NL2037533B1

NL2037533B1 - Characterization of cells

Info

Publication number: NL2037533B1
Application number: NL2037533A
Authority: NL
Inventors: Nerissa Deurloo Yanthi; Sophia Shanko Eriola; Franciscus Van Belkum Alexander; Roelof Stapert Hendrik; J Van Der Linden Adrianus
Original assignee: Shanx Medtech Bv
Priority date: 2024-04-24
Filing date: 2024-04-24
Publication date: 2025-11-10
Also published as: WO2025226152A1

Abstract

The present invention relates to a composition for the detection and/or characterization of a metabolizing cell comprising culturing means, one or more metabolizing cells, chemical sensor material, which detects cell metabolism dependent changes in the concentration of one or more analytes over time, generating a signal, and one or more signal enhancing compounds, which enhance or limit metabolism of the one or more metabolizing cell compared to general growth medium. The invention further relates to a method for characterization of microbial cells present in a sample of a subject, comprising the steps of cultivating the microbial cells present in the sample, measuring changes in the concentration of one or more analytes in the sample over time or on a comparative basis, Wherein the analytes are measured using chemical sensor material Which chemically interacts With the analytes, and characterizing the microbial cells by comparing the metabolism dependent changes over time in the concentration of one or more analytes With a reference indicative for a specific microbial cell. Additionally, the invention relates to determining the drug susceptibility or resistance of one or more microbial cells, present in a sample of a subject, by cultivating the microbial cells present in the sample according to the method of the invention in the presence of at least one drug.

Description

CHARACTERIZATION OF CELLS

The present invention relates to composition for the detection and/or characterization of a metabolizing cell. The invention also relates to a method for characterization of microbial cells present in a sample of a subject, and to a method for determining the drug susceptibility or resistance of one or more microbial cells, present in a sample of a subject, by cultivating the microbial cells present in the sample according to the method of the invention in the presence of at least one drug.

Infectious diseases can be devastating, and sometimes fatal, to the host. Many strategies are available to protect people from infection and to treat an infectious disease once it has developed. One of the most well-known methods of treating infections involves the use of antimicrobial agents. Generally, antimicrobial agents are compounds that kill microorganisms or inhibit their growth without affecting the infected host too much. Antimicrobial medicines can be grouped according to their target microorganism and/or chemical structure. For example, antibiotics are used against bacteria, and antifungals are used against fungi.

Despite their efficacy and the immense impact antimicrobials have had on public health since their discovery and introduction, life threatening infections caused by microbial organisms that are resistant to the currently available therapeutic antimicrobial agents are gaining in prevalence. While the quick onset of antimicrobial resistance was already noted within years of the therapeutic introduction of the first antibiotic penicillin in the 40’s of the previous century, antimicrobial resistance (AMR) is now estimated to be at a pandemic scale, with a recent Lancet study estimating nearly 5 million deaths associated with AMR in 2019,

In order to prevent infection or treat infected subjects with the right antimicrobials, it is of utmost importance to be able to detect, identify and further characterize microbial cells causing the infection. While detection of microbial infection is the first step and has significant clinical value already, the identification and/or characterization of the microbial organism causing the infection is essential for preventing or treating an infection efficiently. It is the identification and/or characterization of microbial cells, and in particular their susceptibility or resistance profile, that enables tailored treatment with suitable antimicrobial agents, preventing ineffective treatment.

Furthermore, bacteria can reproduce very fast through binary fission, and some clinically relevant bacteria can divide every 20 to 30 minutes. Because they can reproduce so quickly, microorganisms can amplify to enormous numbers whilst this rapid proliferation can result in both phenotypic and genomic heterogeneity. If their environment suddenly changes, the community's intrinsic genetic variations make it more likely that some individuals will have or create a genetic background that increases their ability to survive. This standing genetic variation within a large population, coupled with their high reproduction rate, gives microbes a huge advantage over humans when it comes to adapting for survival during episodes of adverse conditions. The rapid and timely detection and identification and characterization of the nature of an infection and/or the pathogen’s susceptibility or resistance profile is therefore of crucial relevance in order to give the person the appropriate treatment as soon as possible.

Known identitication methods of bacteria are plate based culturing, optionally using chromogenic agar, MALDI-TOF or enzyme-linked immunosorbent assays, optionally combined with PCR. Such assays are, however, relatively insensitive, requires skilled personnel, specialized equipment and several (manual) handling steps and/or often require many incubation steps. Also, increasing the selectivity of such methods remains challenging.

It is known in the art that metabolizing cells interact with their extracellular environment through the exchange of various molecules, which can be indirectly monitored by e.g measuring the pH, the O: concentration, or the CO: concentration. Such molecules can be measured directly or using, for example, a molecular or chemical indicator, which exhibit altered fluorescence emission spectra or intensity when exposed to changes in the conditions of the intracellular and/or extracellular environment.

However, the mere detection of metabolizing cells by means of measuring analytes is not sufficient to determine effective (antimicrobial) treatment. Culture based detection and identification of cells is slow and requires at least 24 or in certain cases more than 72 hours from the time a sample is taken. Cells need to be grown in well-defined culture media and the need for transfer and logistics of a sample towards dedicated laboratory equipment is a complicating factor.

Hence, there is a clear need to characterize and/or identify metabolizing cells within a short period of time after sampling to be able to provide effective personalized treatment/prescription. Even more preferably, the detection and characterization of cells should be performed at the Point of

Care (PoC), which is at the time and place of patient care.

The goal of the invention is therefore to provide a composition which can detect, identify and/or characterize metabolizing cells in an accelerated way, accessible at the PoC in order to diagnose and determine correct prevention or treatment measures against infectious or other diseases.

This goal is achieved by a composition for the detection, identification and characterization of a metabolizing cell comprising: culturing means, one or more metabolizing cells, chemical sensor material, which detects cell metabolism dependent changes in the concentration of one or more analytes over time, generating a signal, and one or more signal enhancing compounds, which enhance or limit metabolism of the one or more metabolizing cells compared to general nutrients.

The signal enhancing compounds may be, for example, nutrients which stimulate or inhibit the metabolism of the one or more metabolizing cells, such that the presence and the identity of the metabolizing cells can be determined at an earlier stage when compared to detection and/or identification of these metabolizing cells when not using such metabolism stimulating or inhibiting nutrients. The result of this accelerated detection and identification is that effective prevention and treatment measures can be taken at the PoC.

The composition of the invention enables very rapid detection, identification and characterization of metabolizing cells. In particular, the detection, identification and characterization of metabolizing cells can be achieved in 0.5-12 hours, more in particular within 1- 4 hours. Earlier detection, identification and characterization of microbial cells is particularly beneficial when screening and/or diagnosing aggressive infections, for which an early-stage effective treatment is crucial.

Therefore, the present invention therefore relates to a composition for the detection, identification and characterization of a metabolizing cell comprising: - culturing means, - one or more metabolizing cells, - chemical sensor material, which detects cell metabolism dependent changes in the concentration of one or more analytes over time, generating a signal, and - one or more signal enhancing compounds, which enhance or limit metabolism of the one or more metabolizing cell compared to general nutrients.

In a further aspect, the invention relates to said composition, wherein the enhanced or limited metabolism enables earlier detection of cell metabolism dependent changes in the concentration of one or more analytes over time.

In another embodiment, the invention relates to said composition, wherein the signal enhancing compounds can be selected from a list consisting of: oligosaccharides, disaccharides, monosaccharides, such as dextrose, fructose, glucose, maltose, lactose, sucrose, mannose, arabinose, xylose, galactose, and yeast extract, casamino acid, {L)-norepinephrine, N-hexanoyl-L- homoserine lactone (IUPAC: N-(2-oxohexanoyl)-L-homoserine lactone), acetate, citrate, phosphatidylcholine, gluconate, D-serine. The IUPAC name of (L)-norepinephrine is N-{2-amino- 1-hydroxyethyl)benzene-1,2-diol and the IUPAC name of N-hexanoyl-L-homoserine lactone is N- {2-oxohexanoyl)-L-homoserine lactone.

The signal enhancing compounds are present in a concentration sufficient to enhance or limit the metabolism of the metabolizing cells. Changes in the metabolism can consequently be detected early. Without being bound to theory, the signal enhancing compounds are molecules which stimulate in particular one or more metabolic pathways that result in the production of analytes, the analytes can be detected via the chemical sensor material, such that detection, identification and characterization of metabolizing cells can be achieved at an earlier stage compared to detection identification and characterization of metabolizing cells using general nutrients which are in particular present in a generally accepted concentration and in particular in a generally accepted growth medium.

In a further specific embodiment of the present invention, the signal enhancing compound inhibits the metabolism instead of enhancing the metabolism, when the inhibition of the metabolism enables earlier detection identification and characterization of metabolizing cells.

A further preferred aspect of the invention is that the signal enhancing compounds in the composition of the invention are present in the composition in a concentration of between 0.01 to 100 g/L, preferably between 0.05 to 20 g/L, more preferably between 0.1 to 5 g/L, most preferably between 0.2 to 3 g/L.

The general growth media as used herein are growth media for metabolizing cells, which are commercially available and which are generally used to grow, maintain, store or cultivate metabolizing cells. The general growth medium of the invention is in particular selected from a list consisting of: Mueller-Hinton Broth (MHB), Tryptic Soy Broth (TSB), Lysogeny Broth (LB),

Terrific Broth, Super Optimal Broth (SOB, or Hanahan’s Broth), Super Optimal Broth with

Catabolite Repression (SOC), Brain Heart Infusion (BHI), Thioglycollate Broth, Sabouraud

Dextrose Broth, MacConkey Broth, Triple Sugar Iron Broth intracellular (TSI), Urea Broth,

Sulfide-Indole-Motility Medium (SIM), M9 Minimal, Magic Media Medium, ImMedia Medium, 2X YT Broth, NZCYM Broth, Phage Media, NZ Amine® Broth. The general growth medium may be supplied with agarose to facilitate air-exposed microbial growth on a semi-solid culture medium.

In another embodiment, the invention relates to said composition, wherein the analytes are metabolites. In the context of the present invention, an analyte is a substance or chemical constituent that is of interest in an analytical procedure. However, a metabolite is an intermediate or end product of metabolism. In a specific embodiment the invention relates to said composition, wherein the analyte is one or more compounds which are consumed by (or released during) the metabolism of the metabolizing cell, such that a decrease of such a compound enables detection identification and characterization of the metabolizing cell.

Metabolites can be present inside the cell, the periplasmic space, the intramembrane or the extracellular environment. According to a preferred embodiment of this invention, the metabolites to be detected are present in the extracellular environment.

In a further embodiment the invention relates to said composition, wherein the metabolites, small molecules or {an)ions required for proper cellular function are selected from the list consisting of: H*, OH, O2, CO;, K*, Nat, Ca™, Mg, NO, nucleic acids, peptides, extracellular and/or intracellular proteins, such as, proteases or p-lactamase, ATP, acids such as lactic acids,

NADH, boric acid, citric acid, acetic acid, formic acid, butyric acid and propionic acid.

In yet another embodiment of the invention, the cell metabolism-dependent changes {or lack thereof) over time in the concentration of one or more analytes represent pre-growth, lag phase, growth, division, duplication, stabilization, tolerance, viability and/or senescence and death of the cell. During each of these cell characteristics, the metabolism may undergo changes which 5 can be used to detect identify, characterize metabolizing cells using the composition of the invention.

In a further embodiment, the metabolizing cell is part of a mixture of different cells or a multicellular composition, in particular a biofilm, microcolony, organoids or organ.

In yet another embodiment, the invention relates to the composition of the invention further comprising a drug, in particular, an antibiotic agent. The composition may be used to determine drug or antibiotic susceptibility of a cell, in particular a bacterial cell.

The metabolizing cell as used in the present invention may be a prokaryotic cell or eukaryotic cell, in particular, the metabolizing cell may be an infected cell, in particular a metabolizing cell infected by a virus. More in particular, the metabolizing cell is a bacterial cell, in particular a pathogen. It is beneficial to detect identify and characterize a pathogen in order to determine the presence and the specificity of an infection. In another embodiment, the metabolizing cell is a cancer cell.

In another embodiment, the invention relates to said composition, wherein the eukaryotic cell is an animal cell, in particular a human cell. It can be advantageous to determine the presence identity and character of such eukaryotic cells for diagnostic and/or research purposes. Each of these metabolizing cells can be detected, identified and characterized using the composition of the invention.

The chemical sensor material is in particular used in the composition of the invention to chemically bind analytes or preferably the metabolites, and subsequently produce a signal.

Chemically binding analytes or preferably the metabolites, may involve complexing the analytes or metabolites. The complexing of the analytes or metabolites induces a conformational and/or structural change of the chemical sensor material, which produces a detectable signal.

In a particular embodiment, the invention relates to said composition, wherein the chemical sensor material is one or more luminescent sensor compounds. In particular, the invention relates to the composition, wherein the luminescent sensor compound is selected from the group consisting of fluorophores, phosphorescent molecules, electrofluorescent molecules, thiols, ruthenium(11), osmium(1l), rhenium(I), iridium(lI), platinum(Il) and palladium(Il), rhodamine based dyes, indicators for Ca”, indicators for Na”, indicators for K* , indicators for Cl, potential- sensitive dyes, luciferase-based sensors and mixtures thereof. These compounds are able to detect analytes and/or metabolites consumed or produced by metabolizing cells.

In a more specific embodiment, the luminescent sensor compound is selected from the group consisting of: seminaphtarhodafluor (SNARF-4F), platinum(1l) octaethylporphyrin ketone (PtOEP), OXNANO, ruthenium tris(2,2’-dipyridyl) dichloride hexahydrate (RTDP), platinum{(ID octaethylporphyrin ketone (PtOEPK), platinum (II) meso-tetra(pentafluorophenyl) porphine (PITFPP), tris(2-(dimethylamino)ethyl) 4,4'.4"-(1H-pyrrole-1,2,5-triyi)tribenzoate (TPP-TDMAE), hexaphenylsilole (HPS), potassium-sensing oligonucleotide (PSO), tetra ammonium salt, such as

SBFI or PBF, tetra{tetramethylammonium) salts such as Sodium Green), pyranine, 4-Amino-5-

Methylamino-2’,7’-Difluorofluorescein (DAF-FM), hexapotassium salt such as Calcium Green™-

SN, pentapotassium salt such as Fluo-4 or Magnesium Green!M, tripotassium salt such as Rhod-2, 7-hydroxy-3H-phenoxazin-3-one 10-oxide (Resazurin), tetrazolium compounds such as MTT,

MTS, XTT or WST-1, glycylphenylalanylaminofluorocoumarin (GF-AFC), luciferin, Acridine

Orange, DAPI, ethidium Bromide, PicoGreen, SYBR Green I, SYBR Green U, SYTO 9, cefotaxime + 4-hydroxy-1,8-naphthalimide (CTX-HN) and aptamers, in particular RNA-apatmers and molecular beacons.

Some of these luminescent sensor compounds are further defined by their IUPAC names.

The invention therefore also relates to said composition, wherein the luminescent sensor compound is platinum{Il) octaethylporphyrin ketone (PtOEP, IUPAC: 2,3,7,8,12,13,17,18- octaethylporphyrin-22,24-diide; platinum(2+)), ruthenium tris(2,2’-dipyridyl) dichloride hexahydrate (RTDP, IUPAC: Hexachlororuthentum (Ill) tris(2,2’-bipyridine) hexahydrate), platinum(Il) octaethylporphyrin ketone (PLOEPK, IUPAC: Platinum(Il) 5,10,15,20-tetraethyl- 21H,23H-porphine-2,3-dione), platinum (11) meso-tetra{pentafluorophenyl) porphine (PtTFPP,

IUPAC: Platinum(Il) 5,10.15,20-tetra(pentafluorophenyl)-2 1H,23H-porphine), hexaphenylsilole (HPS, IUPAC: 1,1,2,2,3,3-Hexaphenyl-1-silole}, potassium-sensing oligonucleotide (PSO), tetra ammonium salt, such as SBFI (IUPAC: bis(acetyloxymethyl) 4-[6-[13-[2-[2,4- bis(acetyloxymethoxycarbonyl)phenyl]-5-methoxy- I -benzofuran-6-yl}-1.4,10-trioxa-7,13- diazacyclopentadec-7-y1]-5-methoxy-1-benzofuran-2-yl]benzene-1,3-dicarboxylate) or PBF] (IUPAC: bis(acetyloxymethyl) 4-[6-[16-[2-[2,4-bis(acetyloxymethoxycarbonyl)phenyl]-5- methoxy-1-benzofuran-6-yi}-1,4,10,13-tetraoxa-7,16-diazacyclooctadec-7-y1}-5-methoxy-1- benzofuran-2-yl]benzene-1,3-dicarboxylate), tetra(tetramethylammonium) salts such as Sodium

Green (IUPAC: 5-[[4-]13-[4-[[3-carboxylato-4-(2,7-dichloro-3-oxido-6-oxoxanthen-9- yhbenzoyljamino}-2,5-dimethoxyphenyl]-1,4,10-trioxa-7,13-diazacyclopentadec-7-y1}-2,5- dimethoxyphenyljcarbamoyl}-2-(2,7-dichloro-3-oxido-6-oxoxanthen-9- yh)benzoate;tetramethylazanium)), pyranine (IUPAC: 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt), 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM, IUPAC: 7-amino- 2.7-difluoro-3'.,6'-dihydroxy-6-(methylamino)spirof2-benzofuran-3.9'-xanthene}- 1 -one), hexapotassium salt such as Calcium Green!M-S5N (IUPAC: acetyloxymethyl 2-[N-[2-

(acetyloxymethoxy)-2-oxoethyl]-2-[2-[ 2-[bis[2-(acetyloxymethoxy)-2-oxoethyljamino}-5- nitrophenoxy Jethoxy]-4-[(3',6'-diacetyloxy-2',7'-dichloro-3-oxospiro{2-benzofuran-1,9'-xanthene]-

S-carbonyl)aminolanilino]acetate), pentapotassium salt such as Fluo-4 (IUPAC: 2-[2-[2-[2- [bis(carboxymethyl)amino}-5-(2,7-difluoro-3-hydroxy-6-oxoxanthen-9-yl)phenoxylethoxy}-N- {carboxymethyl)-4-methylanilinojacetic acid) or Magnesium Green™ (IUPAC: pentapotassium;2- [2-(carboxylatomethoxy)-N-(carboxylatomethyD-4-[(2',7'-dichloro-3',6'-dioxido-3-oxospiro[2- benzofuran-1,9'-xanthene]-5-carbonyl)amino]anilinojacetate), tripotassium salt such as Rhod-2 (IUPAC: [9-[4-|bis(carboxymethyl)amino}-3-[2-[2-[bis(carboxymethyl)amino}-5- methylphenoxylethoxy Ipheny1]-6-(dimethylamino)xanthen-3-ylidene}-dimethylazanium;chloride), 7-hydroxy-3H-phenoxazin-3-one 10-oxide (Resazurin, IUPAC: 7-hydroxy-10-oxidophenoxazin- 10-ium-3-one), tetrazolium compounds such as MTT (IUPAC: 3-(4,5-dimethylthiazol-2-y1)-2,5- diphenyltetrazolium bromide), MTS (IUPAC: 3-[4,5 dimethylthiazol-2-y1}-5-[3-carboxymethoxy- phenyl}-2-f4- sulfophenyl]-2H-tetrazolium, inner salt), XTT JQUPAC: 2,3-bis-(2-methoxy-4-nitro-

S-sulfophenyl)-2H-tetrazolium-5-carboxanilide) or WST-1 (IUPAC: 5-(2,4-disulfophenyl)-2-(4- iodophenyl)-3-(4-nitrophenyl)-2H-tetrazolium), glycyt-phenylalanyl-aminofluorocoumarin (GF-

AFC, IUPAC: 2-amino-N-{(25)-1-{4-amino-3-fluoro-2-oxochromen-35-y1)- 1-oxo-3-phenylpropan- 2-yl]acetamide), luciferin (IUPAC: (45)-2-(6-hydrox y-1,3-benzothiazol-2-y1)-4,5-dihydro-1,3- thiazole-4-carboxylic acid), Acridine Orange (IUPAC: 3-N,3-N,6-N,6-N-tetramethylacridine-3,6- diamine), DAPI (IUPAC: 2-(4-carbamimidoylphenyl)-1H-indole-6-carboximidamide), ethidium

Bromide (IUPAC: 5-ethyl-6-phenylphenanthridin-5-ium-3,8-diamine; bromide), PicoGreen (IUPAC: N'-[3-(dimethylamino)propy!]-N,N-dimethyl-N'-[4-[(E)-(3-methyt-1,3-benzothiazol-2- ylidene)methyl]-1-phenylquinolin-1-ium-2-yljpropane-1,3-diamine), SYBR Green I (IUPAC: N,N- dimethyl-N'-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl}- 1-phenylquinolin- 1 -ium-2-yl}-

N'-propylpropane-1,3-diamine), SYBR Green U (IUPAC: N,N-dimethyl-2-[4-[(Z)-(3-methyl-1,3- benzoxazol-2-ylidene)methyl]-1-phenylguinolin-1-1um-2-yl]sulfanylethanamine:iodide), cefotaxime (IUPAC: (6R,7R)-3-(acetyloxymethyl)-7-[[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2- methoxyiminoacetyl]amino]-8-ox0-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid) + 4- hydroxy-1,8-naphthalimide (CTX-HN).

Another aspect of the invention is that the chemical sensor material of the composition of the invention can be in or attached to a matrix, preferably in a hydrogel, bead, micelle, nanoparticle, and or/ virus.

As mentioned before, the detection, characterization and identification of microbial cells responsible for an infection is of vital importance for the treatment of said infection with the appropriate medication, and such a characterization may be performed by measuring the metabolic profile of the microbial cells. Furthermore, the resistance profile of the microbial cells needs to be determined to know which antimicrobial compounds to use in treatment.

Characterization and growth of infecting microbes is generally performed in dedicated laboratories and often cannot be performed at, for example, a general practitioner’s office. The transport of samples takes valuable time and resources, which are sometimes not available. This may lead to treatment with inetfective drugs. For example, flu patients are sometimes given antibiotics, even though flu is caused by viruses which cannot be treated with antibiotics. The unnecessary and ineffective prescription of antibiotics is one of the many factors contributing to the rising incidence of antimicrobial resistance. Furthermore, antibiotics can disrupt the essential commensal bacteria in the human gut, which can, in itself, lead to disease. Therefore, there exists a need for fast and reliable methods to characterize and identify microbes causing infections. Ideally, such methods are simple and do not require a laboratory, so that they may be used and performed at the PoC, such as a general practitioner’s office, a pharmacy or even at home. This may reduce the number of wrong prescriptions, while simultaneously allowing for an earlier treatment with appropriate drugs.

As such, in a further embodiment, the invention relates to a method for characterization of microbial cells present in a sample of a subject. Suitably, the method can be performed with the composition of the present invention, but this is not required.

The invention thus relates to a method for characterization of microbial cells present in a sample of a subject, comprising the steps of: a) cultivating the microbial cells present in the sample, b) measuring changes in the concentration of one or more analytes in the sample over time or on a comparative basis, wherein the analytes are measured using chemical sensor material which chemically interacts with the analytes, and c) characterizing the microbial cells by comparing the metabolism dependent changes over time in the concentration of one or more analytes with a reference indicative for a specific microbial cell.

In the context of the present invention the term identification of a metabolizing cell can be used interchangeably with characterization of a metabolizing cell. The microbial cell can be any metabolizing cell.

In the context of the present invention, cultivating microbial cells should be interpreted as comprising microbial cells in a liquid, preferably water and optionally in the presence of other compounds.

The step of cultivating the microbial cell preferably encompasses culturing, growing and/or maintaining the cell in a growth medium. The growth medium may comprise additional nutrients, such as the signal enhancing compounds as described herein.

The step of measuring changes in the concentration of one or more analytes in the sample over time enables detection of environmental changes resulting from the microbial cells. The changes (or lack thereof) may be the result of growth, division, stabilization, tolerance, viability and/or death of the microbial cell. The changes in the concentrations of analytes are therefore a direct consequence of the metabolism of the microbial cell.

The step of characterizing the microbial cells by comparing the metabolism dependent changes over time with a reference finally allows the person performing the method to determine which microbial species is present in the sample, or are present in the sample at the event of a multi-species sample. This reference may be another measurement made in the same characterization, for example of a reference culture that contains a known microbe. The reference may also be a known reference profile indicative of a certain microbe. These references may be provided together with the means of performing the method.

In a further embodiment, the invention relates to a method for measuring drug susceptibility or resistance of one or more microbial cells. Besides characterizing the microbial species, it is also possible to test their susceptibility or resistance to certain compounds and drugs by adding these before, during or after the cultivation. When cells can continue to metabolize unhindered, they can be considered to be resistant to the added compounds and drugs. Suitably, this can be assessed together or simultaneous with the characterization of the microbial species.

Thus, the invention further relates to a method for determining the drug susceptibility or resistance of one or more microbial cells, present in a sample of a subject, comprising the steps of: a) cultivating the microbial cells present in the sample, b) measuring changes in the concentration of one or more analytes in the sample over time or on a comparative basis, wherein the analytes are measured using chemical sensor material which chemically interacts with the analytes, and c) cultivating the microbial cells present in the sample in the presence of at least one drug, and d) measuring changes in the concentration of one or more analytes in the sample over time or on a comparative basis, wherein the analytes are measured using chemical sensor material which chemically interacts with the analytes, and wherein difference between the changes in the concentration of one or more analytes measured in step b) and d) is indicative of the drug susceptibility or resistance of one or more microbial cells present in the sample.

The drug can also be found in dry or liquid form in the container where the microbial cells will be introduced prior to their cultivating.

The step of cultivating the microbial cell preferably encompasses culturing, growing and/or maintaining the cell in a growth medium. The growth medium may comprise additional nutrients, such as the signal enhancing compounds as described herein. When cultivating the cells once again in step ¢), the growth medium may further comprise one or more drugs against which the susceptibility or resistance is to be assessed. Steps a) and ¢) may be performed in succession but may also be used simultaneously. It is also possible to only perform step c), for example when the data of the cultivation in step a) has already been obtained in a prior performance of the method of the invention.

In another embodiment of the invention, the sample used in these methods of the invention is a clinical sample. Determining the presence and drug susceptibility of cells, like bacteria, in clinical samples allows the reduction of the number of wrong prescriptions and enables earlier treatment with appropriate drugs of subjects having an infection.

In a further preferred embodiment, the difference between the changes in the concentration of one or more analytes measured in step b) and d) is indicative for the growth or inhibition of growth or death of the bacterial species. When testing drugs that are known to inhibit growth of microbial species, this is often directly reflected in growth inhibition or direct cell death. These metrics represent especially clear and unambiguous signals that the tested microbial cells are susceptible to the tested drug.

The step of measuring changes in the concentration of one or more analytes in the sample over time enables detection of intracellular or environmental changes resulting from the microbial cells. The changes may be the result of growth, division, stabilization, tolerance, viability and/or senescence and death of the microbial cell. The changes are measured by using a chemical sensor material which chemically binds the analytes, and subsequently produces a signal which is detectable. Chemically binding analytes or metabolites may involve complexing the analytes. The complexing of the analytes induces a conformational and/or structural change of the chemical sensor material, which produces a detectable signal. Performing the methods by using chemical sensor material of this invention which complexes analytes enables the development of a digital fingerprint of a microbial cell. The digital fingerprint may encompass curve characteristics of the growth, division, stabilization, tolerance, viability and/or death of the microbial cell. Also, complexing analytes enables determining global bacterial metabolic activity instead of single cell analysis.

In a preferred embodiment, the invention thus relates to said method, wherein the chemical sensor material forms a complex with the analytes. The complexing of the analytes or metabolites may induce a conformational and/or structural change of the chemical sensor material, which produces a detectable signal. Detection of analytes using complexing sensors provides a significantly more specific signal and increases the discrimination power of the sensor array and decrease the impact of background signals with respect to conventional methods.

These changes may then be compared between the cultivation steps that did or did not comprise drugs. When no difference can be detected, the microbial cells are likely to be resistant to the tested drug. However, if a difference is detected between the two signals, the microbial cells are likely to be susceptible, as the drug provided has affected the microbial cells.

In a particular embodiment, the invention relates to said method, wherein the measured analytes are metabolites. Changes in metabolite concentrations are indicative of cellular metabolism and activity and their patterns (profiles) of change are particularly suitable for characterizing microbial cells and their potential drug susceptibility. Furthermore, such measurements allow characterization of individual microbial species, which other methods, such as methods for measuring microbial growth, for example by measuring optical density of a microbial culture, do not allow for or are less suitable for.

In a preferred embodiment, these metabolites are present in the extracellular environment.

Detection of the metabolites in the extracellular environment is beneficial because the chemical sensor material does not have to enter cells in order to detect metabolism dependent changes whose presence may influence the genetic makeup of the cell.

In a further preferred embodiment, the metabolites, small molecules or (an)ions required for proper cellular function are selected from the list consisting of: H*, OH", Oa, CO», K*, Nat,

Ca, Mg* NO, nucleic acids, peptides, extracellular and/or intracellular proteins, such as, proteases or B-lactamase, ATP, acids such as lactic acids, NADH, boric acid, citric acid, acetic acid, formic acid, butyric acid and propionic acid.

In another aspect of the invention, the chemical sensor material is one or more luminescent sensor compounds. Such luminescent compounds can be readily detected after and during cultivation. Furthermore, by using luminescent sensor compounds with different emission spectra, several analytes may be measured simultaneously in one sample without the signals interfering.

This in turn allows for more accurate characterisation of the cells present in the tested sample.

In a preferred embodiment, the luminescent sensor compound is selected from the group consisting of fluorophore, phosphorescent molecules, electrofluorescent molecules, thiols, ruthenium(ll), osmium), rhenium{D), iridium(11l), platinum(Il) and palladium(Il), rhodamine based dyes, indicators for Ca”, indicators for Na* , indicators for K*, indicators for CI, potential- sensitive dyes, luciferase-based sensors and mixtures thereof.

The sample to be analysed may be obtained from diverse sources, such as animals, plants, or cell biobank. Infectious disease caused by microbes is not only a problem in humans, but also in animals and plants such as crop plants, domestic pets and livestock. Such animals are also often prescribed antibiotics when sick, and their treatment faces similar challenges as treatment of human patients. Antifungals are used in agriculture to present fungal diseases in certain plant species intended for the food market. As such, in a preferred embodiment, the method of the invention is performed on a sample taken from a mammalian subject. In a further preferred embodiment, the subject is a human.

To characterise the microbe present in a sample, and to determine its susceptibility and resistance to drugs, it is necessary to compare the change in concentration of the analytes measured to a reference. In doing so, the person performing the method of the invention may determine what metabolic profile the microbes in the sample match with. This reference may be another culture with a known microbe that is measured simultaneously, or the reference is one or more prior results fixed in a database. When the measurements are similar, this indicates that the sample culture comprises the microbe present in the reference culture. It is also possible to determine the data profiles generated by reference microbes using the method at a different time point, and comparing the data generated when performing the method of the invention to these pre- determined microbial profiles to characterise the microbe present in the sample. This may allow for faster characterisation of microbes as a reference culture in the same iteration of the method is not required. Therefore, in a preferred embodiment, the reference indicative for a specific microbial cell is a predetermined reference distinctive for a microbial cell.

Many dangerous infectious diseases are caused by bacteria. Bacteria are generally unicellular and may be cultured in a small amount of artificial growth media. Furthermore, many bacterial pathogens and their metabolic properties are well characterised in literature. This makes the method of the invention especially useful for the characterisation of bacterial microbes. As such, in a preferred embodiment, the microbial cells that are measured are bacterial cells. In a further preferred embodiment, the bacterial cells are E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa,

S. aureus, E. cloacae complex, K. oxytoca, C. koseri, E. faecium, E. faecalis, S. agalactiae, S, marcescens cells. These are all well-known, well-characterised, and often encountered bacterial pathogens. However, the method of the invention may also be used to characterise other microbes, such as Staphylococcus saprophyticus, Citrobacter spp. Streptococcus pyogenes (Group A strep),

Serratia spp., Streptococcus pneumoniae, Acinetobacter spp., Candida albicans, Proteus vulgaris,

Morganella morgani, Streptococcus spp., Enterococcus spp., Providencia spp., Streptococcus viridans, Salmonella spp., Haemophilus influenzae, Bacteroides fragilis, Helicobacter pylori,

Listeria monocytogenes, Neisseria gonorrhoeae, Shigella spp., Burkholderia cepacia, Legionella pneumophila, Vibrio cholerae, Clostridium difficile, Bordetella pertussis, Treponema pallidum,

Campylobacter jejuni, Chlamydia trachomatis, Yersinia pestis, Francisella tularensis, Brucella spp., Mycobacterium tuberculosis, Leptospira interrogans, Coxiella burnetii, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Borrelia burgdorferi, Rickettsia rickettsii, Rickettsia prowazekii, Rickettsia typhi, Chlamydophila spp., Acinetobacter baumannii.

This Het is non-exhaustive and is not to be interpreted as Hiniling the method of the invention to the aforementioned microbial species.

The method of the invention, involves assessing one or more metabolism-dependent changes in the concentration of analytes present in the culture. With each additional measured analyte, the accuracy and resolution of the method may be improved. Therefore, in a preferred embodiment of the method of the invention, the metabolism dependent changes over time in the concentration of at least one analyte are measured, preferably two or more analytes are measured, most preferably three or more analytes. When more information on the analytes is available, it becomes increasingly possible to determine exactly which microbes are present in the investigated sample. The concept of determining changes in the concentration of more than one analyte over time is called chemical multiplexing. By choosing the proper analytes and culturing conditions, a specific biological fingerprint or profile may be established for individual species and microbial cells. As such, in a more preferred embodiment of the invention, the metabolism dependent changes in the concentration over time of one or more analytes represent a biological fingerprint of the microbial cell.

This method of invention may be performed using any number and types of analytes, and on a wide range of samples. However, the composition of the invention is particularly suitable for performing the method of the invention. Therefore, in a preferred embodiment, the composition of the invention is used in the method of the invention.

The invention further relates to a kit suitable for performing the method of the invention.

Such a kit suitably comprises means for performing every step of the method of the invention. As such, in one embodiment, the invention relates to a kit of parts for performing the method of the invention, wherein the kit of parts comprises: - cultivating means for cultivating microbial cells; - measuring and recording means for measuring and recording metabolism dependent changes in the concentration over time or on a comparative basis of one or more analytes; - optionally signal enhancing compounds which enhance the metabolism of the microbial cell compared to generally used nutrients.

The cultivating means may comprise any cultivating, measuring, and recording means described for the composition of the invention. It may further comprise any signal enhancing compounds also described for the composition of the invention. As such, in a preferred embodiment, the kit of parts comprising luminescent chemical sensor material, in particular selected from the group consisting of fluorophore, phosphorescent molecules, electrofluorescent molecules, thiols, ruthenium(1l), osmium(1l), rhenium{1), iridium(11]), platinum) and palladium(I1), rhodamine based dyes, indicators for Ca?*, indicators for Na „indicators for K+, indicators for Cl, potential-sensitive dyes, Iuciferase-based sensors and mixtures thereof. The kit may also be used for performing the method of the invention determining the drug susceptibility or resistance of microbial cells. Suitably, in a yet further preferred embodiment, the kit of parts also comprises a drug to be tested. This drug may be therapeutically active compound. More specifically, the drug may be an antimicrobial agent, or an antibiotic. Preferably the kit is easy to use and does not require a laboratory environment.

The invention will be further presented by the following examples that are not intended to limit the invention in any way, and only serve to illustrate the invention and exemplary embodiments thereof. In the Examples, reference is made to the following figures:

Figure 1: Characterization of pH changes and resulting fluorophore intensity change during bacterial growth. Bacterial cultures were performed in 384-well microplates with culture volumes of 50 ul. Fluorescence measurements were taken periodically (5 minute intervals) using a spectrophotometer whilst incubating at 37 °C.

Figure 2: Bacterial growth curves obtained by monitoring the intensity change of a pH sensitive fluorophore which responds to changes in the pH of the global growth medium. Each line represents the average of 9 unique reactions. The red line corresponds to a control reaction, where no bacteria were added to the culture medium. All bacterial cultures are dilutions of pre-cultures in

MHB, The measurements were performed in a 384-microwell-plate which was placed within a microplate reader heated to 37°C. Fluorescence measurements were recorded at 5 min intervals for a total of 16h. The CFUs indicated in the legend, reflect general CFU values seen for these dilutions and are thus not specific. These values provide a good approximation of the expected number of CFUs for each dilution following a pre-culture to an OD of 0.1.

Figure 3: Pseudomonas Aeruginosa identification by comparing pH measurement to O2 measurement.

Figure 4: Detection of metabolism of various bacteria before bacterial growth/duplication has occurred.

Figure 5: Staphylococcus aureus cultured in various commercially available culture broths (Tryptic Soy Broth (TSB), Brain Heart Infusion (BHI), and Mueller-Hinton Broth (MHB). Signal acquired using pH-sensitive chemical sensor.

Figure 6: Klebsiella pneumoniae cultured in MHB, from varying initial bacterial densities, in the presence and absence of supplementary dextrose. Signal acquired using pH-sensitive chemical sensor.

Figure 7: Proteus mirabilis cultured in MHB, from varying initial bacterial densities, in the presence and absence of supplementary dextrose. Signal acquired using pH-sensitive chemical

Sensor.

Figure 8: Antibiotic susceptibility results obtained by using patient urine samples which were tested using the method and composition of the invention. a) Antibiotic susceptibility tests of two unique urine samples tested using a prototype of the composition of the invention. Each urine was first mixed with highly concentrated bacterial nutrients (supplemented with additives) and aliquots were prepared with various antibiotics. Twenty uM of the pH-sensitive fluorophore was added to each aliquot to enable bacterial growth monitoring. Aliquots were injected into unique, isolated chambers of the cartridges, and the cartridge was inserted into a pre-heated (37°C) analyser, where it was periodically measured (every 5 minutes) for a duration of 4 hours. Each trace is an average of three unique culturing means comprising identical cultures / antibiotics. AST results from the prototype were compared with current gold-standard testing methods (Vitek2, bioMerieux,

France) revealing a 100 % correlation in susceptibility results (where comparisons were possible) for these specific samples.

EXAMPLES

Example 1

Monitoring of bacterial growth with a pH dependent fluorophore during culturing.

Suitability of the change in the concentration of one or more analytes in the sample over time to characterise microbial cells was assessed. During growth, microbial cells influence the culture medium through uptake, metabolism, and secretion of compounds. This in turn affects measurable chemical properties of the culture medium. Here, the change in pH through the metabolic activity of microbial cells was measured for 5 well-known microbial species (Escherichia coli, Klebsiella preumoniae, Staphylococcus aureus, Proteus mirabilis, and Pseudomonas aeruginosa), and whether the differences in metabolic activity measured can be used to distinguish between microbial species. The relation between the pH and the fluorophore can be seen in figure 1. As can be seen, the species show different fluorophore activity and corresponding changes in pH in their culture environment when growing, allowing this to be used to differentiate between species. In figure 2, it can be seen that the pH sensitive fluorophore can be used on the same 5 species in Fig. 1 at different initial starting densities. These figures show that even when present at low number in the sample, an observable and distinguishable signal can be obtained in a short time frame.

Example 2

Optimization of time required before a signal can be measured in several bacterial species.

Optimal culturing conditions for getting a rapid signal by which microbial cells in the sample using the method of the invention were investigated. To this end, different nutritional broths were used to produce a signal as fast as possible for the investigated species. Figure 4 demonstrates detection of metabolism of multiple bacteria before bacterial growth/duplication has occurred. Duplication time of E. Coli is every 20 minutes in the laboratory under aerobic, nutrient-rich conditions, yet the presented chemical composition displays faster results in 10 to 15 minutes, due to the enhanced metabolic rate due to the enhancing compounds. Figure 5 shows the growth of Staphylococcus aureus in three different commercially available broths, measured using a pH-sensitive chemical sensor. Figures 6 and 7 show the growth of Klebsiella pneumoniae and Proteus mirabilis cultured in MHB respectively, with and without the addition of 2.5 9% w/v dextrose. It can be seen from these figures that the addition of additional enhancing compounds may produce a stronger and earlier signal, presumably through improved or accelerated metabolic activity.

Example 3

Measurement of bacterial drug response with a pH dependent fluorophore during culturing.

The suitability for the method and composition of the invention for measuring the response of microbes to drugs was investigated. Figure 8 shows two unique urine samples taken from patients which were tested using various antibiotics. Both samples contained E.coli, which can be seen to have a very different antibiotic resistance spectrum between the two samples. The results as measured with the composition of the invention were compared to the results obtained with a state of the art device which is considered the gold standard (Vitek2), and shows an identical resistant pattern. Reliable results using the composition and method of the invention could be obtained in as little as 4 hours whereas the standard procedure took 48 hrs.

Claims

CONCLUSIONS

1. A composition for detecting and/or characterizing a metabolizing cell comprising: - culture media, - one or more metabolizing cells, - chemical sensor material that detects cell metabolism-dependent changes in the concentration of one or more analytes over time, which generates a signal, and - one or more signal-amplifying compounds that increase or limit the metabolism of the one or more metabolizing cells compared to general growth medium.

The composition of claim 1, wherein the enhanced and/or restricted metabolism can detect cell metabolism-dependent changes in the concentration of one or more analytes over time at an earlier stage.

Composition according to claim 1 or 2, wherein the one or more signal amplifying compounds are selected from the list consisting of: oligosaccharides, disaccharides, monosaccharides, such as dextrose, fructose, glucose, maltose, sucrose, mannose, arabinose, xylose, galactose and yeast extract, casamino acid, (L)-noradrenaline, N-hexanoyl-L-homoserine lactone (IUPAC: N-(2-oxohexanoyl)-L-homoserine lactone), acetate, citrate, phosphatidylcholine, gluconate, D-serine.

Composition according to any one of claims 1 to 3, wherein the signal amplifying compounds are present in the composition in a concentration between 0.01 and 100 g/l, preferably between 0.05 and 20 g/l, more preferably between 0.1 and 5 g/l, most preferably between 0.2 and 3 g/l.

The composition of any one of claims 1 to 4, wherein the general growth medium is selected from a list consisting of: Mueller-Hinton Broth (MHB), Tryptic Soy Broth (TSB), Lysogeny Broth (LB), Terrific Broth, Super Optimal Broth (SOB or Hanahan's Broth), Super Optimal Broth with Catabolic Repression (SOC), Brain Heart Infusion (BHI), Thioglycolate Broth, Sabouraud Dextrose Broth, MacConkey Broth, Triple Sugar Iron Broth (TSI), Urea Broth, Sulfide-Indole-Motility Medium (SIM), M9 Minimal, Magic Media Medium, ImMedia Medium, 2X YT Broth, NZCYM Broth, Phage Media, NZ Amine® Broth, in particular wherein the general growth medium is solidified with agar.

6. A composition according to any one of claims 1 to 5, wherein the analytes are metabolites or small molecules or (an)ions required for proper cellular function.

7. The composition of claim 6, wherein the metabolites or small molecules or (an)ions necessary for proper cellular function are present in the extracellular environment.

8. Composition according to claim 7, wherein the metabolites or small molecules or (an)ions necessary for proper cellular function are selected from the list consisting of: H*, OH, O2, CO, K*, Nat, Ca*, Mg, NO, nucleic acids, peptides, extracellular and/or intracellular proteins, such as proteases or β-lactamase, ATP, acids such as lactic acid, NADH, boric acid, citric acid, acetic acid, formic acid, butyric acid and propionic acid.

9. The composition of any one of claims 1 to 8, wherein the cell metabolism-dependent changes over time in the concentration of one or more analytes represent pre-growth lag, growth, division, duplication, stabilization, tolerance, viability, senescence and/or senescence or senescence and death of the cell.

10. Composition according to any one of claims 1 to 9, further comprising a medicament, in particular an antibiotic.

The composition of any one of claims 1 to 10, wherein the metabolizing cell is a prokaryotic cell, an archaeal cell or a eukaryotic cell.

The composition of any one of claims 1 to 11, wherein the metabolizing cell is an infected cell, in particular a metabolizing cell infected by a virus.

The composition of claim 11 or 12, wherein the metabolizing cell is a bacterial cell, in particular a bacterial pathogen.

The composition of claim 11 or 12, wherein the eukaryotic cell is an animal cell, in particular a human cell.

The composition of any one of claims 1 to 14, wherein the chemical sensor material is located in or on a matrix, preferably in a hydrogel, bead, micelle, nanoparticle and/or virus.

The composition of any one of claims 1 to 15, wherein the chemical sensor material is one or more luminescent sensor compounds.

The composition of claim 16, wherein the luminescent sensor compound is selected from the group consisting of fluorophores, phosphorescent molecules, electrofluorescent molecules, thiols, ruthenium(II), osmium(II), rhenium(I), iridium(III), platinum(II), and palladium(II), rhodamine-based dyes, Ca indicators, Na indicators, K indicators, CF indicators, potential-sensitive dyes, luciferase-based sensors, and mixtures thereof.

The composition of claim 17, wherein the luminescent sensor compound is selected from the group consisting of: - seminaphthalene fluorine (SNARF-4F), - platinum (IDoctaethylporphyrin ketone (PtOEP), - OXNANO, - ruthenium tris{2,2’-dipyridyl) dichloride hexahydrate (RTDP), - platinum (IDoctaethylporphyrin ketone (PtOEPK), - platinum (11)-meso-tetra(pentafluorophenyl)porphine (PÚTFPP), - tris(2-(dimethylamino)ethyl) 4,4',4"-{1H-pyrrole-1,2,5-triyl)tribenzoate (TPP-TDMAE), - hexaphenylsilol (HPS), - potassium-sensitive oligonucleotide (PSO), - tetraammonium salt, such as SBFI or PBFI, - tetra(tetramethylammonium) salts such as Sodium Green), - pyranine, - 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), - hexapotassium salt such as Calcium Green™-5N, - pentapotassium salt such as Fluo-4 or Magnesium Green™, - tripotassium salt such as Rhod-2, - 7-hydroxy-3H-phenoxazin-3-one 10-oxide (Resazurin), - tetrazolium compounds such as MTT, MTS, XTT, or WST-1, - glycylphenylalanylaminofluorocoumarin (GF-AFC), - luciferin, - Acridine Orange, - DAPL, - ethidium bromide,

- PicoGreen, -SYBR Green 1, - SYBR Green 11, -SYTO9, - cefotaxime + 4-hydroxy-1,8-naphthalimide (CTX-HN).

19. A method for characterizing microbial cells present in a sample from a subject, comprising the steps of: a) culturing the microbial cells present in the sample, b) measuring changes in the concentration of one or more analytes in the sample over time or on a comparative basis, the analytes being measured using a chemical sensor material that chemically interacts with the analytes, and c) characterizing the microbial cells by comparing the metabolism-dependent changes over time in the concentration of one or more analytes to a reference indicative of a specific microbial cell.

20. A method for determining the drug sensitivity or resistance of one or more microbial cells present in a sample from a subject, comprising the steps of: a) culturing the microbial cells present in the sample, b) measuring changes in the concentration of one or more analytes in the sample over time or on a comparative basis, wherein the analytes are measured using a chemical sensor material that chemically interacts with the analytes, and c) culturing the microbial cells present in the sample in the presence of at least one drug, and d) measuring changes in the concentration of one or more analytes in the sample over time or on a comparative basis, wherein the analytes are measured using a chemical sensor material that chemically interacts with the analytes, and wherein the difference between the changes in the concentration of one or more analytes measured in steps b) and d) is indicative of the drug sensitivity or resistance of one or more microbial cells present in the sample to be present.

21. A method according to claim 19 or 20, wherein the analytes are metabolites, small molecules or (anions) necessary for proper cellular function.

22. The method of claim 21, wherein the metabolites or small molecules or (an)ions required for proper cellular function are present in the extracellular environment.

23. A method according to claim 21 or 22, wherein the metabolites or small molecules or (an)ions required for proper cellular function are selected from the list consisting of: H*, OH, Os, CO), K*, Nat, Ca?*, Mg?*, NO, nucleic acids, peptides, extracellular and/or intracellular proteins, such as proteases or β-lactamase, ATP, acids such as lactic acid, NADH, boric acid, citric acid, acetic acid, formic acid, butyric acid and propionic acid.

24. A method according to any one of claims 19 to 23, wherein the chemical sensor material is one or more luminescent sensor compounds.

The method of claim 24, wherein the luminescent sensor compound is selected from the group consisting of fluorophore, phosphorescent molecules, electrofluorescent molecules, thiols, ruthenium(II), osmium(II), rhenium(I), iridium(III), platinum(II), and palladium(II), rhodamine-based dyes, Ca₂ indicators, Nat indicators, K₁ indicators, Cl indicators, potential-sensitive dyes, luciferase-based sensors, and mixtures thereof.

26. A method according to any one of claims 19 to 25, wherein the subject is a mammal, in particular wherein the subject is a human.

27. A method according to any one of claims 19 to 26, wherein the reference indicative of a specific microbial cell is a predetermined reference distinctive of a microbial cell.

28. The method of any one of claims 19 to 27, wherein the microbial cells are bacterial cells.

The method of any one of claims 28, wherein the bacterial cell is E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa, S. aureus, E. cloacae complex, K. oxytoca, C. koseri, E. faecium, E. faecalis, S. agalactiae, S. marcescens.

30. A method according to any one of claims 19 to 29, wherein the one or more analytes are measured (semi-)continuously, continuously or intermittently, preferably intermittently.

31. A method according to any one of claims 20 to 30, wherein the difference between the changes in concentration of one or more analytes measured in steps b) and d) is indicative of growth, growth inhibition or death of the microbial species.

32. A method according to any one of claims 19 to 31, wherein metabolism-dependent changes over time in the concentration of at least one analyte are measured, preferably two or more analytes are measured, most preferably three or more analytes.

33. The method of claim 19-32, wherein the metabolism-dependent changes in the concentration over time of one or more analytes represent a biological fingerprint of the microbial cell.

34. A composition according to any one of claims 1 to 18 for use in the method according to any one of claims 19 to 33.

A kit for carrying out the method according to any one of claims 19 to 33, the kit comprising: - culture means for culturing microbial cells; - measuring and recording means for measuring and recording metabolism-dependent changes in the concentration over time of one or more analytes; - optionally, signal-amplifying compounds that improve the metabolism of the microbial cell compared to commonly used nutrients.

36. The kit of claim 35 further comprising luminescent chemical sensor material, in particular selected from the group consisting of fluorophore, phosphorescent molecules, electrofluorescent molecules, thiols, ruthenium(II), osmium(II), rhenium(I), iridium(I), platinum(I), and palladium(II), rhodamine-based dyes, Ca¥ indicators, Na' indicators, K* indicators, Cl indicators, potential-sensitive dyes, luciferase-based sensors, and mixtures thereof.

37. The kit of claim 35 or 36, further comprising a medicament.