly variable (FIG. 11B).
[00220] Due to observed elevated mutagenesis induced by both aztreonam and ciprofloxacin, we perform WGS on all 60 clones (30 treated with aztreonam, 30 treated with ciprofloxacin). Antibiotic-induced mutagenesis has been shown to differentially occur within a treated population,106 so we called all variants to PAO1 5% subcl onal frequency to fully capture its effects. Trinucleotide spectra of unique mutations for all independent replicates were plotted and then replicates treated with the same drug (i.e. all aztreonam-treated or ciprofloxacin-treated) were compiled to make de facto signatures, similar to MP AO1 - mutS clones. Both signatures show enrichment of OT in NCG and GCT contexts, OG in GCT contexts, OA in CCA, CCG, and GCA contexts, and general mutagenesis of T>C and T>G (FIG. 12, “AZ” and “CIP”). We additionally performed NMF on this dataset and stably extracted two signatures (FIG. 12, “VI” and “V2”), which generally resemble the lab-produced signatures. However, VI has notably more enrichment of C>T in GCT and TCG and C>A in CCG and GCA contexts (FIG. 12). Lab- constructed signatures and VI qualitatively resemble human SBS6 (FIG. 12, “SBS6”), with similar enrichment in C>T in NCG and GCT contexts.
[00221] We subsequently analyzed the cosine similarity of all clinical isolate spectra to these new signatures. Upon inclusion of new signatures, our predicted MMR-deficient isolates cluster differently into three distinct groups based on degree of similarity to the ciprofloxacin- and aztreonam-induced signatures (FIG. 13, dark grey stars). All isolates enriched in SBS6 and 15 (approximately 20% of all isoaltes) cluster together and show enrichment in ciprofloxacin- and aztreonam-induced signatures, but to a lesser degree. This cluster additionally shows greater similarity to VI than V2 (FIG. 13, light grey star). Taken together, these data show that antibiotics can induce a specific mutational signature in P. aeruginosa, and these signatures are present in appreciable amount of clinical isolates.
[00222] We demonstrate that antibiotic-treatment can produce a specific mutational signature in vitro. Previous work has characterized antibiotic-induced mutagenesis through selection and sequencing of reporter genes, such as rifampicin reversion followed by rpoB Sanger sequencing.106 Instead, we took a WGS approach, which allows for full surveying of mutational consequences in the genome and the elucidation for trinucleotide mutational signatures associated with treatment.
[00223] Similarity to our aztreonam- and ciprofloxacin-induced signatures was observed in about 20% of all analyzed clinical isolates. However, these clinical isolates all demonstrated higher similarity to human SBS6.
Example 6: Antibiotic-induced mutational signatures for additional classes of antibiotics [00224] Methods
[00225] P aeruginosa strain MPA01 was streaked on LB and incubated overnight. From this plate, independent biological replicates (independent colonies) of MPAO1 were grown in 5 mL LB overnight. Biological replicates were serially passaged in 1/10 MIC (concentration unchanging) of each antibiotic for 10 total passages and incubated at 37°C, 200 rpm for 24 hours each passage. After 10 passages, all replicates were processed for genomic DNA isolates and whole genome sequenced using Illumina short read sequencing. Sample libraries were prepared using Illumina DNA Prep Kit and IDT lObp UDI indices. Demultiplexing, quality control (only reads with Q>30 kept), and adapter trimming were performed using Illumina bcl-convert (v3.9.3). Each sample produced a minimum of 400 Mbp high quality reads (2 x 151 bp), with an average depth of coverage of 60X of the ~6.3 Mbp genome. Paired end reads were aligned to the PAO1 reference genome (NCBI accession #NC_002516.2) and variants were called to reference using breseq (vO.36.1).286 All variants called were at 5% frequency. Variant call format (VCF) files containing all variants called to PAO1 for each sample were deduplicated using bcftools2 1 , removing any variant shared with all other biological replicates within that treatment group from all samples in that group, to create unique VCFs for each sample. Unique VCFs were loaded into and parsed in R using the tidyverse suite (for single-base substitutions) or the MutationalPattems package (for indels).288
[00226] Single-base substitutions'. For each mutation, the reference base was retrieved from the PAO1 reference genome sequence along with flanking reference bases on both the 3’ and 5’ ends to produce the trinucleotide mutation context. Where necessary, trinucleotide contexts were converted to their reverse complement to reflect one of the canonical 6 types of bases changes (C>{A,G,T} or T>{A,C,G}). Mutation spectra were plotted in their 96-trinucleotide context. Individual spectra and spectra combined by treatment group from all 30 replicates/treatment were compiled using tidyverse package dplyr and de facto mutational signatures from each treatment plotted with ggplot2. [00227] Insertions/deletions'. To obtain more complex contexts surrounding indels, a BSgenome reference package was constructed for PAO1 with the BSgenomeForge package using the NCBI RefSeq assembly accession number GCF 000006765.1 and the GenBank accession number AE004091.2 as the circular sequence identifier. The MutationalP atterns package was used to retrieve indel mutation contexts based on the size of the indel, the number of sequence unit repeats at the indel location, and if the indel occurred in the presence of flanking microhomology sequences. Tidyverse was again used to combine individual spectra by treatment group. MutationalP atterns was used to compile both individual and combined indel mutation spectra.
[00228] Results & Discussion
[00229] WGS and mutational signature analysis revealed that each antibiotic treatment induced a distinct and reproducible pattern of single base substitution (SBS) and insertiondeletion mutations (indels) in Pseudomonas aeruginosa genomes. In all treatment groups, we observed enrichment in OT transitions in NCG and GCN contexts, particularly in ACG and GCG. We additionally observe T>C transitions enriched in ATC, ATG, CTC, and CTG across all samples. Notably, these trinucleotide contexts are different than those previously seen in OT and T>C enrichment associated with the P aeruginosa mutational signature caused by DNA mismatch repair deficiency. In addition, almost all samples show enrichment in single cytosine insertions at sites with no repeats. We suggest that these mutations are caused by general replicative stress caused by antibiotic treatment and SOS response, such as replication fork stalling and TLS polymerase recruitment.
[00230] Importantly, there are notable differences in both indel and SBS spectra across treatment groups. We specifically chose antibiotics for this experiment that overlapped within drug class, structural categories, and mechanism of action to investigate if these factors impacted downstream induced mutagenesis, and more importantly, if we could tell them apart based on mutation spectrum. The two drugs most closely related, ceftazidime and cefiderocol, produced distinguishable mutation spectra. Ceftazidime-treatment results in enrichment of cytosine insertion in regions with one repeat, whereas cefiderocol-treatment favored cytosine insertion in regions with no repeats. In addition, we observed slight elevation of insertions of 5+bp in regions with 1 repeat. Ceftazidime-treated replicates showed elevation in C>G transversions in ACG and GCC, whereas cefiderocol -treated replicates did not. We suggest that these differences could be due to cytosine deamination in CpG islands or general reactive oxidative stress. [00231] Comparing these two treatment groups to the other beta-lactams, piperacillin- treatment resembles the spectra of ceftazidime-treatment but shows higher enrichment of T>Cs. However, meropenem-treatment uniquely results in enrichment in single thymine deletions, single thymine insertions, 3bp deletions in regions with 6+ repeats, 5+bp deletions, and 5+bp deletions in regions with microhomology. Additionally, meropenem-treatment results in enrichment of C>Ain SCA contexts and overall T>G transversions. Meropenem-treatment also uniquely shows lower C>T in ACG. We suggest that this profile could be due to higher oxidative stress induced by meropenem treatment.
[00232] Aztreonam binds to penicillin-binding protein similar to beta-lactams but is a monobactam and is slightly different structurally. Despite having the same mechanism of action, aztreonam induced a unique mutational signature characterized by enriched C>As in VNR contexts, C>G in a GCG context, T>Gs in a ATN context, and uniquely T>As in a GTT context. Aztreonam-treatment also uniquely results in a strong signature of 3bp deletions in 6+ repeats, 5+bp deletions in 2 repeats, and 5+bp deletions in regions of 5+ repeats of microhomology. Based on this profile, we suggest that aztreonam could be inducing replicative fork stress, oxidative damage and potential bulky adducts fixed by nonhomologous end joining. The table below summarizes the combined mutational signatures described herein:
[00233] Overall, we demonstrate that antibiotic treatment reproducibly induces a specific mutational signature in /I aeruginosa that is distinguishable across specific treatments. We suggest that these mutation spectra reflect both direct and indirect mechanisms of DNA damage, DNA replication stress, and the activity of error-prone repair pathways under antibiotic pressure.
EQUIVALENTS
[00234] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[00235] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[00236] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth. [00237] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
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Claims

WHAT IS CLAIMED IS:
1. A method for predicting the risk of antibiotic resistance in a subject diagnosed with or suffering from a Pseudomonas infection comprising sequencing Pseudomonas nucleic acids isolated from a biological sample obtained from the subject; generating a mutation spectrum from the sequenced Pseudomonas nucleic acids; and determining that the subject is at risk for antibiotic resistance when a Pseudomonas antibiotic-induced mutational signature is detected in the mutation spectrum.
2. A method for selecting a subject infected with a Pseudomonas species for treatment with an antibiotic therapy comprising sequencing Pseudomonas nucleic acids isolated from a biological sample obtained from the subject; generating a mutation spectrum of the Pseudomonas nucleic acid sequences; detecting a Pseudomonas antibiotic-induced mutational signature in the mutation spectrum; and selecting an antibiotic therapy based on the detected Pseudomonas antibiotic- induced mutational signature.
3. The method of claim 1 or 2, wherein generating the mutation spectrum comprises generating a plurality of single base substitution (SBS) contexts and a plurality of insertion/deletion (indel) profiles from the sequenced Pseudomonas nucleic acids.
4. The method of claim 3, wherein each indel profile of the plurality of indel profiles comprise an indel size, a number of sequence unit repeats at an indel location, and the presence or absence of flanking microhomology sequences at an indel location.
5. The method of claim 3 or 4, wherein the plurality of SBS contexts comprise a plurality of single base changes in a trinucleotide context, wherein the plurality of single base changes in the trinucleotide context comprises one or more transitions/substitutions selected from the group consisting of OA, OG, OT, T>A, T>C, T>G, G>T, G>C, G>A, A>T, A>G, and A>C.
6. The method of any one of claims 1-5, wherein the Pseudomonas antibiotic- induced mutational signature comprises one or more of:
(i) an increase in OT transitions in NCG and GCN contexts relative to a reference Pseudomonas nucleic acid sample,
(ii) an increase in G>A transitions in CGN and NGC contexts relative to a reference Pseudomonas nucleic acid sample,
(iii) an increase in T>C transitions in ATC, ATG, CTC, and CTG contexts relative to a reference Pseudomonas nucleic acid sample,
(iv) an increase in A>G transitions in GAT, CAT, GAG, and CAG contexts relative to a reference Pseudomonas nucleic acid sample, and
(v) an increase in cytosine or guanine insertions in non-repeat regions of the sequenced Pseudomonas nucleic acids relative to a reference Pseudomonas nucleic acid sample.
7. The method of claim 6, wherein the increase in T transitions occurs in ACG and GCG contexts and the increase in G>A transitions occurs in CGT and CGC contexts.
8. The method of any one of claims 1-7, wherein the Pseudomonas antibiotic- induced mutational signature further comprises one or more of:
(i) an increase in OG transversions in ACG and GCC contexts relative to a reference Pseudomonas nucleic acid sample,
(ii) an increase in G>C transversions in CGT and GGC contexts relative to a reference Pseudomonas nucleic acid sample, and
(iii) an increase in cytosine or guanine insertion in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample.
9. The method of any one of claims 1-7, wherein the Pseudomonas antibiotic- induced mutational signature further comprises one or more of: (i) an increase in T>C and A>G substitutions relative to a reference Pseudomonas nucleic acid sample,
(ii) an increase in OG transversions in ACG and GCC contexts relative to a reference Pseudomonas nucleic acid sample,
(iii) an increase in G>C transversions in CGT and GGC contexts relative to a reference Pseudomonas nucleic acid sample, and
(iv) an increase in cytosine or guanine insertion in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample.
10. The method of any one of claims 1-7, wherein the Pseudomonas antibiotic- induced mutational signature further comprises one or more of:
(i) an increase of 5+ base pair insertions in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample,
(ii) OG transversions in ACG and GCC contexts that are comparable to a reference Pseudomonas nucleic acid sample, and
(iii) G>C transversions in CGT and GGC contexts that are comparable to a reference Pseudomonas nucleic acid sample.
11. The method of any one of claims 1-7, wherein the Pseudomonas antibiotic- induced mutational signature further comprises one or more of:
(i) an increase in C>A substitutions in SCA contexts relative to a reference Pseudomonas nucleic acid sample,
(ii) an increase in G>T substitutions in TGS contexts relative to a reference Pseudomonas nucleic acid sample,
(iii) an increase in T>G and A>C substitutions relative to a reference Pseudomonas nucleic acid sample,
(iv) a decrease in C >T substitutions in ACG contexts and G>A substitutions in CGT contexts relative to a reference Pseudomonas nucleic acid sample, and (v) an increase in at least one of (a) single thymine or adenine deletions, (b) single thymine or adenine insertions, (c) 5+ base pair deletions, (d) 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units, and (e) 5+ base pair deletions in regions with microhomology, relative to a reference Pseudomonas nucleic acid sample.
12. The method of any one of claims 1-7, wherein the Pseudomonas antibiotic- induced mutational signature further comprises one or more of:
(i) a decrease in 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units relative to a reference Pseudomonas nucleic acid sample, and
(ii) an increase in cytosine or guanine insertions in repeat regions of the sequenced Pseudomonas nucleic acids that include one repeating sequence unit relative to a reference Pseudomonas nucleic acid sample.
13. The method of any one of claims 1-7, wherein the Pseudomonas antibiotic- induced mutational signature further comprises one or more of:
(i) an increase in single nucleotide deletions relative to a reference Pseudomonas nucleic acid sample,
(ii) an increase in 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units relative to a reference Pseudomonas nucleic acid sample,
(iii) an increase in OG transitions in GCT contexts and G>C transitions in AGC contexts relative to a reference Pseudomonas nucleic acid sample,
(iv) an increase in OA transitions in CCA, CCG, and GCA contexts and G>T transitions in TGG, CGG and TGC contexts relative to a reference Pseudomonas nucleic acid sample, and
(v) an increase in T>C and/or T>G substitutions relative to a reference Pseudomonas nucleic acid sample.
14. The method of any one of claims 1-7, wherein the Pseudomonas antibiotic- induced mutational signature further comprises one or more of: (i) an increase in OG transitions in GCT contexts and G>C transitions in AGC contexts relative to a reference Pseudomonas nucleic acid sample,
(ii) an increase in OA transitions in CCA, CCG, and GCA contexts and G>T transitions in TGG, CGG and TGC contexts relative to a reference Pseudomonas nucleic acid sample,
(iii) an increase in T>C and/or T>G substitutions relative to a reference Pseudomonas nucleic acid sample,
(iv) an increase in at least one of : (a) C >A substitutions in VNR contexts, (b) G>T substitutions in YNB contexts, (c) C >G substitutions in a GCG contexts, (d) G>C substitutions in CGC contexts, (e) T > G substitutions in ATN contexts, (f) A>C substitutions in NAT contexts, (g) T>A substitutions in GTT contexts, and (h) A>T substitutions in AAC contexts, relative to a reference Pseudomonas nucleic acid sample, and
(v) an increase in at least one of: (a) 3 base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 6+ repeating sequence units, (b) 5+ base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 2 repeating sequence units, and (c) 5+ base pair deletions in repeat regions of the sequenced Pseudomonas nucleic acids that include 5+ repeating sequence units with microhomology, relative to a reference Pseudomonas nucleic acid sample.
15. The method of any one of claims 6-14, wherein the reference Pseudomonas nucleic acid sample is obtained from a Pseudomonas control strain that is susceptible to antibiotic therapy, is a wild type MPA01 strain, and/or is a non-hypermutator strain.
16. The method of any one of claims 1-15, wherein the biological sample comprises skin tissue, throat swabs, stool, urine, blood, lung tissue, stomach tissue, or urinary tract tissue.
17. The method of any one of claims 1-16, wherein the Pseudomonas antibiotic- induced mutational signature is caused by prior exposure to an antibiotic.
18. The method of claim 17, wherein the antibiotic is a P-lactamase inhibitor, a monobactam, a quinolone, an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, or an antimicrobial peptide (AMP).
19. The method of any one of claims 2-18, wherein the antibiotic therapy comprises one or more antibiotics selected from the group consisting of a monobactam, a quinolone, an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, or an antimicrobial peptide (AMP).
20. The method of any one of claims 8-10 or 14-18, further comprising administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, a carbapenem, an AMP, or an aminoglycoside.
21. The method of any one of claims 11 or 15-18, further comprising administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a quinolone, an AMP, or an aminoglycoside.
22. The method of any one of claims 12 or 15-18, further comprising administering to the subject an effective amount of one or more antibiotics selected from the group consisting of a monobactam, a quinolone, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, or an antimicrobial peptide (AMP).
23. The method of any one of claims 13 or 15-18, further comprising administering to the subject an effective amount of one or more antibiotics selected from the group consisting of an aminoglycoside, a carbapenem, a cephalosporin, a penicillin-based beta-lactam, a monobactam, or an antimicrobial peptide (AMP).
24. The method of any one of claims 18-19, or 22-23, wherein the monobactam is selected from among aztreonam, azactam, tigemonam, nocardicin A, BAL30072, and tabtoxin.
25. The method of any one of claims 18-22 or 24, wherein the quinolone is selected from among Enrofloxacin (Baytril), Ciprofloxacin (i.e., Cipro and Proquin), Enoxacin (i.e., Penetrex), Gatifloxacin (i.e., Gatiflo, Tequin and Zymar), Gemifloxacin (i.e,. Factive), Levofloxacin (i.e., Levaquin), Lomefloxacin (i.e., Maxaquin), Moxifloxacin (i.e., Avelox), Norfloxacin (i.e., Noroxin), Ofloxacin (i.e., Floxin), Prulifloxacin, Sparfloxacin (i.e., Zagam), Trovafloxacin/Altrofloxacin (i.e., Trovan), Danofloxacin (i.e., A180), Difloxacin (i.e., Dicural), Marbofloxacin (i.e., Orbax), Orbifloxacin (i.e., Zeniquin), Cinoxacin (i.e., Cinobac), Rosoxacin, Fleroxacin, Pefloxacin, Rufloxacin, Balofloxacin, Grepafloxacin, Pazufloxacin, Temafloxacin, Tosufloxacin, Besifloxacin, Clinafloxacin, Garenoxacin, Sitafloxacin, Ibafloxacin, Pradofloxacin, and Sarafloxacin.
26. The method of any one of claims 18-20, or 22-25, wherein the carbapenem is selected from among Thienamycin, doripenem, ertapenem, biapenem, tebipenem, Meropenem, Meropenem-vaborbactam, Imipenem, and Imipenem-relobactam.
27. The method of any one of claims 18-19, or 22-26, wherein the cephalosporin is selected from among Cefadroxil, Cefazolin, Cephalexin, Cephradine, Cefaclor, Cefotetan, Cefoxitin, Cefprozil, Cefuroxime, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefoperazone- sulbactam, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftazidime-avibactam, Ceftibuten, Ceftriaxone, Cefepime, Ceftaroline-fosamil, Ceftobiprole/medocaril, Cefiderocol, Ceftolozane- tazobactam, and moxalactam.
28. The method of any one of claims 18-19 or 22-27, wherein the penicillin-based beta-lactam is selected from among amoxicillin, amoxicillin/clavulanic acid, ampicillin, benzylpenicillin, benzathine benzylpenicillin, dicloxacillin, flucioxacillin, oxacillin, cioxacillin, nafcillin, carbenicillin, ticarcillin, temocillin, mecillinam, phenoxymethylpenicillin, mezlocillin, piperacillin, and piperacillin-tazobactam.
29. The method of any one of claims 18-21 or 23-28, wherein the aminoglycoside is selected from among apramycin, Tobramycin, Amikacin, Gentamicin, neomycin, streptomycin, and plazomycin.
30. The method of any one of claims 18-29, wherein the AMP is selected from among Colistin, Dalbavancin, Daptomycin, Oritavancin, Polymyxin b, Teicoplanin, Telavancin, Vancomycin, Murepavadin, PMX-30063, Friulimicin B, PLG0206, IDR1, Omiganan, LTX-109, OP-145, DPK-060, NP101, NP108, Novexatin, Pl 13, and Ctx(Ile21)-Ha.
31. The method of any one of claims 1-7, wherein the Pseudomonas infection is a cystic fibrosis (CF) infection, a respiratory tract infection (RTI), a urinary tract infection (UTI), a pressure sore infection, a burn infection, a wound infection, a bloodstream infection or an intraabdominal infection (IAI).
32. The method of any one of claims 1 or 3-31, wherein the Pseudomonas infection is caused by a Pseudomonas species selected from among P. aeruginosa, P fluorescens, P putida, P cepacia, P stutzeri, P maltophilia, and P putrefaciens.
33. The method of any one of claims 2-31, wherein the Pseudomonas species is selected from among P. aeruginosa, P fluorescens, P putida, P cepacia, P stutzeri, P maltophilia, and P putrefaciens.
34. The method of any one of claims 1-33, wherein the Pseudomonas nucleic acids are sequenced via whole genome sequencing (WGS).
35. The method of any one of claims 20-34, further comprising sequentially, simultaneously, or separately administering to the subject an effective amount of a betalactamase inhibitor.
36. The method of claim 35, wherein the beta-lactamase inhibitor is selected from the group consisting of clavulanic acid, sulbactam, tazobactam, avibactam, relebactam, RG06080, and RPX7009.
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