WO2004113375A2 - Antimicrobial compositions and uses thereof - Google Patents

Abstract

A polynucleotide encoding a polypeptide having α/β-type SASP activity and a delivery system therefor capable of targeting a bacterium, and an antibiotic, as a combined preparation for simultaneous, separate or sequential use as a medicament.

Description

Antimicrobial Compositions and Uses Thereof

Field of the Invention

The present invention relates to antimicrobial compositions, more particularly to antibacterial compositions, and uses thereof, particularly in the treatment of bacterial infections.

Background to the Invention /β-type SASP are a class of proteins known as the Small Acid-soluble Spore Proteins (SASP) which are produced by spore-forming bacteria during the process of sporulation. Spore-forming bacteria form a relatively small class of bacteria which are capable of producing endospores. Endospores are dormant non-reproductive survival forms of the bacteria which are resistant to inhospitable environments such as high temperatures, harmful chemical agents and damage from UV light. These spore-forming bacteria comprise Bacillus , Clostridia and Sporosarcina species as well as one strain of Thermoactinomyces and other less common species of Sporolactobacillus and Oscillospira . During a process of sporulation a class of proteins known as the small acid-soluble spore proteins (SASP) are produced. SASP are acid-soluble and have low molecular weights of between 5 and llkDa. SASP are reported to have two main roles within bacterial spores: firstly, they act to protect the spore DNA from damage from UV, heat, depurination and many potentially harmful chemical agents; and secondly, SASP provide a source of free amino acids upon spore germination, without which the newly vegetative cells cannot outgrow.

In Bacillus species there are three types of SASP known as α, β and γ type- SASP. The amino acid sequences of α/β- type SASP are highly conserved both within and between species (~70% identity and ~80% similarity, without gaps for Bacillus species) . However these proteins show no sequence similarity to any other protein family and do not contain any motifs characteristic of other DNA binding proteins (Setlow, 1988) . The α/β-type SASP are closely related immunogenically, have molecular weights of approximately 6.2-7.6 kDa and have a significant percentage of hydrophobic amino acids (up to 30%) (Setlow, 1988). The γ type SASP have a molecular weight of 8-11 kDa, are extremely low in large hydrophobic amino acids (<11%) and have a higher isoelectric points than the α/β type SASP from the same species (Setlow, 1988). In any given organism there are two major SASP of the α/β type, as well as many minor α/β type SASP, each encoded by a unique gene (Setlow, 1988) . In contrast, all the organisms which have been examined have only one γ type

SASP and its function is quite different to α/β type SASP, being used primarily to supply amino acids for outgrowth (Hackett and Setlow, 1987) . A list of all the α/β type SASP which have been sequenced to date are given in Appendix 1, together with their related protein sequences. The extent of conserved amino acid residues between these protein sequences is shown in Appendix 2.

Various studies on SASP have focused on characterising the way in which the α/β type SASP protects DNA from UV damage. In one study (Setlow et al 1991) a gene { sspC) encoding an α/β-type SASP was inserted in a plasmid under the control of an inducible promoter to show that α/β- type SASP cause DNA of a vegetative cell to assume spore- like characteristics . It was observed that binding of α/β type SASP to E . coli DNA caused an increase in plasmid negative superhelical density suggesting a concomitant change in DNA structure. It is postulated that a change in conformation of DNA from B-like towards A-like protects the DNA against UV light.

WO02/40678 describes uses of polypeptides having α/β-type SASP activity as a medicament, particularly to inhibit or prevent unwanted cell growth such as bacterial cell growth. WO02/40678 is primarily concerned with replacing the use of conventional antibiotics with polypeptides described therein.

Description of the Invention

It has now been surprisingly found that a polypeptide having α/β-type SASP activity may be used in combination with one or more antibacterial agents for the treatment of bacterial infections.

Accordingly, in a first aspect, there is provided a polypeptide having α/β-type SASP activity and an antibiotic, as a combined preparation for simultaneous, separate or sequential use as a medicament.

The polypeptide would be delivered to target cells by a delivery vector of a specific or generalised nature, or would itself encode a sequence capable of targeting specific cells.

A polypeptide according to the present invention may comprise any peptide, oligopeptide, protein and may exist in monomeric or . multimeric form with or without covalent modification such as post-translational modification including glycosylation. Typical polypeptides according to the present invention comprise the amino acid sequence : mannnssnsnellvpgaeqaidqmkyeiasefgvnlgadttarangsvggei tkrlvqlaeqqlgggtk (SEQ ID NO:l).

Preferably, the polypeptide comprises any one of the amino acid sequences shown in appendix 1, such as that encoded by the sspC gene from Bacillus subtilis, or preferably SspCΔ^11_D13κ, comprising a polypeptide encoded by the sspC gene from Bacillus subtilis modified to delete specific amino acid residues at the N terminal, as shown in appendix 3, specifically:

SspCΔ^11_D13κ makllipqaasaieqmkleiasefgvqlgaettsrangsvggeitkrlvrl aqqnmggqfh

This SspCΔ^11_D13κ protein has been shown to bind spore DNA so strongly that spore outgrowth following germination is markedly inhibited (Hayes and Setlow, 2001) . The α/β-type SASP protein, SspCΔ^11_D13κ, exhibits significant antibacterial activity against E. coli and S . aureus cells, causing up to a 6-log drop in bacterial cell viability within 2 h. Any one of these polypeptides may contain mutations (including additions) and/or deletions such as those produced by random mutagenesis or by site directed mutagenesis or modification, which do not substantially reduce the α/β type SASP activity thereof. Despite the high degree of sequence conservation between natural SASP proteins, significant differences in DNA affinities exist (Setlow et al . _r 1992). The potential exists to tailor SASP protein sequences to increase affinity of the protein for target DNA. On this basis it may be possible to utilise the natural variation in SASP or to engineer SASP to optimise targeting of different species of bacteria and/or desired genes within any given organism.

In a further aspect, the invention provides the

SspCΔ^11_D13κ polypeptide, or the gene encoding this polypeptide, for use as a medicament, preferably in accordance with the invention as described herein.

A further variant of the wild-type B . subtilis α/β-type SASP, SspC, has been found to show a >20-fold increase in binding affinity for DNA over that of SspC^wt (Kos an and Setlow, 2003) . This variant combines the SspC^ΔN11_D13κ N- terminal changes with a C-terminal extension of three residues. In vi tro the binding of SspC^{ΔN11_D13κ_c3} to DNA suppressed the formation of cyclobutane-type thymine dimmers and promoted the formation of spore photoproduct upon UV irradiation to the same degree as the binding of SspC^t. However, B . subtilis spores lacking major α/β- type SASP and overexpressing SspC^{ΔN11~D13κ-C3} had a 10-fold lower viability. UV irradiation of the complex between SspC^{Δι ~D13κ~c3} and pUC19 DNA generated a spectrum of photoproducts that was the same as that formed with the wild type SspC, indicating that SspC^{ΔN11~D13κ_c3} has the same effets on DNA in vi tro as does SspC^t, and thus likely has the same effect on DNA structure as SspC^wt (Kosman and Setlow, 2003) . The a/b-type SASP protein, SspC^{ΔN11_D13κ~c3} also exhibits significant antibacterial activity against E. coli. Generally, α/β type SASP activity may be measured by evaluating the effect of the polypeptide on DNA conformation. α/β type SASP activity may therefore be defined as the ability to convert DNA from a B-like conformation towards an A-like conformation. Methods for measuring α/β-type SASP activity are described below.

(a) A reference for describing the change in conformation from B- to A-like is Mohr et al . , 1991. Changes in circular dichroism spectra have long been regarded as sensitive criteria for DNA conformations and distinctions between the main families of secondary structure are unambiguous (Mohr et al . _r 1991). Interaction of both eukaryotic (calf thymus) DNA and prokaryotic DNAs with α/β-type SASP (in particular experiments using SspC from B . subtilis) induces spectroscopic features characteristic of A-DNA. Fourier- transform infrared (FTIR) spectroscopy provides an independent means of evaluating the conformational state of DNA complexed with α/β-type SASP. The FTIR spectra of concentrated solutions of calf thymus DNA show a principal absorption band at 1225 cm^~l which arises from the antisymmetric O-P-0 phosphate stretching vibration

(Mohr et al 1991) . This band shifts to 1246 cm^-1 with SspC-calf thymus. Such behaviour is characteristic of a B- to A-transition, although it should be noted that hydration effects alone can also influence the position of this O-P-0 stretching band. Therefore an additional indication of B- to A- transition can be used, comprising the appearance in the FTIR spectrum of the SASP-DNA complex (a 1:1 ratio) of an absorption band at 1185 cm--'-. This is a specific marker for the A conformation of DNA since neither the B- or C- form of DNA produce an infrared band at 1185 cm^_1(Phole and Fritzsche, 1980). Hydration effects do not influence or affect the analysis of the 1185 cm-1 band. FTIR results show that, although dehydration can cause DNA to change conformation from B- to A-like, SASP promote this conformation change such that it reaches completion with significantly less reduction in humidity than is required for the process with DNA alone (Mohr et al, 1991) .

(b) Also, SASP bound to DNA will protect DNA from degradation by DNase (Setlow et al . _r 1992). Two assays are possible to show that SASP bound to DNA in vitro protects a nucleic acid from nuclease digestion. The first, an electrophoretic assay, is the most straightforward. Briefly, nucleic acid (including pUC19 and pUBllO) is incubated with various amounts of SASP for

1 hour at 37°C. At this point DNase I (or S . aureus nuclease) is added and incubation carried out for a further 15 min before adding SDS/EDTA followed by NaCl and ethanol to precipitate the DNA. The precipitated DNA is analysed by agarose (2%) (for polynucleotides) or acrylamide (oligonucleotides) gel electrophoresis . Protection of both pUC19 and pUBllO is evident at a ratio of SASP to DNA of 1:1 and is maximal at a ratio of 4:1. Analysis of DNase protection for four other α/β-type SASP indicate that these proteins also confer DNAse resistance to this plasmid. SASP-I from Bacillus cereus and SASP-A show similar patterns of protected bands wherease SASP-α and -β from Clostridia bifermentans give different patterns (Setlow et al . , 1992). The second assay is an acid precipitation assay.

(c) SASP bound to DNA protects the DNA against cleavage by restriction enzymes, particularly those with specificity for GC-rich sequences (Setlow et al . , 1992). Restriction enzyme digestions of pUC19 DNA bound by SspC

(8:1 ratio of SspC to DNA) were carried out and digests analysed by agarose gel electrophoresis . For enzymes rich in AT sequence i.e. Darl (TTTAAA) inhibition was <10%. Increasing levels of GC content in the restriction ' enzyme recognition site led to increased protection by SASP with those enzymes recognizing GC-rich sequences (i.e. Kpnl GGTACC) being inhibited >75%.

(d) Also SASP increase negative superhelical density of plasmids in the presence of topoisomerase I. The method for assaying this effect is given in Nicholson et al . , 1990b. In summary, 1 μg samples of plasmid (pUC19 or pUBllO) are incubated overnight in a 20 μl volume reaction mixture at 4°C with various amounts of SspC, followed by topoisomerase I addition and further incubation for 2 h at 37°C. After deproteinization, samples are analysed by electrophoresis on agarose gels containing chloroquine (2 μg per ml) . The average value of negative supertwists can be determined by comparing the position of the bands on the agarose gel with a set of standards prepared by incubating plasmid DNA with topoisomerase in the presence of differing amounts of ethidiu bromide (Nicholson and Setlow, 1990) . Maximum SspC binding results in introduction of a large number of negative supertwists in both plasmids. With 12 μg SspC added to the plasmid DNA approximately 18 and 38 supercoils are introduced in pUC19 and pUBllO, respectively. Since pUC19 is approximately 60% the size of pUBllO, the superhelical density induced in both plasmids by SspC binding is similar. Note that the binding of protein HU to DNA which does not induce a B- to-A conformation change in DNA only induces ~40% the number of negative supertwists per unit of DNA as does SspC (Nicholson et al . , 1990).

(e) Also, SASP bound to DNA protects against the formation of cyclobutane-type thymine dimers upon UV irradiation, but promotes formation of spore photoproduct, an adduct between adjacent thymine residues

(Nicholson et al . , 1991). Yields of pyrimidine dimers and spore photoproduct (SP) were <0.2% and 8% of total thymine, respectively when DNA saturated with SASP was irradiated at 254 nm with 30kJ/m2. In the absence of SASP the yields were reversed - 4.5% and 0.3%, respectively (Nicholson et al . , 1991). Yields of SP in vivo i.e. in spores and thymine dimers in vegetative cells are similar and extremely high (>25% of total thymine) (Donnellan and Setlow, 1965) . UV irradiation of DNA in vitro also ordinarily produces fluorescent bipyrimidine adducts, cyclobutane type cytosine dimers and also cyclobutane dimers between cytosine and thymine as well as a 6-4 bipyrimidine adduct. The yields of all types of photoproduct are greatly reduced upon irradiation, in vitro, of DNA bound by α/β-type SASP (Fairhead and Setlow, 1991) .

(f) It has also been demonstrated that α/β type SASP reduce the rate of depurination of DNA in vi tro at least 20-fold. Three different procedures for measuring DNA depurination in vitro are given in Fairhead et al . , 1993.

Several α/β-type SASP have been examined to determine their antibacterial activity. The following table shows the decrease in E. coli cell viability in the presence of various SASP:

Time sspD saspC saspC3

(min) (Control) sspD (Control) saspC (Control) saspC3

0 3000000 3000000 4600000 4600000 2900000 2900000

30 7000000 1000 11000000 4000 7000000 470000

60 17000000 2000 16000000 1500 16000000 800

90 32000000 1500 37000000 1400 25000000 3600

120 35000000 1300 50000000 1500 37000000 3600

240 180000000 4700 170000000 3000 200000000 5300

Time sspCl sspl SU-2

(min) (Control) sspCl (Control) sspl (Control) SU-2

0 3900000 3900000 4100000 4100000 1300000 1300000

30 4000000 60000 15000000 2000 2200000 9000

60 10000000 160000 16000000 2100 5000000 11000

90 17000000 150000 18000000 2100 14000000 11000

120 28000000 40000 21000000 1200 17000000 22000

240 270000000 28000 41000000 5000 200000000 57000

Table 1: Cultures of E. coli cells containing pET24d with sspD (Bacillus subtilis) , saspC {Bacillus megaterium) , saspC3 {B . megaterium) , sspCl { Clostridium perfringens) , sspl (C. perfringens) , or SU-1

( Sporosarcina ureae) encoding genes inserted were grown to an OD₆oo o approximately 0.2. Each culture was split and one sub-culture was induced with IPTG (1 mM) . Aliquots were removed at 30 min intervals and cells plated out to enumerate no. of viable bacterial cells.

In a further aspect, the present invention provides use of a polypeptide having α/β-type SASP activity and an antibiotic for the production of a combined preparation for simultaneous, separate or sequential use in the treatment of bacterial infection. The present invention also provides a method of treatment of a human or animal subject having a bacterial infection, which method comprises administering to the subject an effective amount of a polypeptide having α/β- type SASP activity and an antibiotic, simultaneously, separately or sequentially.

The combined preparation of the present invention may inhibit or prevent bacterial cell growth.

In a further aspect, the present invention provides a polynucleotide encoding a polypeptide having α/β-SASP activity and an antibiotic, as a combined preparation for simultaneous, separate or sequential use as a medicament. A delivery system is provided for the polynucleotide which is capable of targeting a bacterium. In this way, the polypeptide having α/β-type SASP activity may be expressed in the target bacterium by the polynucleotide thereby resulting in inhibition or prevention of growth of the bacterial cell. The antibiotic has antimicrobial activity against the target bacterial cell. In this way, the antibacterial action of both the expressed polypeptide and antibiotic may be used in combination to treat infection by a population of the bacterial cells.

The polynucleotide may be DNA or RNA, depending on the delivery system used. Whilst it is preferred for reasons of stability and ease of manipulation that the polynucleotide is DNA, if RNA is used it eliminates the possibility of SASP inhibiting its own production. In a particularly preferred embodiment, the DNA comprises the sspC gene from B . subtilis, more preferably the modified sspC gene encoding SspCΔ^11_D13κ. Degeneracy of the genetic code allows mutations which do not alter the amino acid sequence of the expressed production of the DNA.

The polynucleotide may be used for the preparation of a medicament for inhibiting or preventing cell growth in a number of ways. In one embodiment, the medicament comprises the polynucleotide, typically formulated for administration to a subject. In another embodiment, the polynucleotide is used to manufacture a medicament comprising the polypeptide. In a further embodiment, the medicament may be manufactured inside the target cell as the polypeptide.

Accordingly, the present invention provides use of a polynucleotide encoding a polypeptide having α/β-type SASP activity and an antibiotic for the production of a combined preparation for simultaneous, separate or sequential use in the treatment of infection by a bacterium.

In a further aspect, the present invention provides use of (i) a polynucleotide encoding a polypeptide having α/β-type SASP activity and a delivery system therefor capable of targeting a bacterium; and ( ii ) an antibiotic with antimicrobial activity against the bacterium, for the production of a combined preparation for simultaneous, separate or sequential use in the treatment of infection by the bacterium.

In a further aspect the present invention provides a method of treatment of a human or animal subject having an infection by a bacterium which method comprises administering to the subject an effective amount of a polynucleotide encoding a polypeptide having α/β-type SASP activity and an antibiotic separately, simultaneously or sequentially.

In a further aspect the present invention provides a method of treatment of a human or animal subject having an infection by a bacterium which method comprises administering to the subject an effective amount of (i) a polynucleotide having α/β-type SASP activity and a delivery system therefor capable of targeting a bacterium; and { ii ) an antibiotic with antimicrobial activity against the bacterium, separately, simultaneously or sequentially.

In a particularly advantageous embodiment of the present invention, a polynucleotide encoding a protein with α/β type SASP activity is delivered to bacterial cells by a bacterial virus known as a bacteriophage . In general each genera of bacteria, and often species, has its own range of bacteriophages which are able to adsorb, inject viral DNA and multiply within. Some bacteriophages are able to infect only one particular strain of bacteria. Thus, using a bacteriophage as a delivery system ensures that no bacteria, other than those targeted, will be infected.

There are various types of bacteriophage, including lysogenic phages such as lambda, filamentpus phages, or lytic phages which, during in vitro experiments at least, have not been found able to lysogenise bacteria. Bacteriophages can comprise single stranded DNA or RNA, to which SASP is unable to bind, as well as the more common double stranded DNA such as lambda. It is preferred to use a bacteriophage which cannot establish, or cannot stably maintain lysogeny in target cells. It is preferred to inactivate at least one of the genes encoding products involved in the lytic process, particularly a holin gene.

Inactivation of a lysis gene is conveniently achieved by inserting into the gene, or replacing all or part of the lysis gene with, the polynucleotide according to the present invention. If production of the polypeptide is controlled by the phage' s native late promoter, this can have a further advantage in that expression of lysis genes occurs sufficiently late in the life cycle of the phage that many phage particles can be produced in a host cell before the polypeptide is expressed by the polynucleotide .

Typical lysis genes include the S gene of E. coli bacteriophage lambda or the holin gene of S . aureus bacteriophage φll. These genes encode a holin, which is a protein that forms pores in the host cell which then allows other lytic enzymes produced by the bacteriophage to cause lysis. A polynucleotide of the present invention may be inserted within, or partially or completely replace the holin gene and may come under control of the respective phage' s late promoter, such as the P_R' promoter of E. coli , or any other selected promoter. Analogously, the polynucleotide may be inserted in one of the other genes involved in the lytic cycle such as the R gene of E. coli phage lambda or the lytA gene of S . aureus phage φll. The R gene product is a lytic transglycosylase and the lytA gene product is a peptidoglycan hydrolase. In this case, the holin gene may or may not be additionally disrupted. Equivalent genes in other types of bacteriophage can be used in an analogous way as locations for the polynucleotide, particularly when targeting bacteria other than E . coli or S . aureus .

In a further embodiment, the polynucleotide can be located anywhere on the bacteriophage chromosome and placed under control of an alternative bacteriophage or bacterial promoter. Optionally, production of one or more proteins involved in lysis could still be inhibited. Alternatively, the lytic cycle could be left to run its course. For example, it is possible to use bacterial promoters which react to cues found in a host under infection conditions such as temperature sensitive promoters, the P3 promoter of the Staphylococcus aureus agr locus, or other promoters involved in two component sensor regulator pathways: for example the Pi promoter of S. aureus is upregulated during growth in vivo . Further examples include promoters active under microaerophilic conditions, under low iron conditions or those stimulated by host specific factors such as nicotinic acid or magnesium ions.

In a further aspect, the virus may be modified to increase or alter its host specificity. In the case of bacteriophages, these may be engineered to infect cell types other than bacteria by modifying the tail to generate different affinities and/or ability to infect cells. For example, it has been shown that mammalian cell tropism can be conferred on filamentous bacteriophage by presenting a ligand that binds to a mammalian cell surface molecule on the coat protein of the bacteriophage (Larocca et al 1998). For example, it has been demonstrated that when a phage M13) is engineered to display genetically the growth factor ligand, FGF2 (as a fusion to its minor coat protein pill) , it acquires the ability to deliver a gene to mammalian cells through the FGF receptor resulting in transduced cells (Larocca et al . , 1999). Other workers have also reported similar findings using phage that display a single chain antibody (scFvc) directed against ErbB2, a member of the EGF (epidermal growth factor) receptor family (Poul and Marks, 1999) . Selection of phage engineered for receptor-mediated gene transfer to mammalian cells can be enhanced by screening phage libraries for functional ligands capable of delivering DNA to cells (Kassner et al . , 1999).

A barrier to Caudovirales (tailed bacteriophages) infecting cells other than their natural host is the lack of an appropriate receptor present on the surface of the target bacterium to which the phage can adsorb. By addressing this it is possible to create phages which contain the same modified DNA (i.e. SASP containing) but which can target broad host ranges. For example, a phage may be modified to allow it to target a receptor which is common in several species of bacteria. Alternatively, the modified phage DNA may be packaged into identical phage heads which have been given a variety of tails each expressing an affinity for receptors expressed by different bacteria. Bacteriophages can also express antibody fragments as fusion proteins. For example the filamentous phage M13 has been engineered to express a g3p-fusion protein comprising a Helicobacter pylori- antigen-binding single-chain variable fragment (ScFv) (Cao et al . , 2000). This ScFv-phage decreased the cfu of all tested strains of H. pylori . It may also be possible to cause a target bacterium to express a chosen receptor. For example, it has already been shown that Pseudomonas species can be modified to express LamB receptors, which are the receptor for lambda bacteriophage (de Vries et al . , 1984). The gene, lamB, encoding these protein receptors is introduced into Pseudomonas by means of a plasmid and inserts into the Pseudomonas chromosome by homologous recombination. Whilst it is not always practicable to transform cells with plasmids it is possible to deliver the lamB gene to any Gram negative bacteria by means of a modified lysogenic bacteriophage specific to the target. The lamB gene should be under the control of a strong bacterial promoter and the phage should be altered so that lysogeny is always established. Administration of this type of phage, then, will render Pseudomonas species liable to infection by subsequently administered SASP/lambda. Other such modified phages can be produced for each target species and will act to broaden the host range of any given bacteriophage containing SASP.

In these ways, it is possible to extend the range of bacteria that a SASP containing phage can target, at least within the broad categories of Gram positive or Gram negative bacteria.

The antibiotic used in the combined preparation of the invention may be any antibiotic useful in the treatment of bacterial infection. In one embodiment, the antibiotic is a broad spectrum antibiotic. The antibiotic according to the invention may be a single antibiotic or a combination of more than one antibiotic. Where a combination of antibiotics is used, each antibiotic may be administered simultaneously, separately or sequentially in relation to the other antibiotics and in relation to the polypeptide or polynucleotide of the invention.

Examples of common pathogenic bacteria are listed below together with antibiotics that may be used to treat these pathogens. Examples of bacteriophages which are able to infect these bacteria and which may be modified to carry an α/β-type SASP gene are also listed below. However, the present invention is not limited to the bacteriophages or antibiotics listed below as one skilled in the art could easily determine other antibiotics or phages useful in combination with a protein with α/β-type SASP activity.

Pathogen and

Antimicrobial agent or antimicrobial group

Ac±netobacter baumannii and A. lwoffx

Penicillin, chloramphenicol, a combination of aminoglycoside and ticarcill, amikacin, ampicillin/sulbactam, caftazidime

Ciprofloxacin, imipenem

Bacillus anthracis

Amoxicillin, ciprofloxacin, doxycycline, penicillin

Bordetella pertussis

Erythromycin, trimethoprim-Sulfamethoxazole

Campylohacter jeju l

Macrolides (erythromycin, clarithromycin, or azithromycin Fluoroquinolones (ciprofloxacin, levofloxacin, gatifloxacin, ) or moxifloxacin

Clostridium dificile Metronidazole, bacitracin, glycopeptides (e.g. vancomycin)

Enterobacter cloacae

3rd generation cephalosporins, gentamicin, tobramycin; carbenicillin, amikacin, aztreonam, imipenem

Enterococcus faecalis and E. faeclum and E. zakezaki

Glycopeptides (e.g. dalbavancin, daptomycin, ramoplanin, ) Streptogramins (e.g. quinopristin+dalfopristin)

Escherxchia coli trimethoprim-sulfamethoxazole (abbrev. TMO-SMO) , ampicillin; 1st or 3rd generation cephalosporins, ciprofloxacin, Ertapenem, aminoglycosides, aztreonam, a penicillin + a penicillinase inhibitor

Hemophilus influenza : chloramphenicol' or 3rd generation cephalosporins; ampicillin, Ertapenem, TMO-SMO, cefaclor, cefuroxime

Klebsiella pneumonia

1st or 3rd generation cephalosporins; cefotaxime, moxalactam, amikacin, chloramphenicol

Listeria monocytogenes

Ampicillin, ampicillin + an aminoglycoside, penicillin TMO-SMO

Mycobacteria isoniazid (INH) + rifampin or rifabutin given along with pyrazinamide +/or ethambutol

Neisseria : penicllin G; chloramphenicol, amoxicillin, a sulphonamide, Spectinomycin, ceftriaxone, cefuroxime or cefoxitin, Ciprofloxacin, rifampin

Propionibacter acnes

Tetracycline, clindamycin, erythromycin, metranidozole Pseudomonas aeruginosa tobramycin or gentamycin (+/- carbenicillin, aminoglycosides) , amikacin, cefazidime, aztreonam, imipenem

Salmonella :

Chloramphenicol, ampicillin, TMO-SMO, ciprofloxacin

Shigella (various)

Ciprofloxacin, TMO-SMO, ampicillin, chloramphenicol

Staphylococci penicillin G, 1st generation cephalosporins, imipenem, Erythromycin, a penicillinase-resisting penicillin; amoxicillin + clavulanic acid, Glycopeptides (e.g. dalbavancin, daptomycin, ramoplanin, vancomycin, teicoplanin) , oxazolidonones (e.g. Linezolid) , tetracycline, Mupirocin, Gentamycin, Neomycin, bacitracin, fusidic acid

Streptococcus penicillin G; 1st generation cephalosporins, Mupirocin erythromycin, chloramphenico, Oxazolidonones (e.g. linezolid)

Vibrio cholera tetracycline; TMO-SMO, chloramphenicol

Yersinia pestis streptomycin, tetracycline, chloramphenicol

Bacteriophages of respective bacterium

Bacteriophages of Acinetobacter

E6, E8, E9, E13, E15, 1, 11, 66

Bacteriophages of Bacillus anthracis

AP631

Bacteriophages of Bordetella

134

Bacteriophages of Caiαpylohacter

Vfi-6, (syn= V19) , Vfv-3, V2, V3, V8 , V16, (syn= Vfi-1) , V19, V20(V45), V45, (syn= V-45) Bacteriophages of Chlamydia φCPAR39, PhiCPGl, 2, chpl

Bacteriophages of Clostridia

C , CA2 , CA3 , CPT1 , CPT4 , cl , c4 , c5 , HM7 , Hn/Ai , Hι₈/ ι , H₂₂/S₂₃ , H₁₅₈/Aι , K₂/Ai , K₂ι/S₂₃ , M_L, NA2^tox_, Pf2 , Pf3 , Pf4 , S₉/S3 , Su/Ax , S₄₄ /S₂₃ , α2 , 41 , 112 /S₂₃ , 214 /S₂₃ , 233/A_x, 234 /S₂₃ , 235/S₂₃, II-l , II-2 , I I-3

Coliphages

AC30, CVX-5, Cl, DDUP, ECl, EC2, E21, E29, FI, F26S, F27S, Hi, HK022, HK97, (syn=ΦHK97), HK139, HK253, HK256, K7, KU1, N15, ND-1, no.D, PI, P2, PA-2, q, S2, Tl, (syn= α), (syn= P28), (syn= T-l) , (syn= Ti) , T3, T3C, T4, T5, (syn= T-5), (syn= T₅) , T7, UC-1, VT2-Sa, w, β4, γ2, λ, (syn= lambda), (syn- Φλ), ΦD326, φγ, Φ06, Φ7, Φ10, φ80, χ, (syn=χι), (syn= φχ), (syn=φχχ) , 2, 4, 4A, 6, 8A, 102, 150, 168, 174, 186, 933

Bacteriophages of Enterobacter

C3, WS-EO20, WS-EP26, WS-EP28, φmp, 667/617, 886 .

Bacteriophages of JSJnterocoσci

DS9, H24, M35, P3, P9, SB101, S2, 2BII, 5, 182a, 705, 873, 881, 940, 1051, 1057, 21096C

Bacteriophages of Helicobacter HP1

Bacteriophages of Haemophilus

S2, HP1, Flu, Mu, N3

Bacteriophages of Klehsiella

FC3-11, K1₂B, (syn= K12B) , Kl₂₅, (syn= K125) , K1₄₂B, (syn= K142), (syn= K142B) , Klι₈₁B, (syn= K1181), (syn= K1181B) , Kl_765/ι, (syn= K1765/1) , K1₈₄₂B, (syn= K1832B) , KI937B, (syn= K1937B) , LI, φ28, 7, 60, 92, 231, 483, 490, 632

Bacteriophages of Listeria

A005, A006, A020, A500, A502, A511, A118, A620, A640, B012, B021, B024, B025, B035, B051, B053, B054, B055, B056, B101, B110, B545, B604, B65, C70, D44, HS047, H10G, H8/73, H19, H21, H43, H46, H107, H108, H110, H163/84, H312, H340, H387, H391/7, H684/74, H924A, PS, U153, φMLUP5, (syn= P35) , 00241, 00611, 02971A, 02971C, 5/476, 5/911, 5/939, 5/11302, 5/11605, 5/11704, 10, 52, 184, 243, 575, 633, 699/694, 744, 900, 1090, 1317, 1444, 1652, 1806, 1807, 1921/959, 1921/11367, 1921/11500, 1921/11566, 1921/1246, 1921/12582, 1967, 2389, 2425, 2671, 2685, 3274, 3550, 3551, 3552, 4276, 4277, 4292, 4477, 533, 5348/11363, 5348/11646, 5348/12430, 5348/12434, 10072, 11355C, 11711A, 12029, 12981, 13441, 90666, 90816, 93253, 907515, 910716

Bacteriophages of Mycobacteria

AG1, AL_X, ATCC 11759, A2, B.C₃, BG2 , BK, BK₅, butyricum, B-1, B5, B7, B30, B35, Clark, Cl, C2, DNAIII, DSGA, DSP_X, D4, D29, GS4E, (syn= GS₄E) , GS7, (syn= GS-7), (syn= GS₇) , IPα, lacticola, Legendre, Leo, L5, LG, (syn=ΦL-5), MC-1, MC-3, MC-4, minetti, MTPHll, Mx4, MyF₃P/59a, phlei, (syn= phlei 1) , phlei 4, Polonus II, rabinovitschi, Rvl, Rv2, smegmatis, TM4, TM9, TM10, TM20, Y7, Y10, φ630, IB, IF, 1H, 1/, 67, 106, 1430

Bacteriophages of Ne±sseria

Group I, group II, NP1

Bacteriophages of Propionobacteria

P-a-1, P-a-2, P-a-3, P-a-4, P-a-5, P-a-6, P-a-7, P-a-i P-a-9, PB2, TL110B7

Bacteriophages of Mycoplasma arthritidls

MAVI

Bacteriophages of Pseudomonas af, A7, B3, B33, B39, BI-1, C22, D3, D37, D40, D62, D3112, F7, F10, g, gd, ge, gf, Hwl2, Jbl9, KFl, L°, OXN- 32P, 06N-52P, PCH-1, PC13-1, PC35-1, PH2, PH51, PH93, PH132, PMW, PM13, PM57, PM61, PM62, PM63, PM69, PM105, PM113, PM681, PM682, P04, PP1, PP4, PP5, PP7, PP64, PP65, PP66, PP71, PP86, PP88, PP92, PP401, PP711, PP891, Pssy41, Pssy42, Pssy403, Pssy404, Pssy420, Pssy923, PS4, PS-10, Pz, SD1, SL1, SL3, SL5, SM, φC5, φCll, φCll-1, φC13, φ,Cl5, φCTX, φMO, φX, φ04, φll, φ240, 2, 2F, 5, 7m, 11, 13, 13/441, 14, 20, 24, 40, 45, 49, 61, 73, 148, 160, 198, 218, 222, 236, 242, 246, 249, 258, 269, 295, 297, 309, 318, 342, 350, 351, 357-1, 400-1

Bacteriophages of Salmonella c, C236, C557, C625, C966N, El, E34, Felix, F0, g, GV, G5, G173, h, IRA, Jersey, L, MB78, P22-1, P22-, P22-12, Sabl, Sab2, Sab2, Sab4, Sanl, San2, San3, San4, San6, San7, Sanδ, San9, Sanl3, Sanl4, Sanl6, Sanlδ, Sanl9, San20, San21, San22, San23, San24, San25, San26, SasLl,

SasL2, SasL3, SasL4, SasL5, S1BL, SII, Vill, Vir, ViVI, fl, 1, 2, 3a, 3al, 8, 14, 23, 25, 31, 46, 102, 163, 175, 1010

Bacteriophages of Shigella

Bll, DDVII, (syn= DD7 ) , FS_D2b, (syn= W₂B) , FS₂, (syn= F₂) , (syn= F2), FS₄, (syn= F₄) , (syn= F4, FS₅, (syn= F₅) , (syn= F5), FS₉, (syn= F₉) , (syn= F9) , Fll, P2, P2-SO-S, SG₃₆, (syn= SO-36/G), (syn= G36) , SG₃₂₀₄, (syn= SO-3204/G) , SG₃₂₄₄, (syn= SO-3244/G) , SH_l (syn= HI), SH_Vn, (syn= HVII), SHix, (syn= HIX) , SH_XI, SH_XII, (syn= HXII) , SKI, KI, (syn= Si), (syn= Ssl) , SKVII, (syn= KVII) , (syn= S_Vn) , (syn= SsVII), SKIX, (syn= KIX) , (syn= S, (syn= SsIX) , SKXII, (syn= KXII) , (syn= S_XII) , (syn= SsXII) , STi, ST_m, ST_rv, STvi, ST_V1I, S70, S206, U2-SO-S, 3210-SO-S, 3859-SO-S, 4020-SO-S,2, 37, φ3, φ5, φ7, φ8, φ9, φlO, φll, φl3, φl4, φl8 , φ80

Bacteriophages of Staphylococcus

ACI, AC2, A6"C", A9"C", b⁵⁸¹, CA-1, CA-2, CA-3, CA-4, CA- 5, Dll, K, L39x35, L54a, M42, Nl, N2, N3, N4, N5, N7 , N8, N10, Nil, N12, N13, N14, N16, P3, P14, Ph6, Phl2, Phl4, PVL, PV83, U4, U15, SI, S2, S3, S4, S5, tIII-29S, Twort, X2, Zi, φB5-2, φD, w, 11, (syn= φll), 12, (syn= φl2) , 13, (syn= φl3) , (syn= φll-M15) , 15, 17, 28, 28A, 29, 31, 31B, 37, 42, 42D, (syn= P42D) , 44A, 47, 48, 51, 52, 52A, (syn= P52A) , 52B, 53, 55, 69, 71, (syn= P71) , 71A, 72, 75, 76, 77, 79, 80, 80a, 81, 82, 82A, 83A, 84, 85, 86, 88, 88A, 89, 90, 92,93, 95, 96, 102, 107, 108, 111, 129-26, 130, 130A, 155, 157, 157A, 165, 187, 275, 275A, 275B, 356, 456, 459, 471, 471A, 489, 581, 676, f812, 898, 1139, 1154A, 1259, 1314, 1380, 1405, 1563, 2148, 2638A, 2638B, 2638C, 2731, 2792A, 2792B, 2818, 2835, 2848A, 3619, 5841, 12100

Bacteriophages of Streptococcus

AT298, A5, alO/Jl, al0/J2, al0/J5, alO/J9, A25, BTll, b6, CA1, CP-1, c20-l, C20-2, DP-1, Dp-4, DTI, ET42, elO, F_A101, F_EThs, F_κ, F_κκ101, F_KL10, F_KP74, F_κll, F_L0Ths, F_γ101, φl, Fio, F₂₀140/76, g, GT-234, HB3, (syn= HB-3) , HB-623, HB-746, M102, O1205, fO1205, PST, P0, Pi, P2, P3, P5, P6, P8, P9, P9, P12, P13, P14, P49, P50, P51, P52, P53, P54, P55, P56, P57, P58, P59, P64, P67, P69, P71, P73_Λ P75, P76, P77, P82, P83, P88, sc, sch, sf, Sfill, (syn= SFill), (syn= φSFill) , (syn= FSfill) , (syn= fSfill) , sfil9, (syn= SF119) , (syn= φSFil9) , (syn= φSfil9) , Sfi21, (syn= SFi21), (syn= φSFi21) , (syn= φSfi21) , ST_G, STX, st2, ST₂, ST₄, S3, (syn= φS3) , s265, 1A, IB, φXz40, φl7, φ42, φ57, φ80, φ81, φ82, φ83, φ84, φ85, φ86, φ87, φ88, φ89, φ90, φ91, φ92, φ93, φ94, φ95, φ96, φ97, φ98, φ99, φlOO, φlOl, φl02, φ227, φ7201, wl, w2, w3, w4, w5, w6, w8 , wlO, 1, 6, 9, 10F, 12/12, 14, 17SR, 19S, 24, 50/33, 50/34, 55/14, 55/15, 70/35, 70/36, 71/ST15, 71/45, 71/46, 74F, 79/37, 79/38, 80/J4, 80/J9, 80/ST16, 80/15, 80/47, 80/48, 101, 103/39, 103/40, 113, 120, 124,121/41, 121/42, 123/43, 123/44, 124/44, 337/ST17

Bacteriophages of Vibrio fsl, fs-2, hv-1, OXN-52P, P13, P38, P53, P65, P108, Pill, TP1, VP3, VP6, VP12, VP13, 70A-3, 70A-4, 70A-10, 72A-1, 108A-3, 109-B1, 110A-2, 138, 145, 149, (syn= φl49), 163, IV, (syn= group IV)

Bacteriophages of Yersinia

R, Y, PI, Yer2AT

There are additional bacterial pathogens and their respective phages which are too numerous to list and whilst there is no significant problem with antibiotic resistance in these bacteria, nevertheless they make excellent candidates for treatment with α/β-type SASP: combined with one or more antibiotics. Thus, all bacterial infections caused by bacteria for which there is a corresponding phage can be treated using the present invention.

The animals to be treated by the present invention include but are not limited to man, domestic pets, livestock and pisciculture, and all instances where an antibiotic is used. While the present invention can be used to treat any bacterial infection in an animal in combination with any antibiotic, it would be particularly useful as a therapy in infections caused by drug- resistant bacteria. The routes of administration include but are not limited to: topical, oral, aerosol or other device for delivery to the lungs, nasal spray, intravenous, intramuscular, intraperitoneal, intrathecal, vaginal, rectal, lumbar puncture, and direct application to the brain and/or meninges .

The uses for which the combined preparation of the present invention may be suitable include but are not limited to treating topical infections, dental caries, respiratory infections, eye infections or localised organ infection.

The present invention extends to pharmaceutical compositions incorporating one or more of the components described herein suitably formulated to treat any of the conditions described via any of the routes of administration indicated.

The combined preparation of the invention may be provided as a kit including each component of the combined preparation together with any additional components required. Alternatively, the combined preparation may be provided as an individual dose formulated in accordance with the route of administration required. Pharmaceutically-acceptable excipients, diluents or carriers may be used in combination with the components of the combined preparation.

Excipients which can be used as a vehicle for the delivery of the phage will be apparent to those skilled in the art. For example, the free phage could be in lyophilized form and be dissolved just prior to administration, for example by IV injection. The dosage of administration is contemplated to be in the range of about 10⁶ to about 10¹³ pfu/per kg/per day, and preferably about 10¹² pfu/per kg/per day. The phage are administered until successful elimination of the pathogenic bacteria is achieved.

With respect to the aerosol administration to the lungs, the SASP-phage is incorporated into an aerosol formulation specifically designed for administration to the lungs by inhalation. Many such aerosols are known in the art, and the present invention is not limited to any particular formulation. An example of such an aerosol is the TOBI inhaler produced by Chiron, which comprises tobramycin. The concentrations of the propellant ingredients and emulsifiers are adjusted if necessary based on the phage being used in the treatment. The number of phage to be administered per aerosol treatment will be in the range of 10⁶ to 10¹³ pfu, and preferably 10¹² pfu.

With respect to topical administration, the present invention may be mixed with a topical antibiotic such as mupirocin, possibly in the formulation which comprises Bactroban. Alternatively the present invention may be mixed with mupirocin or any other topical antibiotic in any cream or ointment or water or other base, adjusted if necessary based on the phage and antibiotic being used in the treatment. REFERENCES

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APPENDIX 1

A list of all the α/β type SASP which have been sequenced to date together with their related protein sequences

Bacillus subtilis

SASP A mannnsgnsn nllvpgaaqa idqmkleias efgvnlgadt tsrangsvgg eitkrlvsfa qqn gggqf

SASP B manqnssndl lvpgaaqaid qmkleiasef gvnlgadtts rangsvggei tkrlvsfaqq qmggrvq

SASP C aqqsrsrsn nnndllipqa asaieqmkle iasefgvqlg aettsrangs vggeitkrlvr laqqnmggq fh

SASP D masrnklvvp gveqaldqfk levaqefgvn lgsdtvaran gsvggemtkr lvqqaqsqln gttk

Bacillus megaterium.

SASP A mantnklvap gsaaaidqmk yeiasefgvn lgpeataran gsvggeitkr lvqmaeqqlg gk

SASP C manyqnasnr nssnklvapg aqaaidqmkf eiasefgvnl gpdatarang svggeitkrl vqlaeqnlgg ky

SASP Cl annnssnnn ellvygaeqa idqmkyeias efgvnlgadt tarangsvgg eitkrlvqla eqqlgggrf

SASP C2 mannkssnnn ellvygaeqa idqmkyeias efgvnlgadt tarangsvgg eitkrlvqla eqqlgggrsk ttl

SASP C3 martnklltp gveqfldqyk yeiaqefgvt Igsdtaarsn gsvggeitkr lvqqaqahls gstqk

SASP C4 mannkssnnn ellvygaeqa idqmkyeias efgvnlgadt tarangsvgg eitkrlvqla eqqlgggrf

SASP C5 mansrnkssn elavhgaqqa idqmkyeias efgvtlgpdt tarangsvgg eitkrlvqma eqqlgggrsk sis

SASP C-l mannnssnnn ellvygaeqa idqmkyeias efgvnlgadt tarangsvgg eitkrlvqla eqlgggrf

SASP C-2 mannkssnnn ellvygaeqa idqmkyeias efgvnlgadt tarangsvgg eitkrlvqla eqlgggrskt tl

Bacillus cereus

SASP 1 mgknnsgsrn evlvrgaeqa Idqmkyeiaq efgvqlgadt tarsngsvgg eitkrlvama eqqlggranr

SASP 2 srstnklav pgaesaldqm kyeiaqefgv qlgadatara ngsvggeitk rlvslaeqql ggyqk

SASP C5 lfiniqrye sdtneilisa ttstieqmky eiafelgvtl gpdtshhlqm vriggeitkr lvrmaekqlt gqyrlh

Bacillus stearothermophilus

SASP 1 mpnqsgsnss nqllvpgaaq vidqmkfeia sefgvnlgae ttsrangsvg geitkrlvsf aqqqmgggvq

Bacillus firmus

SASP A mannnssnql vvpgvqqald qmkyeiasef gvqlgpdata rangsvggei tkrlvqmaeq qmggyqk

Clostridium bifermentans

SASP α ttnnnntkav peakaalkqm kleianelgi snydtadkgn mtarqngyvg gymtkklvem aeqqmsgqqr

SASP β stkkavpeak aalnqmklei anelglsnye svdkgnltar qngyvggymt kklvemaerq msgk

Clostridium. perfringens

SASP 1 mskslvpeak nglskfknev arelgvpfsd yngdlssrqc gsvggemvkr mveayesqik SASP Cl msqhlvpeak nglskfknev aaemgvpfsd yngdlsskqc gsvggemvkr mveqyekgi SASP C2 msqhlvpeak nglskfknev anemgvpfsd yngdlssrqc gsvggemvkr mvekyeqsmk

Sporosarcina halophila

SASP 1 mannnssnel vvpgvqqald qmkyeiaqef gvqlgadsts rangsvggei tkrlvqmaeq qfggqqygqq qk

Sporocarcina ureae

SASP 1 tnnnnsnsn qllvpgvqqa inqmkeeian efgvnlgpds tsrangsvgg eitkrlvrqa qsqmngytk ω

SASP 2 mpnnnssnql lvpgvqqaln qmkeeiasef gvqlgpdass rangsvggei tkrlvrqaqs qmngytk hermoactinomyces thalpophilus

SASP 1 maqqgrnrss nqllvagaaq aidqmkfeia qefgvtlgad ttsrangsvg geitkrlvsl aqqqlgggts f

APPENDIX 2

An alignment of α/β type SASP protein sequences known to date, also showing alignment of two modified SASP C proteins (SASP-CΔ^11_Dl3κ and SASP-CΔ^11_D13K_C3) .

SASP A (1) mannnsgnsnnllvpgaaqaidqmkleiasefgvnlgadttsrangsvggeitkrlvsfaqqnmgggqf

SASP B (1) manqnssndllvpgaaqaidqmkleiasefgvnlgadttsrangsvggeitkrlvsfaqqqmggrvq

SASP C (1) maqqsrsrsnnnndllipqaasaieqmkleiasefgvqlgaettsrangsvggeitkrlvrlaqqnmggqfh

SASP D (1) masrnklvvpgveqaldqfklevaqefgvnlgsdtvarangsvggemtkrlvqqaqsqlngttk

SASP A (2) mantnklvapgsaaaidqmkyeiasefgvnlgpeatarangsvggeitkrlvqmaeqqlggk

SASP C (2) manyqnasnrnssnklvapgaqaaidqmkfeiasefgvnlgpdatarangsvggeitkrlvqlaeqnlggky

SASP Cl (2) mannnssnnnellvygaeqaidqmkyeiasefgvnlgadttarangsvggeitkrlvqlaeqqlgggrf

SASP C2 (2) mannkssnnnellvygaeqaidqmkyeiasefgvnlgadttarangsvggeitkrlvqlaeqqlgggrskttl

SASP C3 (2) martnklltpgveqfldqykyeiaqefgvtlgsdtaarsngsvggeitkrlvqqaqahlsgstqk

SASP C4 (2) mannkssnnnellvygaeqaidqmkyeiasefgvnlgadttarangsvggeitkrlvqlaeqqlgggrf

SASP C5 (2) mansrnkssnelavhgaqqaidqmkyeiasefgvtlgpdttarangsvggeitkrlvqmaeqqlgggrsksls >

SASP C-l (2) mannnssnnnellvygaeqaidqmkyeiasefgvnlgadttarangsvggeitkrlvqlaeqlgggrf

SASP C-2 (2) mannkssnnnellvygaeqaidqmkyeiasefgvnlgadttarangsvggeitkrlvqlaeqlgggrskttl

SASP 1 (3) mgknnsgsrnevlvrgaeqaldqmkyeiaqefgvqlgadttarsngsvggeitkrlvamaeqqlggranr

SASP 2 (3) msrstnklavpgaesaldqmkyeiaqefgvqlgadatarangsvggeitkrlvslaeqqlggyqk

SASP 1 (4) mpnqsgsnssnqllvpgaaqvidqmkfeiasefgvnlgaettsrangsvggeitkrlvsfaqqqmgggvq

SASP A (5) annnssnqlvvpgvqqaldqmkyeiasefgvqlgpdatarangsvggeitkrlvqmaeqqmggyqk

SASP 1 (6) mannnssnelvvpgvqqaldqmkyeiaqefgvqlgadstsrangsvggeitkrlvqmaeqqfggqqygqqqk

SASP 1 (7) mtnnnnsnsnqllvpgvqqainqmkeeianefgvnlgpdstsrangsvggeitkrlvrqaqsqmngytk

SASP 2 (7) mpnnnssnqllvpgvqqalnqmkeeiasefgvqlgpdassrangsvggeitkrlvrqaqsqmngytk

SASP 1 (8) maqqgrnrssnqllvagaaqaidqmkfeiaqefgvtlgadttsrangsvggeitkrlvslaqqqlgggtsf

SASP CΔ^11_D13K (9) makllipqaasaieqmkleiasefgvqlgaettsrangsvggeitkrlvrlaqqnmggqfh SASP CΔ^11_D13K-^C3 (10) makllipqaasaieqmkleiasefgvqlgaettsrangsvggeitkrlvrlaqqnmggqfhgqq

(2) Bacillus megaterium (6) Sporosarcina halophila

(3) Bacillus cereus (7) Sporocarcina ureae

(4) Bacillus stearothermophilus (8) Thermoactinomyces thalpσphilus

(9 and 10) Modified versions of SASP-C from B . subtilis

Residues conserved in Bacillus and Sporosarcina SASP as well as the Thermoactinomyces SASP.

SASP α (11) ttnnnntkavpeakaalkqmkleianelgisnydtadkgnmtarqngyvggymtkklvemaeqqmsgqqr

SASP β (11) stkkavpeakaalnqmkleianelglsnyesvdkgnltarqngyvggymtkklvemaerqmsgk

SASP 1 (12) mskslvpeaknglskfknevarelgvpfsdyngd—Issrqcgsvggemvkrmveayesqik

SASP Cl (12) msqhlvpeaknglskfknevaaemgvpfsdyngd—lsskqcgsvggemvkrmveqyekgi

SASP C2 (12) msqhlvpeaknglskfknevanemgvpfsdyngd—Issrqcgsvggemvkrmvekyeqsmk

00000 0 * * * * * 0 * *** 0 * *0 0

Key

( 11 ) Clostridium bifermentans

( 12 ) Clostridium perfringens

* Residues conserved in Bacillus, Sporosarcina and Clostridia SASP as well as the Thermoactinomyces SASP. r

0 Residues conserved in Clostridia SASP ^

APPENDIX 3

DNA sequence of sspC Δ^π"D13K gene encoding modified SASP C originating from Bacillus subtilis strain 168 (obtained from Subtilist at the Institut Pasteur) .

atggctaaat tactaattcc tcaagcagct tcagctattg aacaaatgaa 50 acttgaaata gcttctgagt ttggtgttca attaggcgct gagactacat 100 ctcgtgcaaa cggttcagtt ggtggagaaa tcactaaacg tttagttcgc 150 ttagctcaac aaaacatggg cggtcaattt cattaattta tgagggggat 200 aattcccctc tcttttttaa gtcttctcta aatccatac 239

Note:

The sspCA^n'OUK gene extends from 1-186 ( inclusive)

Claims

CLAIMS :

1. A polynucleotide encoding a polypeptide having α/β- type SASP activity and a delivery system therefor capable of targeting a bacterium, and an antibiotic, as a combined preparation for simultaneous, separate or sequential use as a medicament.

2. A polynucleotide and antibiotic according to claim 1, wherein the antibiotic has antimicrobial activity against the target bacterium.

3. A polynucleotide and antibiotic according to claim 1 or claim 2, wherein the polynucleotide encodes a polypeptide which comprises the amino acid sequence: mannnssnsnellvpgaeqaidqmkyeiasefgvnlgadttarangsvggei tkrlvqlaeqqlgggtk (SEQ ID N0:1).

4. A polynucleotide and antibiotic according to claim 1 or claim 2, wherein the polynucleotide encodes a polypeptide which comprises any one of the amino acid sequences shown in appendix 1.

5. A polynucleotide and antibiotic according to any preceding claim, wherein said polynucleotide is RNA.

6. A polynucleotide and antibiotic according to any preceding claim, wherein said polynucleotide is DNA.

7. A polynucleotide and antibiotic according to claim 6, wherein said DNA comprises the sspC gene from B . subtilis .

8. A polynucleotide and antibiotic according to claim 7, wherein said sspC gene is modified to encode the protein SspCΔ^11_D:L3κ.

9. A polynucleotide and antibiotic according to any preceding claim, wherein said antibiotic is a combination of antibiotics .

10. A polynucleotide and antibiotic according to claim 9, wherein each antibiotic of the combination of antibiotics is for simultaneous, separate or sequential use in relation to the other antibiotics and in relation to the polypeptide.

11. A polynucleotide and antibiotic according to any preceding claim, wherein the delivery system comprises a bacteriophage .

12. A polynucleotide and antibiotic according to claim

11, wherein the bacteriophage is a non-lysogenic bacteriophage .

13. A polynucleotide and antibiotic according to claim

12, wherein the non-lysogenic bacteriophage comprises a lysogenic bacteriophage with at least one inactivated lysis gene.

14. A polynucleotide and antibiotic according to claim

13, wherein the at least one inactivated lysis gene is a holin gene.

15. A polynucleotide and antibiotic according to claim 13 or claim 14, wherein the at least one lysis gene is inactivated by insertion of the polynucleotide.

16. A polynucleotide and antibiotic according to any one of claims 11 to 15, wherein the bacteriophage has been modified to increase or alter its host-specificity.

17. A polynucleotide and antibiotic according to any preceding claim, wherein said combined preparation is suitable for topical administration.

18. A polynucleotide and antibiotic according to any one of claims 1 to 16, wherein said combined preparation is suitable for administering via the following routes: oral, aerosol or other devices for delivery to the lungs, nasal spray, intravenous, intramuscular, intraperitoneal, intrathecal, vaginal, rectal, lumbar puncture, and direct application to the brain and/or meninges .

19. Use of a polynucleotide encoding a polypeptide having α/β-type SASP activity and an antibiotic for the production of a combined preparation according to any one of claims 1 to 18 for simultaneous, separate or sequential use in the treatment of infection by a bacterium.

20. Use according to claim 19, which is for human therapy.

21. Use according to claim 19, wherein said combined preparation is suitable for use in domestic pets, livestock and pisciculture.

22. A polypeptide having α/β-type SASP activity and an antibiotic, as a combined preparation for simultaneous, separate or sequential use as a medicament.

23. A polypeptide and antibiotic according to claim 22, wherein said polypeptide comprises the amino acid sequence : mannnssnsnellvpgaeqaidqmkyeiasefgvnlgadttarangsvggei tkrlvqlaeqqlgggtk (SEQ ID NO:l).

24. A polypeptide and antibiotic according to claim 22, wherein said polypeptide comprises any one of the amino acid sequences shown in appendix 1.

25. A polypeptide and antibiotic according to claim 22, wherein said polypeptide comprises the amino acid sequence encoded by the sspC gene from Bacillus subtilis.

26. A polypeptide and antibiotic according to claim 22, wherein said polypeptide comprises the protein SspCΔ^11"

D13K

27. A polypeptide and antibiotic according to any one of claims 22 to 26, wherein said polypeptide contains mutations and/or deletions which do not substantially reduce the α/β-type SASP activity thereof.

28. A polypeptide and antibiotic according to any one of claims 22 to 27, wherein said antibiotic is a combination of antibiotics.

29. A polypeptide and antibiotic according to claim 28, wherein each antibiotic of the combination of antibiotics is for simultaneous, separate or sequential use in relation to the other antibiotics and in relation to the polypeptide.

30. A polypeptide and antibiotic according to any one of claims 22 to 29, wherein said combined preparation is suitable for topical administration.

31. A polypeptide and antibiotic according to any one of claims 22 to 29, wherein said combined preparation is suitable for administering via the following routes: oral, aerosol or other devices for delivery to the lungs, nasal spray, intravenous, intramuscular, intraperitoneal, intrathecal, vaginal, rectal, lumbar puncture, and via direct application to the brain and/or meninges .

32. Use of a polypeptide having α/β-type SASP activity and an antibiotic for the production of the combined preparation according to any one of claims 22 to 31 for simultaneous, separate or sequential use in the treatment of bacterial infection.

33. Use according to claim 32, which is for human therapy.

34. Use according to claim 32, wherein said combined preparation is suitable for use in domestic pets, livestock and pisciculture.