WO2011065854A1

WO2011065854A1 - Enterococcal phage peptides and methods of use thereof

Info

Publication number: WO2011065854A1
Application number: PCT/PT2010/000050
Authority: WO
Inventors: Miguel Ângelo DA COSTA GARCIA; Madalena Maria Vilela Pimentel; Carlos Jorge SOUSA DE SÃO JOSÉ
Original assignee: Technophage, Investigação E Desenvolvimento Em Biotecnologia, Sa; Bluepharma - Industria Farmacêutica, S.A.
Priority date: 2009-11-24
Filing date: 2010-11-24
Publication date: 2011-06-03
Also published as: PT104837A

Abstract

The present invention is directed to isolated and chimeric polypeptides of Enterococcal bacteriophage origin having antibiotic activity and use thereof in the treatment and control of bacterial infections. In some specific aspects, the present invention is directed to the use of a novel antibacterial derived from bacteriophage 168 and chimeric constructs thereof, and their use for the treatment and control of infections caused by gram-positive bacteria, including Enterococcus faecalis.

Description

ENTEROCOCCAL PHAGE PEPTIDES AND METHODS OF USE THEREOF

REFERENCE TO RELATED APPLICATION

This application claims the benefit of Portuguese Provisional Application No. 104 837, Titled "ESTUDO DA ACTIVIDADE DE LISINAS CODIFICADAS POR BACTERIOFAGOS QUE INFECTAM ENTEROCOCCUS SP'\ filed November 24, 2009, which is incorporated herein by reference in its entirety.

1. FIELD OF THE INVENTION

[0001] The present invention is directed to isolated and chimeric polypeptides of

Enterococcal bacteriophage origin having antibiotic activity and use thereof in the treatment and control of bacterial infections. In some specific aspects, the present invention is directed to the use of a novel antibacterial peptide derived from bacteriophage 168 and chimeric constructs thereof, including chimeric constructs with an antibacterial peptides derived from bacteriophage 170, and their use for the treatment and control of infections caused by gram-positive bacteria, including Enterococcus faecalis.

2. BACKGROUND OF THE INVENTION

|0002] Bacteriophage (phage) are viruses that specifically infect and lyse bacteria. Phage therapy, a method of using whole phage viruses for the treatment of bacterial infectious diseases, was introduce by Felix d'Herelle, who discovered phage around 1920. In the beginning of the 20^th century, there were various studies of the application of phage for therapy in humans as well as in animals. In 1940 Eli Lilly Company produced 7 phage products for human use, including phage preparations for treating different sicknesses caused by Staphylococcus sp., E. coli, and other pathogenic bacteria. These preparations were utilized to treat infections that cause abscesses, purulent wounds, vaginitis, acute chronic upper-respiratory tract infections, and mastoid infections.

[0003] However, with the arrival of antibiotics in the 1940's, the development of phage based therapeutics declined in the Western world. One of the most important factors that contributed to the decline of interest in phage therapy in the Western world was the problem of credibility. The reduction in the number of appropriately conducted studies and the lack of well- established protocols and standardizations interfered with the rigorous documentation of the value of phage therapy. Many problems related to the production of phage samples/specimens also complicated the initial study/research related to phage therapy. Diverse stabilizers and preservatives were used in attempts to increase the viability of the phage therapeutics. However, without a good understanding of the biological nature of phage and their stability in response to various physical and chemical agents, many of the ingredients added to prolong the viability of the phage preparations resulted in a negative effect on the viability of the phage, and in some cases proved to be toxic to humans. Another problem related to phage production was the purity grade of the commercial preparations of these viruses. The phage therapy preparations, including those originating from well-established companies in the United States and other countries, consisted of raw lysates of the host bacteria treated with the phage of interest. Thus, the preparations had bacterial components, including endotoxins, that could have adverse effects in patients treated with these preparations, particularly those receiving intravenous administration. However, the use of bacteriophage for therapeutic ends continued jointly with, or in place of antibiotics, in Eastern Europe and in the former Soviet Union where access to antibiotics was limited.

|0004j With the rise of antibiotic resistant strains of bacteria, interest in phage based therapeutics has gained broader interest. Even though novel classes of antibiotics may be developed, the prospect that bacteria eventually will develop resistance to the new drugs has intensified the search for non-chemotherapeutic means for controlling and treating bacterial infections. There are three general strategies for using phage-based therapies in a clinical environment: 1 ) the use of active, virulent phage; 2) the use of endolysins or purified lysins isolated from bacteriophage; and 3) the use of a structural protein of the identified phage as a metabolic inhibitor of key enzymes for the synthesis of bacterial peptidoglycan.

[0005] Among the most promising of the strategies currently in development are phage lysins. Preparations of purified endolysins can be used as therapeutic agents, per se, or combined with classic antibiotics. The addition of exogenous lysins to susceptible gram-positive bacteria can cause complete lysis in the absence of bacteriophage (Loeffler et al.. 2001 , Science 294:2170- 2172; Shuch et al., 2002, Nature 418:884-889). Microscopic images of bacteria treated with a lysin indicate that these en2ymes exercise their lethal effect by digesting peptidoglycan, leading to the formation of holes in the cell wall. Compared with the external environment, the inside of a bacterium is hypertonic, and when the bacterial wall loses its structural integrity the result is the extrusion of the cytoplasmic membrane and hypertonic lysis.

[0006| While penicillin and antibiotics of the Cephalosporin class inhibit the synthesis of peptidoglycan causing lysis of the bacterial cell wall during cell division, the phage lysins destroy the peptidoglycan directly, exercising their lytic effect seconds after being administered. The lysins can also destroy the cell wall of bacteria that are not growing and are insensitive to many antibiotics. When simultaneously administered, two lysins, or two lysin catalytic domains, that have differing targets may attack the peptidoglycan in multiple regions, presenting a synergistic effect. Increased antibiotic resistance has turned attention to phage lysins as antibacterial agents, as well as to the development of chimeric constructs of such lysins.

[0007] Chimeric lysins have been constructed by re-combining catalytic domains of different lysins, in attempts for example to produce lysins with different bacterial and catalytic specificities (Fischetti VA, 2010, '''Bacteriophage endolysins: A novel anti-infective to control Gram-positive pathogens '" Intl J. Med. Microbiol. 300(6): 357-62). Prior work with such constructs generally has focused on lysins directed against Pneamococcus and Staphylococcus spp. For example, catalytic domains of lytic enzymes for S pneumoniae phage were swapped to create a lysin having the same binding domain for pneumococci, but able to cleave a different peptidoglycan bond (Garcia et al., 1990, "Modular organization of the lytic enzymes of

Streptococcus pneumoniae and its bacteriophages," Gene 86, 81 -88; Weiss et al., 1999, "Group A Streptococcus carriage among close contacts of patients with invasive infections," Am. J. Epidemiol. 149, 863-868). Similar studies later were carried out for Staphylococcus phage lysins (Monoharadas et al., 2009, "Antimicrobial activity of a chmeric enzybiotic towards

Staphylococcus aureus i. Biotechnol. 139, 1 18- 123.). Nonetheless, the use of phage lysins has only much more recently been applied to treating Enterococcus spp., with no chimeric

Enterococcal phage lysins having been described by other than the instant inventors. (See, e.g., WO 2010/090542 and WO 2010/041970, each hereby incorporated by reference in its entirety). Moreover, most lysins investigated to date are specific to species (or subspecies) of bacteria from which they are derived. For example, it has been shown that lysins isolated from streptococcal phage only kill certain streptococci and that lysins produced by pneumococcal phage only kill pneumococci (Fishcetti, 2005, Trends in Microbio 13:491-496).

[0008| Enterococcus ranks 4^lh in N. America and 5^th in Europe as the pathogen causing blood infections, and the incidence of antibiotic-resistant enterococci has progressively been increasing in the United States and Europe over the past decade (Deshpande, LM, et al. 2007, "Antimicrobial resistance and molecular epidemiology of vancomycin-resistant enterococci from North America and Europe: a report from SENTRY antimicrobial surveillance program," Diagn. Microbiol. 58: 163-1 70). Indeed, increased use of vancomycin for treating methicillin-resistant Staphylococcus spp. has been a leading cause for the increase in vancomycin-resistant enterococci (VRE) (Chavers, LS, et al., 2003. "Vancomycin-resistant enterococci: 15 years and counting," J. Hosp. Infect. 53 : 1 9-171 ).

[0009] Enterococci can cause a variety of infections, including endocarditis, bacteremia, meningitis, and surgical would infections, and also are capable of colonizing environmental surfaces, including medical equipment, for prolonged periods (Arias, CA, et al., 2008,

"Emergence and management of drug-resistant enterooccal infections," Expert Rev. Anti Infect. Ther. 6(5): 637-655). Common species of Enterococcus include E. faecalis and E. faecium, both gram-positive bacteria that colonize the mouth, vagina, and lower intestines. While they normally cause no adverse effects in humans, high-level antibiotic resistance can lead to these organisms becoming a significant source of nosocomial infections, particularly in

immunocompromised patients (Murray, BE, 1990, "The life and times of the enterococcus," Clin. Micorbiol. Rev. 3:46-65). Nonetheless, even five years ago, there were no therapeutic evaluations available regarding any E. faecalis bacteriophages (Uchiyama J., et al., 2008, "In silico and in vivo evaluation of bacteriophage OEF24C, a candidate for treatment of

Enterococcus faecalis infections," Applied and Environmental Microbiology. 74(13): 4149- 4163); and the first lysin having antibacterial action against Enterococcus was discovered only six years ago (Yoong, et al. "Identification of a Broadly Active Phage Lytic Enzyme with Lethal Activity against Antibiotic-Resistance Enterococcus faecalis and Enterococcus faecium " 2004, J. of Bacterid. 186(14): 4808-4812).

[0010J Accordingly, there is a clear need for further investigation of Enterococcal phage lysins as potential therapeutic and prophylactic agents of use, in vivo, to manage and treat pathogenic bacteria, including pathogenic enterococci. Further, there is an increasing need to discover novel lysin en2ymes that may be used to treat the increasing number of Enterococci bacterial species that have developed antibiotic resistance, as well as a need to develop lysin constructs that permit species and/or strain cross-reactivity. In particular, the isolation and/or development of novel lysins with lytic killing or antibacterial activity towards Enterococci and other bacteria, beyond the specific species from which the lysins are derived, would be especially valuable.

3. SUMMARY OF THE INVENTION

[00111 The present invention is directed to isolated and chimeric polypeptides of

Enterococcal bacteriophage origin having antibiotic activity and use thereof in the treatment, prevention, and control of bacterial infections, particularly Enterococcal infections. In one aspect, the present invention is directed to chimeric polypeptides comprising the catalytic domains of two or more Enterococcal bacteriophage endolysins, where the chimeric

polypeptides show increased lytic performance towards Enterococcus bacteria compared to the native bacterophage endolysins. Increased lytic performance includes an increased spectrum of activity against Enterococcus bacteria species and/or strains; as well as an increased ability to kill and/or inhibit the growth and reproduction of Enterococcus bacteria. The catalytic domains of the chimeric polypeptides may be from the same or different bacteriophages, which may have the same or different bacterial hosts, e.g., bacterial hosts of the same or different species, or of the same or different bacterial strain. In some embodiments, the native endolysins are from bacteriophages that natively infect E. faecalis, in particular strains E. faecalis 1518/05 and E. faecalis 926/095.

[0012J In some embodiments, the chimeric polypeptides of the invention include a

CHAP domain of an endolysin from bacteriophage F 168/08, in particular a CHAP domain from Lysl 68 (SEQ ID NO:7). In some embodiments, the CHAP domain is combined with an amidase domain of an endolysin from bacteriophage F 170/08, in particular an amidase-2 domain from Lysl 70 (SEQ ID NO:5).

{0013J In some specific embodiments, the CHAP domain comprises or consists of the amino acid sequence SEQ ID NO:7, or a fragment thereof, having antimicrobial or antibiotic activity against Enterococcus sp., particularly, E. faecalis and/or E. faecium. In other embodiments, a peptide is used that corresponds to the CHAP domain and comprises a fragment, variant or derivative of SEQ ID NO:7, wherein the fragment, variant or derivative has antibiotic activity or antimicrobial activity {e.g., lytic killing activity) against a Gram-positive bacteria, e.g., Enterococcus sp.. particularly, E. faecalis and/or E. faecium. In a specific example in accordance with this embodiment, the invention provides for peptides having an amino acid sequence with at least 60%, 65%, 70%, 75%, 85%, 95%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to an amino acid sequence of the same length (i.e., consisting of the same number of residues) and having amino acid sequence SEQ ID NO:7, or a fragment thereof. (0014J In some specific embodiments, the amidase domain comprises or consists of the amino acid sequence SEQ ID NO:5, or a fragment thereof, having antimicrobial or antibiotic activity against E. faecalis. In other embodiments, a peptide is used that corresponds to the amidase domain and comprises a fragment, variant or derivative of SEQ ID NO:5, wherein the fragment, variant or derivative has antibiotic activity or antimicrobial activity (e.g., lytic killing activity) against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E. faecalis and/or E. faecium. In a specific example in accordance with this embodiment, the invention provides for peptides having an amino acid sequence with at least 60%, 65%, 70%, 75%, 85%», 95%, 90%, 95%. 96%, 97%, 98%, 99%, or greater sequence identity to an amino acid sequence of the same length (i.e., consisting of the same number of residues) and having amino acid sequence SEQ ID NO:5, or a fragment thereof.

(0015] In some more specific embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence SEQ ID NO: 9, or a fragment thereof, having antimicrobial or antibiotic activity against Enterococcus sp., particularly, E. faecalis and/or E. faecium. In other embodiments, the chimeric polypeptide comprises a fragment, variant or derivative of SEQ ID NO:9, wherein the fragment, variant or derivative has antibiotic activity or antimicrobial activity (e.g., lytic killing activity) against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E. faecalis and/or E. faecium. In a specific example in accordance with this embodiment, the invention provides for chimeric polypeptides having an amino acid sequence with at least 60%, 65%, 70%, 75%, 85%, 95%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to an amino acid sequence of the same length (i.e. , consisting of the same number of residues) and having amino acid sequence SEQ ID NO:9, or a fragment thereof.

[0016J In another aspect, the present invention is directed to peptides obtained from bacteriophage F 168/08, which peptides exhibit antibiotic activity against a Gram-positive bacterium, e.g., Enterococcus sp.. particularly, E. faecalis and/or E. faecium, as well as to chimeric constructs thereof, including chimeric constructs with antibacterial peptides obtained from bacteriophage F 170/08. In some specific embodiments, the peptide of the invention comprises or consists of the amino acid sequence SEQ ID NO:7. In other embodiments, the peptide of the invention comprises a fragment, variant, or derivative of SEQ ID NO: 7, wherein said fragment, variant or derivative has antibiotic (e.g., lytic killing activity) activity against a Gram-positive bacteria, e.g., Enterococcus sp.. particularly, E. faecalis and/or E. faecium. In a specific example in accordance with this embodiment, the invention provides for peptides having an amino acid sequence with at least 60%. 65%, 70%, 75%, 85%, 95%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to a second amino acid sequence of the same length (i.e., consisting of the same number of residues), wherein the second amino acid sequence is SEQ ID NO: 7, or a fragment thereof.

[0017] The invention also encompasses polynucleotides that encode the polypeptides of the invention. The invention also relates to a vector comprising said nucleic acid. In one specific embodiment, said vector is an expression vector. The invention further provides host cells containing a vector comprising polynucleotides encoding the polypeptides of the invention.

[0018] In a specific embodiment, the invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding a peptide of phage 168, or active fragment thereof, which polypeptide or fragment exhibits antibiotic activity (e.g., lytic killing activity) against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E. faecalis and/or E. faecium. In a specific example in accordance with this embodiment, the invention provides for a nucleic acid comprising or consisting of the nucleic acid sequence SEQ ID NO:8, or a fragment thereof. The invention also relates to a vector comprising said nucleic acid. In one specific embodiment, said vector is an expression vector. The invention further provides host cells containing a vector comprising polynucleotides encoding the polypeptides of the invention.

[0019] In another specific embodiment, the invention provides a chimeric nucleic acid comprising a nucleic acid sequence encoding a peptide of phage 168 corresponding to a catalytic domain, or active fragment thereof, combined with a catalytic domain of a heterologous lysin protein. In a specific example in accordance with this embodiment, the invention provides for a nucleic acid comprising or consisting of the nucleic acid sequence SEQ ID NO: 10, or a fragment thereof. The invention also relates to a vector comprising said chimeric nucleic acid. In one specific embodiment, said vector is an expression vector. The invention further provides host cells containing a vector comprising polynucleotides encoding the chimeric polypeptides of the invention.

[0020] The invention encompasses methods for the evaluation of antibiotic activity of isolated peptides and chimeric polypeptides (e.g., killing based on the antimicrobial activity and/or lytic activity of the peptides and polypeptides of the invention). Antibiotic activity may be assessed by any method known in the art and/or described herein. In certain embodiments, antibiotic activity is assessed by culturing Gram-positive bacteria according to standard techniques (e.g., in liquid culture or on agar plates), contacting the culture with peptides and/or polypeptides of the invention and monitoring cell growth after said contacting. For example, in a liquid culture, the bacteria, e.g., E. faecalis. may be grown to an optical density ('OD") representative of a mid-point in exponential growth of the culture; portions of the culture exposed to one or more concentrations of one or more peptides and/or polypeptides of the invention and the OD monitored relative to a control culture. Decreased OD relative to a control culture is representative of a peptide and/or polypeptide exhibiting antibiotic activity (e.g., exhibits antimicrobial and/or lytic killing activity). Similarly, bacterial colonies can be allowed to form on an agar plate, the plate exposed to a peptide and/or polypeptide of the invention, and the subsequent growth of the colonies evaluated compared to control plates. Decreased size of colonies, or decreased total numbers of colonies indicate a peptide and/or polypeptide with antibiotic activity.

[0021] The present invention encompasses methods for the production of peptides and polypeptides of the invention or active fragments thereof, particularly for use in pharmaceutical compositions, e.g., antibiotic or antimicrobial compositions. In certain embodiments, the peptides and polypeptides of the invention are isolated directly from cell cultures (e.g. bacterial cell cultures) infected with bacteriophage 168 or bacteriophage 170, using standard techniques known in the art and/or described herein. In other embodiments, the peptides and polypeptides of the present invention are produced by recombinant means using an expression vector comprising a nucleic acid sequence encoding a peptide or polypeptide of the invention, e.g., SEQ ID NO: 6, 8 or 10, or an active fragment, derivative, or variant thereof (i.e., which active fragment has antibiotic activity). (0022] The peptides and polypeptides of the invention or fragments thereof can be produced by any method known in the art for the production of a polypeptide, in particular, by chemical synthesis or by recombinant expression techniques. In a specific embodiment, the invention relates to a method for recombinants producing a lysin peptide or chimeric polypeptide of the invention, or active fragment thereof, said method comprising: (i) constructing a nucleic acid encoding said peptide or polypeptide; (ii) culturing in a medium a host cell comprising said nucleic acid, under conditions suitable for the expression of said peptide or polypeptide; and (iii) recovering said peptide or polypeptide from said medium. In certain embodiments, the nucleic acid sequence encoding the lysin peptide or chimeric polypeptide of the invention is operably linked to a heterologous promoter, meaning combination with a promoter not naturally found with the sequence.

[0023J The present invention encompasses pharmaceutical compositions comprising isolated peptides and/or chimeric polypeptides derived from bacteriophage 168, in particular isolated peptides or chimeric polypeptides having antimicrobial and/or antibiotic activity. The pharmaceutical compositions of the invention may additionally comprise a pharmaceutically acceptable carrier, excipient, or stabilizer. In specific embodiments, the pharmaceutical compositions comprise a polypeptide having the amino acid sequence of SEQ ID NO: 7. In another embodiment, the pharmaceutical compositions comprise a polypeptide that is a variant, derivative or fragment of SEQ ID NO: 7, wherein the variant, derivative or fragment retains antimicrobial activity against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E. faecalis and/or E. faecium. In other specific embodiments, the pharmaceutical compositions comprise a chimeric polypeptide having the amino acid sequence of SEQ ID NO:9. In another embodiment, the pharmaceutical compositions comprise a chimeric polypeptide that is a variant, derivative or fragment of SEQ ID NO:9, wherein the variant, derivative or fragment retains antimicrobial and/or antibiotic activity against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E. faecalis and/or E. faecium.

[0024] In specific embodiments, the pharmaceutical compositions of the invention are antibiotic compositions for the treatment, prevention, and/or amelioration of symptoms of a disease or disorder associated with infection by a Gram-positive bacteria in a subject in need thereof. Accordingly, another aspect of the invention relates to a method of treating a bacterial infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition. The subject receiving a pharmaceutical composition of the invention may be a mammal (e.g., bovine, ovine, caprine, equid, primate (e.g., human), rodent, lagomorph) or avain (e.g., chicken, duck, goose). In the context of the present invention, "treatment" refers to both therapeutic treatment and

prophylactic or preventative measures, wherein the object is to eliminate, lessen, decrease the severity of, slow the progression of, or prevent the symptoms or underlying cause (e.g., bacterial infection) associated with the pathological condition or disorder. The pharmaceutical compositions of the present invention may be used in the treatment or management of infections associated with, but not limited to. Gram-positive bacteria, such as. Enterococcus, in particular, Enterococcus faecalis and/or Enterococcus faecium, as well as Staphylococcus aureus (including MRSA), Staphylococcus haemolyticus. Staphylococcus epidermidis. Bacilus subtilis, Bacilus licheniformis. Streptococcus grupo, Micrococcus luteus, Escherichia coli, and combinations thereof. In certain embodiments, the pharmaceutical compositions of the invention are of use in the treatment of conditions associated with infection by vancomycin-resistant strains of

Enterococcus (VRE). The pharmaceutical compositions may also be used to treat conditions or disorders associated with bacterial infections including, but not limited to, endocarditis, bacteremia, diverticulitis, meningitis, urinary tract infection, and surgical wound infections.

[0025] In certain embodiments, the invention provides for the use of lysin peptides or chimeric polypeptides as a single agent therapy. In other embodiments, the lysin peptides and chimeric polypeptides of the present invention may be combined with one or more lysins from a bacteriophage other than bacteriophage 168, and/or other than with lysins from bacteriophage 170. In yet other embodiments, the invention provides for the use of a lysin peptide or chimeric polypeptide, or active fragment, variant, derivative thereof, in combination with a standard or experimental treatment for Gram-positive bacterial infection. In still other embodiments, the invention provides for the use of a lysin peptide, chimeric polypeptide, or active fragment of either, that has been chemically conjugated to still another therapeutic molecule (e.g., peptide or non-peptide cytotoxin). Such combination therapy may enhance the efficacy of the standard or experimental treatment. Examples of therapeutic agents that are particularly useful in

combination with a peptide or polypeptide of the invention are anti-inflammatory agents, standard chemotherapeutic antibiotic agents (e.g., penicillin, synthetic penicillins, bacitracin, methicillin, cephalosporin, polymyxin, cefaclor, cefadroxil, cefamandole nafate, cefazolin, cefixime, cefmetazole, cefonioid, cefoperazone, ceforanide, cefotanme, cefotaxime, cefotetan, cefoxitin, cefpodoxime, proxetil, ceftazidime, ceftizoxime, ceftriaxone, cefriaxone moxalactam, cefuroxime, cephalexin, cephalosporin C, cephalosporin C sodium salt, cephalothin, cephalothin sodium salt, cephapirin, cephradine, cefuroximeaxetil, dihydratecephalothin, moxalactam, loracarbef mafate and chelating agents). The combination therapies encompassed by the invention may be formulated into a single pharmaceutical composition or may be administered in separate compositions as part of an overall treatment regimen.

[0026] The pharmaceutical compositions of the invention may be administered by any method known in the art suitable for administration of an antibiotic compound, e.g., via oral or parenteral (e.g., inhalation, intramuscular, intravenous, or epidermal) delivery.

[0027] The pharmaceutical compositions of the present invention may also be used for traditionally non-therapeutic uses such as antibacterial agents in cosmetics, or in sprays or solutions for use on solid surfaces to prevent the colonization of Gram-positive bacteria (e.g., as a disinfectant or anti-infectant).

[0028] The present invention is also directed to methods for screening peptides for antibiotic activity. In some embodiments the method comprises screening contiguous amino acid sequences of at least 6, 10, 15, 20 or 25 residues in length from SEQ ID NO:5, 7 or 9 for antimicrobial activity, e.g., antimicrobial activity against Enterococcus sp., particularly, E.

faecalis and/or E. faecium, said antibiotic and/or targeting activity measured by the peptide's or polypeptide's ability to inhibit bacterial growth in agar or liquid culture.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 shows the lytic activity of lysins Lysl68, Lysl 70, and Lysl 70-168, tested for four different amounts, in 98 clinic strains of Enterococcus.

[0030] FIG. 2 shows Lysl 68 and Lysl 70 activity as the percent reduction in turbidity for each of three E. faecalis strains, E. faecalis 926/05, E. faecalis 1518/05, and E. faecalis 1915/05, after addition of 5μg/mL of each lysin.

[0031] FIG. 3 shows a cell viability assay for each of three E. faecalis strains, E. faecalis

926/05, E. faecalis 1518/05, and E. faecalis 1915/05, measured as CFU/mL at the initial (T₀) and end (T₉₀) of the turbidity reduction assay. (0032] FIG. 4 shows a therapeutic evaluation for Lysl 68 and Lysl 70 in the hearts of 3 female Wistar rats, where buffer was used as negative control and the treatment was carried out after 24 hours of heart infection.

[0033] FIG. 5 shows a therapeutic evaluation of Lys 168 and Lysl 70 in the blood of 3 female Wistar rats, where buffer was used as negative control and the treatment was carried out after 24 hours of heart infection.

[0034] FIG. 6 shows a therapeutic evaluation of Lys 168 and Lys 170 in the hears of 1 male and 3 female Wistar rats, where the male heart and buffer were used as negative controls and the treatment was carried out after 19-24 hours of heart infection.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1 DEFINITIONS

[0035] As used herein, the terms "polypeptide", "peptide," and "protein" are used interchangeably to refer to an amino acid sequence of any length. In general, however, "peptide" refers to shorter sequences, e.g., a fragment of a full-length polypeptide or protein, including a functional fragment corresponding to an enzymatic domain that retains its functionality separate from the rest of the polypeptide or protein from which it is derived. "Protein" generally refers to an amino acid sequence expressed and found naturally by an organism in nature; while

"polypeptide" generally refers to a recombinant and/or chimeric product, but also can include the general meanings of "protein" and "peptide."

[0036] As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 1 75 amino acid residues, or at least contiguous 200 amino acid residues of the amino acid sequence of a second polypeptide. In a specific embodiment, the fragment is a functional fragment in that it retains at least one function of the second polypeptide (e.g., antimicrobial or antibiotic activity; or targeting activity).

|0037] As used herein, the term "isolated" in the context of a peptide, polypeptide, or fusion protein refers to a peptide, polypeptide, or fusion protein that is substantially free of cellular material or contaminating proteins from the cell or tissue source from which it is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of a peptide, polypeptide, or fusion protein in which the peptide, polypeptide, or fusion protein is separated from cellular components of the cells from which it is isolated or recombmantly produced. Thus, a peptide, polypeptide, or fusion protein that is substantially free of cellular material includes preparations of a peptide, polypeptide, or fusion protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as "contaminating protein"). When the peptide, polypeptide, or fusion protein is recombinanfly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the peptide, polypeptide, or fusion protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i. e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the peptide, polypeptide, or fusion protein. Accordingly such preparations of a peptide, polypeptide, or fusion protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the peptide, polypeptide, or fusion protein of interest.

[0038] As used herein, the term "isolated" in the context of nucleic acid molecules refers to a first nucleic acid molecule which is separated from other nucleic acid molecules which are present in the natural source of the first nucleic acid molecule. Moreover, an "isolated"" nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized and may be free of other cDNA or other genomic DNA molecules, e.g., where it has been isolated from other clones in a nucleic acid library.

10039| The term "purified", in the context of a lysin or chimeric lysin in accordance with the instant invention, means that the lysin or chimeric lysin construct has been measurably increased in concentration by any purification process, including but not limited to, column chromatography, HPLC, precipitation, electrophoresis, etc., thereby partially, substantially, or completely removing impurities such as precursors or other chemicals involved in preparing the lysin or chimeric lysin. One of ordinary skill in the art will appreciate the amount of purification necessary for a given use. For example, isolated protein meant for use in therapeutic

compositions intended for administration to humans ordinarily must be of high purity in accordance with regulatory standards (e.g., of higher purity than isolated proteins for laboratory use).

[0040) As used herein, the term "derivative" in the context of polypeptides refers to a polypeptide that comprises an amino acid sequence which has been altered by the introduction of amino acid residue substitutions, deletions, or additions. The term "derivative" as used herein also refers to a polypeptide that has been modified, i.e., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, polypeptides may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation,

derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative polypeptide may be produced by chemical

modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative polypeptide may contain one or more non-classical amino acids. A polypeptide derivative may possess a similar or identical function as the polypeptide from which it was derived, or it may possess an improved function. The term "derived" as used in reference to a polypeptide "derived" from an organism may also refer to isolation of a polypeptide directly from said organism (e.g. bacterial cells or phage).

[0041] As used herein, the term "chimeric" refers to a construct derived from two or more heterologous sources. A chimeric gene or chimeric nucleic acid, for example, can comprise sequences derived from a first nucleic acid combined with sequences derived from a second nucleic acid, where the first and second nucleic acids are native to different types of bacteriophage or naturally occur in different polypeptides of a single type of bacteriophage. The sequences from each nucleic acid typically correspond to coding sequences for a functional domain of the respective encoded polypeptides, e.g., a catalytic domain of a lysin. The heterologous nucleic acid sequences may be combined in frame, e.g., by recombinant means, so as to encode a fusion protein or chimeric polypeptide, which can be expressed thereform under appropriate conditions. A chimeric polypeptide can be engineered to include the full sequence of two or more native proteins, or only a portion of either. Chimeric polypeptides generally are created to impart functionality from each of the original proteins to the resulting chimeric polypeptides. The dual (or higher order) functionality of fusion proteins is made possible by the fact that protein functional domains are generally modular, such that a linear portion of a polypeptide constituting a given domain, such as catalytic domain, may be removed from the rest of the protein without destroying its enzymatic capability. A chimeric nucleic acid or chimeric polypeptide comprising sequences derived from two or more different lysin genes or

polypeptides can be referred to as a "chimeric lysin" or "chimeric lysin construct".

[0042] As used herein, the term "endolysin" is used interchangeably with the term

"lysin". Endolysins are double-stranded DNA bacteriophage-encoded proteins, produced towards the end of a lytic life cycle of the bacteriophage, and designed to attack the

peptidoglycan cell wall of the host bacterium, in order to allow release of the progeny phage particles. Endolysins are also capable of degrading peptidoglycan when applied exogenously to a cell wall, e.g., as isolated and/or recombinant polypeptides, usually resulting in rapid lysis of the bacterial cell wall. Gram-positive phage lysins usually have a modular domain structure, with the N-terminal domain containing the catalytic domain and the C-terminal domain containing a binding or targeting domain that binds to a specific substrate of the host bacterium cell wall. Enzymatic activities of the catalytic domains include, e.g., an endo-β-Ν- acetylglucosaminidase or N-acetylmuramidase activity (lysozyme activities), which act on the carbohydrate moiety of a bacterial cell wall; an endopeptidase activity, which acts on the peptide cOrss-bridge; or an N-acetylmuramoyl-L-alanine amidase activity (amidase activity), which attacks the amide bond connecting the glycan strand and peptide moieties.

[0043] As used herein, a "CHAP domain" refers to a conserved amidase domain found in several phage-encoded peptidoglycan hydrolases and stands for "cysteine, histidine-dependent amidohydrolases/peptidases." See, e.g., Rigden D, et. al., Trends Biochem Sci. 2003 May 28(5): 230-4. It is found in a superfamily of amidases, including GSP amidase and peptidoglycan hydrolases. The family includes at least two different types of peptidoglycan cleavage activities: L-muramoyl-L-alanine amidase and D-alanyl-glycyl endopeptidase activity. CHAP domains generally contain conserved cysteine and histidine residues and hydrolyze γ-glutamyl-containing substrates. These cysteine residues are believed to be essential for the activity of several of these amidases, and their thiol groups appear to function as the nucleophiles in the catalytic

mechanisms of all enzymes containing this domain. CHAP domains are often found in association with other domains that cleave peptidoglycan, e.g., acting in a cooperative manner to cleave specialized substrates. See also, Bateman A, et al.. Trends Biochem Sci. 2003 May 28(5): 234-7.

[0044] As used herein, the term "host cell" refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. It can also refer to a cell infected, particularly naturally infected, with a particular bacteriophage. Progeny of a host cell may not be identical to the parent cell transfected with the nucleic acid molecule, or infected with the phage, due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule or phage genetic material into the host cell genome.

[0045] As used herein, the term "in combination" refers to the use of more than one prophylactic and/or therapeutic agent. The use of the term "in combination" does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject with a disease or disorder. A first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent (different from the first prophylactic or therapeutic agent) to a subject with a disease or disorder.

[0046] As used herein, the terms "nucleic acids" and "nucleotide sequences" include

DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA). combinations of DNA and RNA molecules, chimeric DNA and RNA molecules, or hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules. Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases. Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes. The nucleic acids or nucleotide sequences can be single- stranded, double-stranded, may contain both single-stranded and double-stranded portions, and may contain triple-stranded portions, but preferably are double-stranded DNA.

|0047] As used herein, the terms "prophylactic agent" and "prophylactic agents" refer to peptides, polypeptides, and/or proteins of the invention, which can be used in the prevention, delay of the onset of, slowing the progression of, amelioration, or management of one or more symptoms of a disease or disorder, or of the underlying cause of the disease or disorder, associated with infection by a Gram-positive bacteria and, in particular, associated with infection by an Enterococcus.

[0048] As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to peptides, polypeptides, and/or proteins of the invention that can be used in the treatment, management, or amelioration of one or more symptoms of a disease or disorder, or of the underlying cause of the disease or disorder, associated with infection by a Gram-positive bacteria and. in particular, associated with infection by an Enterococcus.

[0049J As used herein, the term "therapeutically effective amount" refers to that amount of a therapeutic agent sufficient to result in amelioration of one or more symptoms of a disease or disorder (e.g., a disease or disorder associated with infection by Gram-positive bacteria and, in particular, associated with infection by an Enterococcus) in a subject; or to result in a reduction in total bacterial burden in said subject, in particular a reduction in total Enterococcal bacterial burden.

[0050| As used herein, the terms "treat", "treatment" and "treating" refer to the amelioration of one or more symptoms associated with an infection by Gram-positive bacteria, in particular, associated with an infection by Encterococcus or to the reduction in total bacterial burden, in particular, a reduction in total Enterococcal bacterial burden, resulting from the administration of one or more peptides, polypeptides, and/or proteins of the invention.

[00511 The term "antibiotic activity" refers to the ability to kill and/or inhibit the growth or reproduction of a microorganism and can be used interchangeably with "antimicrobial activity". In certain embodiments, antibiotic or antimicrobial activity is assessed by culturing Gram-positive bacteria according to standard techniques (e.g., in liquid culture or on agar plates), contacting the culture with peptides, polypeptides, and/or proteins of the invention and monitoring cell growth after said contacting. For example, in a liquid culture, the bacteria, e.g., E. faecalils or E. faecium, may be grown to a optical density ("OD") representative of a midpoint in exponential growth of the culture; the culture exposed to one or more concentrations of one or more polypeptides of the invention; and the OD monitored relative to a control culture. Decreased OD relative to a control culture is representative of a polypeptide exhibiting antibiotic activity (e.g., exhibiting lytic killing activity). Similarly, bacterial colonies can be allowed to form on an agar plate, the plate exposed to a polypeptide of the invention, and subsequent growth of the colonies evaluated related to control plates. Decreased size of colonies, or decreased total numbers of colonies, indicate a polypeptide with antibiotic activity. A fragment, variant, or derivative of a lysin polypeptide having antibiotic or antimicrobial activity refers to the fragment having the catalytic ability to bring about host bacterial cell death and/or to bring about inhibition of growth or reproduction thereof, or to the fragment having such catalytic ability as well as targeting activity towards the host, as defined below.

|0052] The term "targeting activity" refers to the ability of a lysin polypeptide to direct catalytic activity, such as antibiotic or antimicrobial activity, to a given bacterial host cell.

Targeting activity may be associated with a particular region or domain of the polypeptide, such that, e.g., a chimeric construct comprising a targeting domain of a first lysin polypeptide, native to a first host species, can direct the catalytic activity, such as the antibiotic activity, of the chimeric construct to bacterial cells of first host species. As used herein, targeting activity "towards" a particular host cell or bacterial species is used interchangeably with the related expressions targeting activity "to" or "against" the host cell or bacterial species. "Targeting domain" as used herein refers to a functional domain of a lysin polypeptide capable of directing the lysin polypeptide to a host cell, , e.g., E. faecalis, thereby facilitating lytic action upon the host cell. A "targeting domain", for example, can correspond to the cell wall binding domain of a lysin polypeptide.

[0053] Generally, where fragments, variants, or derivatives of a lysin polypeptide isolated from phage 168 are concerned, "antimicrobial activity" or "antibiotic activity" refers to both or either functionality, that is. to the catalytic and/or targeting activities to bring about cell death of gram-positive bacteria, e.g., E. faecalis, the native host for phage 168. 5.2 Lysin Polypeptides

[0054] In one aspect, the invention is directed to polypeptides isolated from a phage that infects gram-positive bacteria. The polypeptides have antimicrobial (e.g., lytic) and/or targeting activity against one or more strains of E. faecalis. In one embodiment, polypeptides are provided that exhibit antimicrobial and/or targeting activity against vancomycin-resistant strains of Enterococcus (VRE). In addition, polypeptides having antimicrobial and/or targeting activity against one or more bacterial pathogens such as Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, S. haemolyticus, S. epidemidis, Bacilus subtilis, Bacilus licheniformis, Streptococcus grupo. Micrococcus luteus. and Escherichia coli are provided herein.

[0055) Preferably, the polypeptide of the invention is isolated from bacteriophage 168, which infects the host E. faecalis. Herein, the term "bacteriophage 168" is used interchangeably with the terms "bacteriophage F 168/08" or "phage 168." In one embodiment, the polypeptide is an isolated lysin peptide from phage 168, the lysin peptide comprising the amino acid sequence of SEQ ID NO:7 or a fragment thereof havng antimicrobial activity against E. aecalis. In another embodiment, the peptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 7, or a fragment thereof, which peptide exhibits antibiotic and/or targeting activity against E. faecalis. Sequence identity with respect to the peptide or polypeptide sequences disclosed herein is defined as the percentage of amino acid residues that are identical in a candidate sequence of the same length {i.e., consists of the same number of residues) as the amino acid sequences of the present invention. The present invention also encompasses variants, derivatives, and/or fragments of SEQ ID NO: 7, retaining antimicrobial activity and/or targeting activity to at least one Gram-positive bacterial strain or species.

[0056] In another aspect, the invention is directed to isolated peptides, polypeptides, and/or proteins of the present invention recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to therapeutic agents, e.g., small molecules, heterologous polypeptides, or catalytic domains of heterologous polypeptides, to generate fusion proteins or chimeric polypeptides. The fusion does not necessarily need to be direct, but may occur through linker sequences or through chemical conjugation. Non-limiting examples of therapeutic agents to which the peptide, polypeptides, or protein of the invention may be conjugated are peptide or non-peptide cytotoxins (including antimicrobials and/or antibiotics), tracer/marker molecules (e.g., radionuclides and fluorophores), and other antibiotic compounds as known in the art.

(0057] In some embodiments, the present invention is directed to chimeric polypeptides comprising the catalytic domains of two or more Enterococcal bacteriophage endolysins, where the chimeric polypeptides show increased lytic performance towards Enterococcus bacteria compared to the native bacterophage endolysins. The catalytic domains of the chimeric polypeptides may be from the same or different bacteriophages, which may have the same or different bacterial hosts, e.g., bacterial hosts of the same or different species, or of the same or different bacterial strain. In some embodiments, the native endolysins are from bacteriophages that natively infect E. faecalis, in particular strains £. faecalis 1518/05 and E. faecalis 926/095. |0058] In a particular embodiment, the invention in directed to chimeric polypeptides where at least one domain of a polypeptide isolated from phage 168, or a fragment thereof, is combined with at least one domain of a heterologous protein. Preferable chimeric constructs include the fusion of a catalytic domain of a lysin isolated from phage 168 {e.g., Lysl 68) with a catalytic domain of a lysin isolated from phage 170 {e.g., Lysl 70), which infect hosts of the Enterococcus species. Herein, the term "bacteriophage 170" is used interchangeably with the terms "bacteriophage Fl 70/08" or "phage 170". The resulting chimeric lysin constructs are renamed Lysl 70-168. Preferably Lysl 70-168 comprises an amidase domain of Lysl 70 and a CHAP domain of Lys 168.

[0059] In one embodiment, the chimeric polypeptide Lys 170- 168 comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 9, which chimeric polypeptide exhibits antibiotic or antimicrobial activity against E. faecalis. Sequence identity with respect to the chimereic polypeptide sequences disclosed herein also is defined as the percentage of amino acid residues that are identical in a candidate sequence of the same length {i.e., consists of the same number of residues) as the amino acid sequences of the present invention. The present invention also encompasses variants, derivatives and/or fragments of SEQ ID NO: 9 retaining

antimicrobial activity and/or antibiotic activity. In particularly preferred embodiments, the chimeric polypeptides and variants, derivatives, and/or fragments thereof improve the properties of Lysl 68 and/or Lysl 70, e.g., in terms of increased solubility, yield, stability, and/or lytic performance, such as including an increased lytic spectrum of activity towards Enterococcus species and/or other Gram-positive bacteria.

5.3 ANTIBIOTIC COMPOSITIONS

|0060] The isolated and chimeric polypeptides of the present invention may be administered alone or incorporated into a pharmaceutical composition for the use in treatment or prophylaxis of bacterial infections caused by gram-positive bacteria, including Enterococcus faecalis. In such embodiments, the pharmaceutical composition may be an antibiotic composition. The polypeptides may be combined with a pharmaceutically acceptable carrier, excipient, or stabilizer. Examples of pharmaceutically acceptable carriers, excipients and stabilizers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysin; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™ as known in the art. The pharmaceutical composition of the present invention {e.g., antibiotic composition) can also include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients.

[0061] A polypeptide of the present invention may also be combined with one or more therapeutic and/or prophylactic agents useful for the treatment of infection with gram-positive bacteria {e.g. one or more antibiotics and/or lysins as are known in the art). Therapeutic agents that may be used in combination with the polypeptide of the invention include standard antibiotics agents, anti-inflammatory agents, and antiviral agents.

[0062] Standard antibiotics that maybe used with pharmaceutical compositions comprising polypeptides of the invention include, but are not limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, rifamycin, naphthomycin, geldanamycin, ansamitocin, carbacephems. imipenem, meropenem, ertapenem, faropenem, doripenem, panipenem/betamipron, biapenem, PZ-601 , cephalosporins, cefacetrile, cefadroxil. cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cefonicid, cefprozil, cefuroxime, cefuzonam, cefrnetazole, cefotetan, cefoxitin, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, ceftnenoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, latamoxef, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, flomoxef, ceftobiprole, azithromycin, clarithromycin, dirithromycin, erythromycin,

roxithromycin, aztreonam, pencillin and penicillin derivatives, actinomycin, bacitracin, colistin, polymyxin B, cinoxacin, flumequine, nalidixic acid, oxolinic acid, piromidic acide, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin. rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, garenoxacin, gemifloxacin, stifloxacin, trovalfloxacin, prulifloxacin, acetazolamide, benzolamide,

bumetanide, celecoxib, chlorthalidone, clopamide, dichloφhenamide, dorzolamide,

ethoxyzolamide, furosemide, hydrochlorothiazide, indapamide, mafendide, mefruside, metolazone, probenecid, sulfacetamide, sulfadimethoxine, sulfadoxine, sulfanilamides, sulfamethoxazole, sulfasalazine, sultiame, sumatriptan, xipamide, tetracycline, chlortetracycline, oxytetracycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline,

rolitetracycline and any combination thereof. Preferred antibiotics for use in the management, prevention, and/or treatment of enterococcal infections, particularly antibiotic resistant enterococcal infections, include β-lactams, aminoglycosides, glycopeptides, lipoglycopeptides, lipopeptides such as daptomycin, oxazolidinones such as linezolid, glycylcyclines such as tigecycline, and pristinamycins such as quinupristin-dalfopristin. In certain embodiments, the combination of one or more polypeptides of the invention and one or more antibiotics as known in the art may enhance {e.g., additively or synergistically) the therapeutic effect of the

polypeptide of the invention for a given infection, such as an Enterococcal infection.

[0063] The chimeric polypeptides of the present invention comprise a combination where heterologous lysins are recombinantly fused, preferably where a catalytic domain of a lysin isolated from phage 168 is joined to a catalytic domain of a heterologous lysin, such as a lysin from phage 170. Preferably, the lysin construct comprises an amidase-2 domain of the lysin from phage 170 and a CHAP domain from the lysin from phage 168, which both natively infect Enlerococcus species. Without wishing to be bound by theory, it is believed that the chimeric construct in accordance with the instant invention targets gram-positive bacteria, including

Enterococcus faecalis, the native host for phages 170 and 168, whereupon the phage 1 70 and 168 catalytic domains act synergistically to destroy the host cell wall, causing lysis and bacterial death, with increased lytic activity as well as an increased lytic spectrum. Accordingly, the present invention provides chimeric lysin constructs that permit strain and species cross- reactivity in accordance with a goal of the invention. In some particularly preferred

embodiments, this cross-reactivity serves to improve the lytic performance of the chimeirc polypeptides on certain gram-positive bacteria, including E. faecalis, compared to the lytic activity of Lysl 70 and/or Lysl 68.

[0064 J The polypeptides of the present invention also may be combined with one or more lysins isolated from a bacteriophage other than bacteriophage 170 and 168, in particular, another Enterococcus phage lysin. Lysins, in general, either have amidase, endopeptidase, muramidase, or glucosaminidase activity. Therefore, the combination of catalytic domains of lysins, especially those of different enzymatic activities, is contemplated by the presented invention, which in preferred embodiments produces lysins with increased lytic performance towards

Enterococcus bacteria.

[0065] The pharmaceutical compositions can be administered using various modes of administration. For example, they may be administered by inhalation, in the form of a suppository or pessary, topically (e.g., as a lotion, solution, cream, ointment, or dusting powder), epidermally (e.g., by use of a skin patch), orally (e.g., as a tablet, (e.g., a tablet containing excipients such as starch or lactose), a capsule, ovule, elixir, solution, or suspension, optionally containing flavoring or coloring agents and/or excipients), or they can be injected parenterally, for example intravenously, intramuscularly, or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration, the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

[0066] For topical application to the skin, the polypeptides of the present invention may be combined with one, or a combination of carriers, which include but are not limited to, an aqueous liquid, an alcohol base liquid, a water soluble gel, a lotion, an ointment, a nonaqueous liquid base, a mineral oil base, a blend of mineral oil and petrolatum, lanolin, liposomes, proteins carriers such as serum albumin or gelatin, powdered cellulose carmel, and combinations thereof. A topical mode of delivery may include a smear, a spray, a time-release patch, a liquid-absorbed wipe, and combinations thereof. The polypeptide of the invention may be applied to a patch either directly or in one of the carriers. The patches may be damp or dry, wherein the lysin or chimeric lysin is in a lyophilized form on the patch. The carriers of topical compositions may comprise semi-solid and gel-like vehicles that include a polymer thickener, water, preservatives, active surfactants, emulsifiers, antioxidants, sun screens, and/or a solvent or mixed solvent system. U.S. Patent No. 5,863,560 discloses a number of different carrier combinations that can aid in the exposure of skin to a medicament.

|0067] As indicated, the therapeutic agent of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray, e.g., presentated from a pressurized container, pump, spray, or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2- tetrafluoroethane (HFA

134A.TM.) or 1 ,1,1,2,3,3,3-heptafluoropropane (HFA 227EA.TM.), carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray, or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g.

sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the agent and a suitable powder base such as lactose or starch.

[0068] For administration in the form of a suppository or pessary, the therapeutic compositions may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment, or dusting powder. The therapeutic agent of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route. For ophthalmic use, the compounds can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH-adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.

Alternatively, they may be formulated in an ointment such as petrolatum. [0069] For administration in tablet form, the tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, and glycine, disintegrants such as starch (preferably corn, potato ,or tapioca starch), sodium starch glycollate, croscarmellose sodium, certain complex silicates, and/or granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be included.

[0070] Dosages and desired drug concentrations of the pharmaceutical compositions of the present invention may vary depending on the particular use. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. In vitro and in vivo animal experiments can provide reliable guidance for the determination of effective doses in human therapy, such as those provided in the Examples below. Interspecies scaling of effective doses can be performed by one of ordinary skill in the art following the principles described by Mordenti, J. and Chappell, W., "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Ding Development, Yacobi et al., Eds., Pergamon Press, New York 1989. pp42-96 (hereby incorporated by reference in its entirety).

5.4 THERAPEUTIC USE

[0071 ] The polypeptides of the present invention have antibiotic activity against a number of gram-positive bacteria, including Enterococcus faecalis, and including several vancomycin-resistant strains of Enterococcus bacteria. Therefore, the polypeptides of the present invention may be used in methods of treating infections associated with bacteria against which it has lytic activity (e.g., antibiotic or antimicrobial activity) in both humans and animals. In one embodiment, compositions of the present invention may be used to treat an infection caused by one or more of the following Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus (including MRSA), S. haemolyticus, S. epidermidis, Bacilus subtilis, Bacilus

licheniformis, Streptococcus grupo, Micrococcus luteus, and Escherichia coli. In certain embodiments, the polypeptides of the invention may also exhibit antibiotic or antimicrobial activity (e.g., lytic killing activity) again Gram-negative bacteria or bacteria that are not classified as either Gram-positive or Gram-negative. In such embodiments, the polypeptides of the invention may be used to treat, prevent, and/or manage infections associated with non-Gram- positive bacteria.

[0072] Examples of diseases that are caused by infection of gram-positive bacteria that may be treated with pharmaceutical compositions of the present invention include, but are not limited to, endocarditis, bacteremia, diverticulitis, meningitis, urinary tract infection, and surgical wound infections, as well as post-operative endophtalmitis, other infections of the central nervous system, other wound infections (e.g., diabetic foot ulcers), pneumonia, osteomylelitis, sepsis, and mastitis.

5.5 DISINFECTANT AND ANTI-INFECTIVE USE

[0073] Nearly all bacterial pathogens infect at a mucous membrane site (upper respiratory, lower respiratory, intestinal, urogenital, and/or ocular). The mucous membranes themselves are often the reservoir, sometimes the only reservoir, for many pathogenic bacteria found in the environment (e.g. pneumococci, staphylococci, enterococci, and streptococci).

There are very few anti-infectives that are designed to control the carrier stage of pathogenic bacteria. However, studies have shown that by reducing or eliminating this reservoir in environments such as hospitals and nursing homes, the incidence of infections by these bacteria will be markedly reduced.

[0074] The polypeptides of the present invention may be used in anti-infective

compositions for controlling gram-positive bacteria, including E. faecalis, in order to prevent or reduce the development of serious infections. In addition to use in compositions for application to mucous membranes, the lysins or lysin constructs of the present invention may also be incorporated into formulations such as sprays or ointments for controlling colonization of Gram- positive bacteria on the skin and other solid surfaces, including medical devices like catheters.

5.6 DIAGNOSTIC METHODS

[0075] The present invention also encompasses diagnostic methods for determining the causative agent in a bacterial infection. In one embodiment, the method comprises culturing bacteria isolated from a bacterial infection and measuring the susceptibility to the antimicrobial polypeptides of the present invention, wherein susceptibility to the polypeptide indicates the presence of certain Gram-positive bacteria and the lack of susceptibility indicates the presence of non-responsive bacteria (e.g., non-responsive Gram-negative or non-responsive Gram-positive bacteria). The bacteria may be collected from, e.g., pus, urine, exudate from a wound, vaginal secretions, or any other bodily fluid infected with the bacteria.

5.7 AMINO ACID V ARIANTS

[0076] The invention also encompasses variants of the lysin polypeptides, or active fragments, or derivatives thereof, isolated from bacteriophage 168. In certain embodiments, the invention encompasses an amino acid sequence variant of SEQ ID NO:7, or active fragment or derivative thereof. Amino acid sequence variants of the polypeptides of the invention can be created such that they are substitutional, insertional, or deletion variants. Deletion variants lack one or more residues of the native protein which are not essential for function (e.g., antimicrobial and/or targeting activity). Insertional mutants typically involve the addition of material at a nonterminal point in the polypeptide. Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative

substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0077] Once general areas of the gene are identified as encoding the particular lysin protein, or active fragment, as described herein, point mutagenesis may be employed to identify with particularity which amino acid residues are important in the antibiotic activities. Thus, one of skill in the art will be able to generate single base changes in the DNA strand to result in an altered codon and a missense mutation.

[0078] Preferably, mutation of the amino acids of a protein creates an equivalent, or even an improved, second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without detectable loss of function (e.g., antibiotic and/or targeting activity). In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, interaction with a peptidoglycan within the outer coat of a Gram-positive bacteria. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics; for example: isoleucine (+4.5); valine (+4.2); leucine (+3. 8) ; phenylalanine (+2.8); cysteine/cystine (+2.5): methionine (+1.9): alanine (+1 .8); glycine (-0.4); threonine(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (- 1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. Like hydrophobicity, values of hydrophilicity have been assigned to each amino acid: arginine (+3.0); lysine (+3.0): aspartate (+3.0 + 1 ); glutamate (+3.0 ± 1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1 ); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). Equivalent molecules may be obtained by substitution of one amino acid for another where their

hydrophilicity indices are within ± 2, preferably ± 1 , or most preferably ± 0.5 of each other.

5.8 CHIMERIC CONSTRUCTS

[0079] The invention also encompasses chimeric polypeptides comprising a sequence corresponding to a catalytic domain of a first Enterococcal bacteriophge endolysin and a sequence corresponding to a catalytic domain of a second Enterococcal bacteriophage endolysin. The catalytic domains may be an amidase domain, preferably an amidase-2 domain, and/or a CHAP domain, for example an amidase-2 domain of one lysin fused to the CHAP domain of a heterologous lysin.

[0080) In preferred embodiments, the chimeric polypeptide shows increased lytic performance towards Enterococcus bacteria compared to said first and/or said second phage lysin. "Increased lytic performance" may refer to an increased spectrum of activity, e.g., against a larger number or a more diverse group of Enteroccocus species, of E. faecalis strains, E.

faecium strains, and/or of other Gram-positive bacteria. "Increase lytic performance" may also refer to an increased ability to kill and/or inhibit the growth or reproduction of a microorganism, in particular Enterococcus bacteria.

[0081 j The chimeric polypeptide may be constructed using lysins from the same or different bacteriophages. Where the lysins come from different bacteriophages, the different bacteriophages may have bacterial hosts that are of the same or different bacterial species, or the different bacteriophages may have bacterial host that are of the same or different bacterial strain. In some embodiments, the chimeric polypeptides are derived from bacteriophage F 168/08, which chimeric polypeptides exhibit antibiotic activity against a Gram-positive bacterium, e.g., against an Enterococcus species, such as E. faecalis. The chimeric polypeptide may be derived from a lysin isolated from phage 168, or fragment or variant thereof, which is recombinantly fused to a heterologouos lysin. In a particular embodiment, the invention in directed to chimeric polypeptides where at least one domain of a lysin isolated from phage 168, or fragment or variant thereof, is combined with at least one domain of a heterologous lysin, or a fragment or variant thereof. Preferable chimeric constructs include combination of a catalytic domain of a lysin isolated from phage 168, such as a catalytic domain of Lysl68, with a catatlytic domain of a lysin isolated from phage 170, which has antimicrobial or antibiotic activity against E. faecalis, such as a catalytic domain of Lysl 70. Even more preferably, the chimeric lysin comprises a CHAP domain of Lysl 68 recombinantly fused to an amidase or amidase-2 domain of Lysl 70. 100821 In some specific embodiments, the chimeric polypeptides of the invention comprise catalytic domains of lysins from bacteriophages having hosts from different strains of E. faecalis, such as from E. faecalis 926/05 and E. faecalis 1518/05. In particularly preferred embodiments, one endolysin catalytic domain originates from bacteriophage F 170/08 and the other from bacteriophage F 168/08. In even more particularly preferred embodiments, one endolysin is Lysl 70, corresponding to SEQ ID NO: 1 ; and/or one endolysin is Lysl68 corresponding to SEQ ID NO: 3. In other embodiments, the chimeric polypeptide comprises a catalytic domain of an endolysin having an amino acid sequence with 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 1 , which polypeptide exhibits antibiotic and/or targeting activity against Enterococcus sp..

particularly, E. faecalis and/or E. faeciuM: and/or a catalytic domain of an endolysin having an amino acid sequence with 60%. 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%. or greater sequence identity to SEQ ID NO: 3, which polypeptide exhibits antibiotic and/or targeting activity against Enterococcus sp., particularly, E. faecalis and/or E. faecium.

(0083] In certain specific embodiments, the chimeric polypeptide comprises a catalytic domain comprising SEQ ID NO:5, or a fragment thereof having antimicrobial activity against Enterococcus sp., particularly, E. faecalis and/or E. faecium. In other embodiments, the catalytic domain has an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 5 or a fragment thereof, and exhibits antibiotic and/or targeting activity against Enterococcus sp.. particularly, E. faecalis and/or E. faecium. In certain specific embodiments, the chimeric polypeptide comprises a catalytic domain comprising SEQ ID NO: 7, or a fragment thereof having antimicrobial activity against Enterococcus sp., particularly, E. faecalis and/or E. faecium. In other embodiments, the catalytic domain has an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 7 or a fragment thereof, and exhibits antibiotic and/or targeting activity against Enterococcus sp., particularly, E.faecalis and/or E. faecium.

(0084] In certain embodiments, the chimeric polypeptide comprises the amino acid sequence SEQ ID NO:9, or a fragment thereof having antimicrobial or antibiotic activity against at least one Enterococcus species, e.g., E. faecalis. In other embodiments, the chimeric polypeptide comprises a fragment, variant, or derivative of SEQ ID NO:9, wherein the fragment, variant or derivative has antibiotic activity or antimicrobial activity against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E. faecalis and/or E. faecium. Amino acid sequence variants of the chimeric polypeptides of the present invention can be created as described above with respect to isolated polypeptides of the invention, for example, by substitutions, insertions, deletions, and the like, preferably to generate further improved second- (or third- or more) generation molecules. In certain embodiments, the chimeric polypeptide comprises an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO:9 or a fragment thereof, and exhibits antibiotic and/or targeting activity against Enterococcus sp., particularly, E. faecalis and/ 'or E. faecium

Enterococcus sp., particularly, E. faecalis and,' 'or E. faecium. In particularly preferred

embodiments, the chimeric polypeptides and variants, derivatives, and/or fragments thereof, show improved properties, e.g., with respect to increased solubility, yield, stability, and/or lytic performance, such as including an increased lytic spectrum towards Enterococcus species and/or other Gram-positive bacteria, compared to the native isolated polypeptides.

5.9 COMBINATORIAL THERAPY

[0085] The present invention further provides compositions comprising one or more polypeptides of the invention and one or more differing prophylactic or therapeutic agents, and methods for treatment of bacterial infection in a subject in need thereof, (e.g., preventing, treating, delaying the onset of, slowing the progression of, or ameliorating one or more symptoms associated with an infection by gram-positive bacteria) comprising administering to said subject one or more of said compositions. Therapeutic or prophylactic agents include, but are not limited to, peptides, polypeptides, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules. Any agent which is known to be useful, or which has been used, or is currently being used for prevention or treatment of infection by a gram-positive bacteria, or for the prevention, treatment, or amelioration of one or more symptoms associated with an infection by a gram-positive bacteria, can be used in combination with the antibiotic or antimicrobial polypeptide in accordance with the invention described herein.

[0086] In certain embodiments, "in combination" refers to the use of a fusion protein or chimeric polypeptide, wherein an isolated polypeptide of the invention is covalently or non- covalently joined to another polypeptide, as described above. Preferable fusion proteins include chimeric polypeptides of Lysl68 with one or more heterologous lysins. such as Lysl 70 or another lysin from Enterococcus phages. For example, in a specific embodiment, the

combination of a CHAP domain from Lysl 68 with an amidase-2 domain from Lysl 70 produces Lysl 70-168. Some such chimeric constructs show increased lytic performance compared to native Lysl 68 and/or native Lysl 70, e.g., showing an increased lytic spectrum of activity towards Gram-positive bacteria, in particular Enterococcus species such as E. faecalis, and/or towards other Gram-positive bacteria. 5.10 POLYNUCLEOTIDES ENCODING POLYPEPTIDES

[0087] The invention provides polynucleotides comprising a nucleotide sequence encoding a polypeptide of the invention. The invention also encompasses polynucleotides that hybridize under high stringency, intermediate, or lower stringency hybridization conditions, to polynucleotides that encode a polypeptide of the invention. "High stringency conditions" can include, but are not limited to, those that (1 ) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.001 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ, during hybridization, a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1 % bovine serum albumin/0.1 % Ficoll/0.1 %

polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5XSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, SXDenhardt's solution, sonicated salmon sperm DNA (50 μg/mL), 0.1 % SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2XSSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1XSSC containing EDTA at 55°C.

"Moderately stringent conditions" are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2.sup.nd Ed.. New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5XSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 XSSC at about 37-50°C.

(0088] The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, a polynucleotide encoding a polypeptide of the invention may be generated from nucleic acid from a suitable source, e.g., bacteriophage 168, as described in the Examples below. If a source containing a nucleic acid encoding a particular polypeptide is not available, but the amino acid sequence of the polypeptide of the invention is known, a nucleic acid encoding the polypeptide may be chemically synthesized and cloned into replicable cloning vectors using methods well known in the art. [0089] Once the nucleotide sequence of the polynucleotide of the invention is determined, the nucleotide sequence may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate polypeptides having a different amino acid sequences, for example to create variants, fragments, and/or derivatives of the polypeptide, with e.g., amino acid substitutions, deletions, and/or insertions.

[0090] Chimeric polynucleotides of the invention encompass nucleotide sequences encoding a chimeric polypeptide of the invention, such as chimeric polypeptides comprising a catalytic domain of a lysin isolated from phage 168 fused to a catalytic domain of a lysin isolated from a heterologous lysin, preferably a heterologous phage lysine, such as a catalytic domain of a lysin from phage 170. The invention also encompasses polynucleotides that hybridize under high stringency, intermediate, or lower stringency hybridization conditions, e.g., as defined supra, to chimereic polynucleotides that encode a chimeric polypeptide of the invention

[0091] Chimeric polynucleotides may be obtained by recombinant techniques, as are well known and routinely practiced in the art. Recombinant chimeric polynucleotides typically are created by joining two or more genes, or portions thereof, which originally coded for separate proteins. The individual sequences typically correspond to coding sequences for a functional domain of each of the respective proteins, such that the chimeric polypeptide encodes a fusion protein having dual functionality. For chimeric lysins, functional domains may correspond to modular catalytic domains, such as domains having lytic acitivity, including, e.g., amidase activity, and the coding sequences for the different catalytic domains fused together.

[0092] For example, a first coding sequence, or portion thereof, may be joined in frame to a second coding sequence, or portion thereof, which typically is achieved through ligation or overlap extension PCR. Ligation is used with the conventional method of creating chimeric genes, called the "cassette mutagenesis method." In this method, DNA can be cut into specific fragments by restriction endonucleases acting at restriction endonuclease recognition sites, and the specific fragments can be then ligated. A particular fragment can be substituted with a

" *"

- > J - heterologous one having compatible ends in order to ligate it into the parental DNA. See, e.g., Wells et al., Gene 34:315-23 (1985), hereby incorporated by reference in its entirety.

|0093] Alternatively, various approaches involving PCR may be used, such as the overlap extension PCR method. See, e.g.. Ho, S.N., et al (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 77: 51-59, hereby incorporated by reference in its entirely. Several variations of this PCR approach are known and have been used to generate chimeras. One such approach, for example, involves modified overlap extension PCR to create chimeric genes in the absence of restriction enzymes in three steps: (i) a conventional PCR step, using primers partially complementary at their 5' ends to the adjacent fragments that are to be fused to create the chimeric molecule; (ii) a second PCR step where the PCR fragments generated in the first step are fused using the complementary extremities of the primers; and (iii) a third step involving PCR amplification of the fusion product. The final PCR product is a chimeric gene built up with the different amplified PCR fragments. See, e.g.,

Wurch, T. et al ( 1998). A modified overlap extension PCR method to create chimeric genes in the absence of restriction enzymes. Biotechnology Techniques. 12(9):653-657, hereby incorporated by reference in its entirety. Any ligation and/or PCR-based recombinant approaches may be used to create the chimeric polynucleotides of the present invention.

[0094] Alternatively a nucleic acid encoding the chimeric polypeptide may be chemically synthesized. For example, using the desired amino acid sequence of the chimeric polypeptide of the invention, the corresponding nucleotide sequence may be devised, chemically synthesized, and cloned into replicable cloning vectors using, e.g., well known methods in the art. The Examples below provide additional details for creating the chimeric polynucleotide, SEQ ID NO: 10, encoding a chimeric polypeptide of the invention.

5.11 RECOMBINANT EXPRESSION OF MOLECULES OF THE INVENTION

[0095] Once a nucleic acid sequence encoding a molecule of the invention (e.g., a polypeptide of bacteriophage origin, or functional derivative, chimeric construct, variant, or fragment thereof) has been obtained, the vector for the production of the molecules may be produced by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequences for the molecules of the invention and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and Ausubel et al. eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

[0096] An expression vector comprising the nucleotide sequence of a molecule identified by the methods of the invention can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells then can be cultured by conventional techniques to produce the molecules of the invention. The Examples below provide additional details for producing chimeric polypeptides according to SEQ ID NO:9 from chimeric polynucleotides encoding same, after transfection of vectors comprising SEQ ID NO: 10 into competent cells for expression.

[0097J The host cells used to express the molecules identified by the methods of the invention may be either bacterial cells (nonsusceptible to the lysin protein, lysin construct, or fragment thereof of the invention) such as Escherichia coli in certain embodiments. A variety of host-expression vector systems may be utilized to express molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of the molecules of the invention may be expressed and subsequently purified; and also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the molecules of the invention in situ. These include, but are not limited to, microorganisms such as bacteria that are not susceptible to the lysin protein, lysin construct, or fragment of the invention {e.g., E. coli and B. subtilis in some embodiments) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing coding sequences for molecules encompassed by the invention; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing sequences encoding molecules encompassed by the invention; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing sequences encoding molecules encompassed by the invention; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing sequences encoding molecules encompassed by the invention; or mammalian cell systems (e.g., COS, CHO, BH , 293, 293T, 3T3 cells, lymphotic cells (see U.S. 5,807,715), Per C.6 cells (human retinal cells developed by Crucell)) harboring recombinant expression constructs containing sequences encoding molecules encompassed by the invention operatively linked to promoters, e.g., promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; or the vaccinia virus 7.5K promoter).

(0098] In bacterial systems not susceptible to the lysin protein, lysin construct, or fragment of the invention, a number of expression vectors may be advantageously selected depending upon the use intended for the molecule being expressed. For example, when a large quantity of such a protein is to be produced, e.g., for the generation of pharmaceutical compositions of a polypeptide, vectors which direct the expression of high levels of fusion protein products and that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2: 1791. hereby incorporated by reference in its entirety), in which the coding sequence may be ligated individually into a vector in frame with a lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509; each of which is hereby incorporated by reference in its entirety); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix of glutathione- agarose beads, followed by elution in the presence of free gluta-thione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites, so that the expressed product can be released from the GST moiety. The Examples below provide additional details for producing chimeric polypeptides according to SEQ ID NO:9 from chimeric polynucleotides encoding same, using E. coli BL21 as the expression system.

|0099] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The polypeptide coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[00100] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the polypeptide coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene, e.g., then may be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the inserted polypeptide molecule in infected hosts (e.g., see Logan & Shenk. 1984, Proe. Natl. Acad. Sci. USA 81 :355-359, hereby incorporated by reference in its entirety). Specific initiation signals may also be required for efficient translation of inserted coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These translational control signals and/or initiation codons can be of a variety of origins, both natural and synthetic, including exogenous sources. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, Bittner et al., 1987, Methods in Enzymol. 153:51 -544, hereby incoporated by reference in its entirety).

[00101] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express a polypeptide of the invention may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1 -2 days in an enriched media, and then switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may be advantageously used to engineer cell lines which express the polypeptides of the invention. Such engineered cell lines may be particularly useful in screening and evaluation of bacterial species susceptible to the polypeptides of the invention.

[00102| A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977. Cell 1 1 : 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48: 202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes, which can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981 , Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981 , Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12: 488-505: Wu and Wu, 1991, 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191 -217; May, 1993, TIB TECH 1 1 (5): 155-215); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147). Methods commonly known in the art of recombinant DNA technology which can be used for application of such selection systems are described, e.g., in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; riegler, 1 90, Gene Transfer and Expression. A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics. John Wiley & Sons, NY.; Colberre-Garapin et al., 1981 , J. Mol. Biol. 150: 1 ;

[00103] The expression levels of a polypeptide of the invention can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning. Vol. 3, Academic Press, New York, 1987). Briefly, when a marker in the vector system expressing a polypeptide of interest is amplifiable, increasing the level of inhibitor present in the host cell culture can increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence encoding the polypeptide of interest, production of the polypeptide also will increase (Crouse et al., 1983, Mol. Cell. Biol. 3 :257).

[00104J Once a polypeptide of the invention has been recombinantly expressed, it may be purified by any method known in the art for purification of polypeptides, for example, by chromatography (e.g., ion exchange, affinity, and/or sizing column chromatography),

centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides or antibodies. The Examples below provide additional details for purifying isolated lysins Lys l 70 (SEQ ID NO: l ) and Lysl68 (SEQ ID NO:3), as well as the chimeric lysin construct Lysl 70-168 (SEQ ID NO:9) after their respective expression in E.coli BL21 cells. |00105] The following examples illustrate but do not limit the invention. Thus, the examples are presented with the understanding that modifications may be made and still be within the spirit and scope of the invention.

6. EXAMPLES

[00106] This work aimed to study the lytic activity of two lysins from phages F 168/08 and F 1 70/08, respectively, both of which infect Enterococcus sp., as well as a chimeric lysin constructed from a catalytic domain of each of these two lysins.

6.1 Example 1 : Isolation of Bacteriophages 168 and 170

[00107] Bacteriophage 168 was isolated from E. faecalis 926/05; bacteriophage 170 was isolated from E. faecalis 1518/05. Genomic DNA can be isolated from a stock of phage 168 and a stock of phage 170, each obtained from a clinical isolate of E. faecalis._. and the genomic DNA of the phages can be extracted, cloned, and sequenced according to protocols as follows.

[00108] Purification of phages 168 and 170

[00109] Preparation of stock phage 168 and stock phage 170 can be carried out using the protocols described in Carlson ., 2005, "Working with bacteriophages: common techniques and methodological approaches," in utter, E. Sulakvelidze, A. (eds.) Bacteriophages: Biology and Applications. 5^th ed. CRC press ("Carlson;" hereby incorporated by reference in its entirety). The stock phage 168 and 170 each can be concentrated by precipitation with PEG according to the protocol described in Yamamato et al., 2004, PNAS 101 :6415-6420 (hereby incorporated by reference in its entirety) and Carlson.

[00110] The phage 168 and phage 170 stocks can be incubated in 1 M NaCl for one hour at 4°C with agitation. Next, PEG 8000 (AppliChem, Cheshire, MA) can be added gradually until a final concentration of 10% (p/v) is reached. The compositions then can be each incubated overnight at 4°C. After the incubation period, each composition can be centrifuged at 10000 x g for 30 minutes at 4°C. The sediments then can be re-suspended in SM (0.05 M Tris-HCl at pH 7.4, 0.1 M NaCl, 10 mM MgS0₄ and gelatin at 1% p/v) and centrifuged again at 1000 rpm at 4°C for 10 minutes. The supernatant containing each suspended phage can saved for further purification. [00111] Purification of phages 168 and 170 can be achieved using a CsCl gradient as described by Carlson. Removal of CsCl from each of the phage stocks can be achieved through dialysis. A dialysis membrane Cellu.Sep H I High Grade Regenerated Cellulose Tubular Membrane (Cellu. Sep, River Street, USA) can be prepared according to the

manufacturer's instructions. The dialysis may consist of a first incubation of 30 minutes in 100 mM Tris-HCl and 3 M NaCl (pH 7.4) at 4°C. This can be followed by a second incubation of 30 minutes in 100 mM Tns-HCl and 0.3 M NaCl (pH 7.4) at 4°C. After dialysis, each of the suspended phages can be removed from the interior of the dialysis bag and stored at 4°C.

100112] Extraction of phage DNA

[00113) Phage 168 and phage 170 DNA can be obtained from each stock phage, respectively, purified on CsCl. To 5 ml of each purified phage can be added 20 mM EDTA at pH 8.0. SDS at 0.5% (p/v) and Proteinase at a final concentration of 40 μg/ml. Each mixture then can be incubated at 56°C for one hour. This may be followed by successive extractions in phenol:chloroform:alcohol at a proportions of 25:24: 1, until the interface between the aqueous and organic phases becomes clear. Each aqueous phase then can be treated with an equal volume of chloroform and centrifuged at 13,0000 x g for 10 minutes at 4°C. Each aqueous phase can be once again removed and the DNA precipitated by adding two volumes of absolute ethanol and incubating for thirty minutes at 20°C. The samples then can be centrifuged at 1 1 ,000 x g for 30 minutes at 4°C. The pellets then can be washed with 70% ethanol at room temperature and re- suspended in 50 μΐ of ultra-pure water (Gibco, California). The DNA concentration for each phage then can be determined by measuring the absorbance at 260 nm in a ND-1000

Spectrophotometer. The integrity of each isolated phage DNA then can be analyzed by electrophoresis on a 1 % agarose gel.

[00114] The phage 168 and 170 DNA each can be sequenced, and the open reading frames (ORFs) coding for amino acid sequences identified, using the tools described under the

Bioinfomatics Analysis section. In addition, the homology of phages 168 and 170 DNA can be compared to existing sequences using the program FASTA3.

[00115] Bioinformatic Analysis

[00116] Analysis of target DN A and amino acid sequences can be carried out by using ExPASy (Expert Protein Analysis System) of the Swiss Institute of Bioinformatics. Additional analysis also can be carried out using the programs Translate Tool, Prosite, and ProtPram. The homology of the target amino acid sequences with sequences in the UniProt Knowledgebase database can be performed using FASTA3. Sequence alignments can be performed using ClustalW. Both programs can be accessed through the European Molecular Biology Laboratory - European Bioinformatics Institute (EMBL-EBI) website. The determination of the secondary structure of the target sequence can be carried out using the program Foldlndex

[00117] Sequencing of the bacteriophage genomes can allow identification of potential open reading frames (ORFs) within the genome. The putative ORFs of bacteriophages can be translated into their corresponding amino acid sequences and the amino acids sequences can be used to search the UniProt Knowledgebase using the program FASTA3. Alignment with other known lysin proteins from other bacteriophages can allow identification of the phage 170 and phage 168 lysin proteins (Lysl 70, SEQ ID NO: 1 and Lys l 68, SEQ ID NO: 3, respectively) and their corresponding gene sequences (SEQ ID NO:2 and SEQ ID NO:4, respectively).

[00118] Amino acid sequence analysis of Lys 170 and Lys 168 indicated an amidase-2 domain (SEQ ID NO:5) and a CHAP domain (SEQ ID NO:7), respectively, in these proteins and their corresponding gene sequences determined (SEQ ID NO:6 and SEQ ID NO:8, respectively). This information was used to construct a chimeric polypeptide, in order to evaluate the possibility of improving lytic activity of the lysins. The strategy employed produced soluble polypeptides, which were used to test lytic activity against E. faecalis and E. faecium. as well as against other Enterococcus sp. and non-Enterococciis sp.

6.2 Example 2: Cloning of Lysl68 and Lysl70

|00119| Lys 168 was isolated from bacteriophage 168 and Lys 170 was isolated from bacteriophage 170. The lysins also can be isolated from recombinant cells expressing the phage proteins from plasmids encoding same.

[00120] Construction of Plasmids

[001211 The lysins Lys 168 (237aa) and Lys 170 (289aa) were amplified using specific primers respectively from bacteriophages F168 and F170; and were cloned in vectors pIVEX 2.3d (Roche) and pTrCHisA (Invitrogen) using E. coli MRF' XLl-blue (Stratagene). E. coli BL21 was used for expression.

100122] The plasmids can be constructed by inserting the sequence of cDNA

corresponding to the isolated lysin of phage 168 (Lys 168), or that of phage 170 (Lys 1 70), into the selected vectors. The PCR reaction can be set up using the following conditions: puReTaq Ready-to-Go PCR Beads (Amersham Biosciences, U.K.), 200 ng of genomic DNA from phage 168 or phage 170, the primers at a final concentration of 0.4 pmol/μΐ and ultra-pure water to a final volume of 25 μΐ. The following thermocycler conditions can be used: 1 minute at 95°C for 1 cycle, 1 minute at 95°C +1 minute at 57°C + l minute at 72°C for 30 cycles, and 5 minutes at 72°C for one cycle.

[00123] The vectors can be digested with restriction enzymes, such as Bam HI, Hindlll and

Bam l and XJiol (Fermentas). The restriction digest mixture can be prepared according to the manufacturer's instructions. The fragments of DNA resulting from the digestion of the vectors as well the amplified DNA of Lys l 68 and Lysl 70 can be run on a 1 % agarose gel. The DNA then can be purified from the gel using the High Pure PCR Product Purification Kit (Roche, Germany) according to the manufacturer's instructions.

|00124] The purified vector DNA and the cDNA encoding Lysl 68 and Lysl 70 can be combined in a ratio of 1 :5 moles along with 1 of T4 DNA ligase (New England Biolabs, Frankfort, Germany) 10 x ligation buffer (50 mM Tris-HCl, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, pH 7.5 at 25°C) and ultra-pure water at a final volume of 20 μΐ. The ligation mixture can be incubated overnight at 22°C followed by transformation of E. coli strain BL21. The transformation can be done according to the protocol described previously.

[00125] Transformants can be selected by plating in a Petri dish containing LB agar and the respective selection marker. Colonies of transformants then can be used to inoculate LB broth containing the appropriate selection marker and incubated overnight. The cultures then can be centrifuged and the DNA extracted as described previously.

[00126J Correct insertion of the cloned fragment can be determined by digestion of the recombinant plasmid using the same restriction enzymes used to construct the plasmids. All methods and procedures for cloning the Lysl 68 and Lysl 70 fragment can be done according to standard protocols. Recombinant plasmids containing the DNA of interest corresponding to the DNA of Lysl 68 or Lysl 70 can be sequenced by Macrogen (Coreia do Sul).

6.3 Construction of Lys 170- 168

[00127] With the aim to construct an endolysin with a larger spectrum of action and knowing that the catalytic activity of amidase-2 represents a larger spectrum action than several other classes of endolysins, a chimeric sequence lysl 70-168 was constructed. It resulted from the junction of the nucleotide sequence corresponding to the amidase-2 domain of Lysl 70 with the nucleotide sequence corresponding to the CHAP (ligation) domain of Lysl 68.

[00128] The technique of Overlap-Extension by Polymerase Chain Reaction (OE-PCR) can be used. See, e.g.. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R. ( 1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 77: 51 -59. The encoded amino acid sequence and corresponding nucleotide sequence of the chimeric gene correspond to SEQ ID NO:9 and SEQ ID NO: 10, respectively.

6.4 Expression of Lys 170, Lys 168 and Lys 170- 168

[00129] Expression vectors were constructed, expressing each of Lysl 68, Lysl70, and the chimeric lysin Lysl 70-168.

[00130] Preparation of Competent Cells

[00131] In order to prepare competent cells, LB broth can be inoculated with competent E. coli and incubated overnight with agitation of 135 rpm at 37°C. The following day, 5 ml from the cultured LB broth can be added to 200 ml of fresh LB broth. The culture can be incubated with agitation at 37°C until an optical density (ODeoo) of 0.7-0.8 is reached. The optical density can be measured on a UV/Vis Spectrometer UVA (Unicam). The cells then can be centrifuged for 20 minutes at 5,000 rpm at 4°C. After removal of the supernatant, the pellet can be re- suspended in 10 ml of a 10% glycerol solution previously cooled in ice. The volume then can be brought to 50 ml by adding more of the 10% glycerol solution and centrifuged again at 5,000 rpm for 20 minutes at 4°C. After removal of the supernatant, the pellet can be re-suspended as described before and centrifuged an additional time. The pellet then can be re-suspended in the residual 10% glycerol solution that remains in the tube after decantation. The samples then can be aliquoted and stored at -80°C.

[00132] Transformation of Competent Cells by Electroporation

[00133] In order to a transform the competent E. coli cells, an aliquot of competent cells can be removed from storage at -80°C and thawed at 4°C for 10-20 minutes. Then 1 μΐ of plasmid DNA can be added to 25 μΐ of competent cells. The suspended cells then can be transferred to an electroporation cuvette (Electroporation Cuvettes Plus model No. 610, BTX, Holliston, USA) and electroporated in a Gene Pulser Xcell System (Bio-Rad, Hertfordshire, U.K.). The parameters that can be used for a 1 mm cuvette are as follows: electric impulse - 10 μΡ, Resistance - 600 Ohms and Voltage - 1800 V. Immediately after electroporation the cells can be resuspended in 1 ml of LB broth and incubated at 37°C for 1 hour with agitation at 135 rpm. Then the cells can be centrifuged at 13,000 rpm for 1 minute at room temperature. The cells then can be resuspended in 50 μΐ of LB broth and plated on a Petri dish containing LB agar (50 μΐ/plate) and the necessary selective markers. The plates can be incubated overnight at 37°C.

[001341 Expression of Lvsl 70. Lvsl 68. and Lvsl 70-168

[00135] The genes lysl 70 and lysl 68, as well as the chimeric gene lysl 70-168, each can be cloned into an expression vector, under the control of an inducible promoter. The construct can be used to transform E. coli BL21 for expression. Transformed bacteria can be used to inoculate 5 ml of 2 x YT broth containing the appropriate selection markers. The cultures can be incubated overnight at 37°C with agitation until a OD (600nm) of 0.6 is obtained. Expression of the lysin can be induced by the addition of 1 mM to 0.5 mM of IPTG. This can be done approximately after incubation for 4 hours at 37°C with agitation. .

[001361 Purification of Lysl 68. Lvs70 and Lysl 70- 168

[00137] After incubation is complete, lysis of the E. coli cells can be achieved by the addition of lysosyme (Sigma- Aldrich ) at a final concentration of 0.1 mg/ml and 1 μΐ of Protease Inhibitor Cocktail Set I (Calbiochem, USA) followed by freezing and thawing. The sample can be exposed to five cycles of freezing and thawing. The sample then can be centrifuged at 14,0000 x g for 10 minutes at 4°C. After centrifugation, the supernatant can be removed and the pellet re-suspended in 500 to of PBS 1 x.

[00138] Alternatively, the liquid cultures can be centrifuged at 1 1 ,000 rpm for 40 minutes at 4 °C. The supernatant can be removed and the pellet resuspended in 5 ml of BugBuster Master mix with Protease Inhibitor Cocktail Set I at a dilution of 1 : 1000. The cells can be lysed according to the manufacturer's instructions. The samples then can be centrifuged at 4°C and 14,000 x g for 10 minutes. After centrifugation, the supernatant can be removed and the pellet resuspended in 5 ml of PBS lx. The samples then can be centrifuged again at 4 °C and 14,000 x g for 1 0 minutes. The pellet containing Lysl 68, Lys l 70, or Lys l 70-168 can be stored at 4 °C. The samples can be analyzed and Lys l 68, Lys l 70, or Lys l 70- 168 purified by SDS-PAGE and Western Blot. 100139] Purification using a Ni-NTA column

100140] Lys l 68, Lys l 70, or Lys 1 70- 1 68 can be purified using a Ni-NTA column

(Qiagen). The Ni-NTA resin can be stored at 4 °C prior to be added to the column. The column then can be washed with 50 ml of wash buffer (50 mM Na2HP0₄, 300 mM NaCl, 20 mM imidazole in 1 L of distilled water at pH 8.0) using a peristaltic pump at medium speed. The cellular extracts prepared according to the previously described protocols then can be loaded with a peristaltic pump set at low speed. The column then can be washed with 50 ml of wash buffer to remove nonspecific proteins and other impurities. The protein then can be eluted from the column using an elution buffer (50 mM Na₂HP0_4. 300 mM NaCl, 250 mM imidazole in 1 L of distilled water at pH 8.0) and collected in 1 .5 ml fractions. All of the fractions can be analyzed by SDS-PAGE.

|00141 ] Dialysis of samples purified bv Ni-NTA Chromatography

(00142] A Cellu.Sep H I High Grade Regenerated Cellulose Tubular Membrane can be prepared according to the manufacturer's instructions. The samples can be dialyzed against 1000 volumes of 50 mM Tris-HCl pH 7.5 overnight at 4 °C with slight agitation. The previous procedure can be repeated again until total incubation time reached 24 hours. After dialysis, the lysin can be removed from the interior of the membrane and stored at 4°C. A sample can be analyzed by SDS-PAGE and Western Blot and the amount of protein quantified using Bradford assay.

J00143] Concentration

[00144] The proteins of interest can be concentrated on an Amicon Centrifugal Filter Device (Millipore, USA) according to the manufacturer's instructions. The Amicon Centrifugal Filter Devices can also be used to exchange the buffer of the protein samples. In this mode, 12 ml of a 100 mM Tris-HCl buffer at pH 7.0 can be added together with a sample of Lys 1 68, Lys 1 70, or Lys 1 70- 1 68. The column then can be centrifuged for 30 minutes at 5,000 rpm at 4 °C.

Concentrated sample of the lysin can be analyzed by SDS-PAGE and/or Western Blot in order to confirm the integrity of the purified protein. The concentration can be determined using a ND- 1000 spectrophotometer measuring the absorbance at 280 nm and using a Bradford assay.

100145] SDS-PAGE

[00146] 15% polyacrylamide gels can be used. The resolving gel can be prepared by adding 6.25 mi Protogel, 3.35 ml Protogel Resolving Buffer (National Diagnostics, Georgia, USA). The stacking gel can be prepared using 650 Protogel, 1.25 ml Protogel Stacking Buffer (National Diagnostics), 3 ml distilled water, 50 41 APS 10%, and 7.5 TEMED. Protein samples to be analyzed then can be denatured by placing them in 6 x denaturing buffer (0.35 M Tris-HCl at pH 6.8, 10.28% DS, 36% glycerol, 0.6 M DTT and 0.012 % bromophenol blue) and heated at 100 °C for 10 minutes. The gel can be run on a Mini-PROTEAN Tetra Cell (Bio-Rad). While the samples are in the stacking gel, the voltage can be maintained at 140 V. After the samples enter the resolving gel, the voltage can be increased to 200 V. Precision Plus Protein™

Standards Dual Color of Bio-Rad and PageRuler™ Prestained Protein Ladder of Fermentas can be used as molecular weight ladders.

[00147] Transfer to Nitrocellulose Membrane

[00148] In order to visualize the protein bands on the SDS-PAGE gel, the gel can be immersed in a solution of Coomassie stain for 1 hour at ambient temperature. The gel then can be transferred to a destaining buffer (10% acetic acid, 10% methanol in 1 L of distilled water) to remove excess stain.

[00149] The gel then can be placed in 1 x transfer buffer (48 mM Tris, 39 mM glycine, 0.04% SDS, 10% methanol, and 1L of distilled water) at ambient temperature. The proteins then can be transferred from the gel to a nitrocellulose Hybond C (GE Healthcare, Germany) using a Mini Transblot Module (Bio-Rad). The transfer can take place at 200 mA for 1 hour.

[00150] Western Blot

[00151] The nitrocellulose membrane can be blocked in PBS 1 x. 5% milk protein, 0.05% TWEEN™ 20 overnight at 4 °C. The membrane then can be washed 5 times in PBS lx, 0.05% Tween 20 at room temperature. The membrane then can be incubated for 1 hour with agitation at room temperature in a solution containing PBS Ix, 2% milk protein, 0.05 % Tween 20, and anti-His6 antibody conjugated to peroxidase diluted 1 :5000 (Roche). The membrane then can be washed three times for 15 minutes in a solution of PBS lx, 0.05% Tween 20 at room temperature. The protein of interest can be detected using the ECL™ Plus Western Blotting Detection System (GE Healthcare) following the manufacturer's instructions. The membrane then can be exposed to Amersham Hyperfilm ECL and developed in an AGFA Curix 60 processor.

Evalulation of Lysin Activity in vitro [00152] The lysin peptides expressed from the recombinant vectors were assayed for lytic activity in vitro on a number of bacterial strains of clinical origin, as shown in Table I below.

Strain Species Sample Type

1553/05

Enterococcus faecalis Urine

556/06 Enterococcus faecalis Urine

73/07

Enterococcus faecalis Urine

857/05

Enterococcus faecalis Urine

882/06

Enterococcus faecalis Urine

926/06

Enterococcus faecalis Urine

958/05

Enterococcus faecalis Urine

1113/06

Enterococcus faecalis Hemoculture

127/06

Enterococcus faecalis Urine

1408/05

Enterococcus faecalis Urine

1409/05

Enterococcus faecalis Urine

1551/05

Enterococcus faecalis Urine

Enterococcus faecalis

1554/05 Urine

1558/05

Enterococcus faecalis Urine

1853/05

Enterococcus faecalis Urine

2/06

Enterococcus faecalis Urine

2093/05

Enterococcus faecalis Urine

3/06

Enterococcus faecalis Urine

307/06

Enterococcus faecalis Urine

43/06

Enterococcus faecalis Urine

44/06

Enterococcus faecalis Pus

563/07

Enterococcus faecalis Hemoculture

750/06

Enterococcus faecalis Hemoculture Strain Species Sample Type

751/06

Enterococcus faecalis Hemoculture

81/06

Enterococcus faecalis Urine

952/06

Enterococcus faecalis Hemoculture

954/06

Enterococcus faecalis Hemoculture

1/07

Enterococcus faecalis Urine

110/07

Enterococcus faecalis Urine

139/07

Enterococcus faecalis Urine

158/07

Enterococcus faecalis Hemoculture

310/07

Enterococcus faecalis Urine

311/07

Enterococcus faecalis Urine

328/07

Enterococcus faecalis Urine

332/07

Enterococcus faecalis Urine

514/07

Enterococcus faecalis Urine

606/07

Enterococcus faecalis Urine

1000/05

Enterococcus faecium Urine

1131/05

Enterococcus faecium Pus

1132/05

Enterococcus faecium Pus

1607/05

Enterococcus f aecium Wound exudate

1729/05

Enterococcus faecium Urine

1793/05

Enterococcus faecium Pus

1795/05

Enterococcus faecium Urine

1866/05

Enterococcus faecium Urine

1903/05 Enterococcus faecium Pus Strain Species Sample Type

969/05

Enterococcus faecium Urine

184/06

Enterococcus faecium Pus

185/06

Enterococcus faecium Catheter

186/06

Enterococcus faecium Pus

187/06

Enterococcus faecium Pus

188/06

Enterococcus faecium Hemoculture

198/06

En terococcus fa ecium Bile

226/06

Enterococcus faecium Urine

267/06

Enterococcus faecium Anal exudate

268/06

Enterococcus faecium Pus

269/06

Enterococcus faecium Hemoculture

388/06

Enterococcus faecium Hemoculture

389/06

Enterococcus faecium Hemoculture

390/06

Enterococcus faecium Hemoculture

729/06

Enterococcus faecium Pleural fluid

515/07

Enterococcus faecium Vaginal exudate

1040/06

Enterococcus sp. Urine

1041/06

Enterococcus sp. Urine

1271/06

Enterococcus sp. Urine

1285/06

Enterococcus sp. Urine

140/07

Enterococcus sp. Urine

1403/06

Enterococcus sp. Ascites

Escherichia coli Urine [00154J Lytic Activity Assay

(00155] Lytic activity of the lysins was tested on bacteria cells of different strains, including the phage host strains, by measuring optical density (OD).

[001561 To study the lytic activity of the isolated or chimeric endolysins, different quantities of each of the pure enzymes (10, 5, 1 , and 0.2 g), in a 5 μΐ volume, can be spotted on the surface of a soft agar medium containing viable target bacteria. The different target bacteria can be grown in appropriate media until OD₆oo ⁼ 0.8-1.0 and concentrated 100-fold in fresh media. One hundred microliters of this cell suspension can be incorporated in 10 ml of soft-agar medium (25 mM Phosphate-Na buffer pH 6.5, 250 mM NaCl, 0.7% agar) and poured in a Petri dish. This procedure can guarantee a homogeneous and dense lawn of target bacteria. The relative lytic activity obtained with the various enzyme amounts can be qualitatively evaluated by registering the dimensions and transparency of lysis halos, after overnight incubation at 37 °C.

[00157] Lytic activity also can be evaluated in a liquid medium (25 mM Phosphate-Na buffer pH 6.5, 250 mM NaCl). Cultures of the Entrerococcus, e.g., can be grown until exponential growth phase (OD6oo ⁼ 0.3-0.4), recovered by centrifugation. and concentrated 2-fold in the liquid medium. Ten micrograms of each purified enzyme (in a 5 μΐ volume) then can be added to one ml of this cell suspension and cell lysis monitored by registering OD₆oo values at different time points after endolysin addition. One milliliter of the cell suspension added to 5 μΐ of enzyme storage buffer can serve as negative control.

[00158J The bacterial strains tested can be isolated from clinical samples (including blood, urine, pus, and medical devices) in different Portuguese hospitals and clinical settings or, in the case of the Micrococcus and Bacillus strains, obtained from ATCC strains.

[00159] As shown in FIG. 1 , lytic activity of lysins Lysl 68, Lys l 70-168, and Lysl 70, was tested for four different amounts of each lysine in 98 clinic strains of Enterococcus, including E. faecalis and E. aecium. Aslo, as shown in FIG. 2, Lysl 68 and Lysl 70 activity was tested as the percent reduction in turbidity for each of three E. faecalis strains, E. faecalis 926/05, E. faecalis 151 8/05, and E. faecalis 1915/05, after addition of μg/mL of each lysin. "C-" indicaes a negative control where no lysin was added. The results obtained with Lysl 68 and Lysl 70 show that strain E. faecalis 191 5/05 is much more sensitive to Lysl 68 activity compared with Lysl 70 (FIG. 2). |00160] As shown in FIG. 3, cell viability assays were conducted for each of the three E. faecalis strains, E. faecalis 926/05, E. faecalis 1518/05, and E. faecalis 1915/05, measured as CFU/mL at the initial (To) and end (T_%) of the turbidity reduction assay. Cell viability was determined by serially diluting and plating the same cell suspensions used in the reduction turbidity assay in the absence (negative control, C-) and presence of 5μg/mL of Lysl 68 and Lysl 70. This test proved that during the lytic activity assay, the cells remained viable. It also showed that both Lysl 68 and Lys l 70 exhibited lower activity against the phage host strains, E. faecalis 926/05 and E. faecalis 1518/05, respectively, compared with previous results (FIG. 1 ). Moreover, Lys l 68 showed greater lytic activity than Lysl 70 against E. faecalis 1915/05.

[001611 Overall results lead to the conclusion that Lysl 68, Lysl 70, and Lysl 70-168 exhibit antibacterial activity towards numerous Enterococcus sp. In addition, Lysl 70-168 appeared to show increased lytic spectrum compared to the native lysins.

6.6 Evalulation of Lysin Activity in vivo

[00162] Lysin activity of lysins Lysl 68 and Lysl 70 was tested in Wister rats, previously infected with 1.5xl 0'cells of E. faecalis 1915/05. 30min after the lysins were administered, their activity was measured as CFU/mL of E. faecalis 1915/05 occurring in the heart and blood of the rats (FIGs. 4-6).

100163] Specifically, FIG. 4 shows a therapeutic evaluation for Lysl 68 and Lysl 70 in the hearts of 3 female Wistar rats, where buffer was used as a negative control and the treatment was carried out after 24 hours of heart infection. FIG. 5 shows a therapeutic evaluation of Lysl 68 and Lysl 70 in the blood of 3 female Wistar rats, where buffer was used as negative control and the treatment was carried out after 24 hours of heart infection. FIG. 6 shows a therapeutic evaluation of Lysl 68 and Lysl 70 in the hearts of 1 male and 3 female Wistar rats, where the male heart and buffer were used as negative controls and the treatment was carried out after 19-24 hours of heart infection.

[00164] Having described the invention with reference to particular compositions, method for detection, and source of activity, and proposals of effectiveness, and the like, it will be apparent to those of skill in the art that it is not intended that the invention be limited by such illustrative embodiments or mechanisms, and that modifications can be made without departing from the scope or spirit of the invention, as defined by the appended claims. It is intended that all such obvious modifications and variations be included within the scope of the present invention as defined in the appended claims. It should be understood that any of the above described one or more elements from any embodiment can be combined with any one or more element in any other embodiment. Moreover, when a range is mentioned, it should be understood that it is contemplated that any real number that falls within the range is a contemplated end point. For example, if a range of 0.9 and 1. 1 g kg is given, it is contemplated that any real number value that falls within that range ( for example, 0.954 to 1.052 g/kg) is contemplated as a subgenus range of the invention, even if those values are not explicitly mentioned. All references referred to herein are incorporated by reference in their entireties. Finally, the above description is not to be construed to limit the invention but the invention should rather be defined by the below claims.

Claims

WHAT IS CLAIMED:

1. A chimeric polypeptide comprising a sequence corresponding to a catalytic domain of a first Enterococcal bacteriophage endolysin and a sequence corresponding to a catalytic domain of a second Enterococcal bacteriophage endolysin, wherein said chimeric polypeptide shows increased lytic performance towards Enterococcus bacteria compared to said first and/or said second Enterococcal bacteriophage endolysin.

2. The chimeric polypeptide of claim 1 , wherein said catalytic domains of said first and said second Enterococcal bacteriophage endolysins are each independently selected from an amidase-2 domain and a CHAP domain.

3. The chimeric polypeptide of claim 1 or 2, wherein said catalytic domain of said first Enterococcal bacteriophage endolysin corresponds to an amidase-2 domain and said catalytic domain of said second Enterococcal bacteriophage endolysin corresponds to a CHAP domain.

4. The chimeric polypeptide of any of claims 1 to 3, wherein said increased lytic performance comprises an increased spectrum of activity against Enterococcus bacteria species and/or strains.

5. The chimeric polypeptide of any of claims 1 to 3, wherein said increased lytic performance comprises increased ability to kill and/or inhibit the growth or reproduction of Enterococcus bacteria.

6. The chimeric polypeptide of any of claims 1 to 5, wherein said first and second endolysins correspond to endolysins from the same bacteriophage.

7. The chimeric polypeptide of any of claims 1 to 5, wherein said first and second endolysins correspond to endolysins from different bacteriophages.

8. The chimeric polypeptide of claim 7, wherein said first and second endolysins correspond to endolysins from different bacteriophages, said different bacteriophages having bacterial hosts of the same bacterial species.

9. The chimeric polypeptide of any of claims 1 to 8, wherein said bacterial species is E, faecalis or E. faecium.

10. The chimeric polypeptide of claim 7. wherein said first and second endolysins correspond to endolysins from different bacteriophages, said different bacteriophages having bacterial hosts of the same bacterial strain.

1 1. The chimeric polypeptide of claim 7, wherein said first and second endolysins correspond to endolysins from different bacteriophages, said different bacteriophages having bacterial hosts of different bacterial strains.

12. The chimeric polypeptide of claim 1 1 , wherein said first endolysin is from a bacteriophage having bacterial host E. faecalis 151 8/05 and said second endolysin is from a bacteriophage having bacterial host E. faecalis 926/05.

13. The chimeric polypeptide of any of claims 1 to 6 and 7 to 12, wherein said first endolysin is from bacteriophage F 1 70/08 and said second endolysin is from bacteriophage F 168/08.

14. The chimeric polypeptide of any of claims 1 to 13. wherein said first endolysin is Lys l 70 corresponding to SEQ ID NO: 1 .

15. The chimeric polypeptide of any of claims 1 to 14, wherein said second endolysin is Lysl 68 corresponding to SEQ ID NO: 3.

16. The chimeric polypeptide of any of claims 1 to 1 3. wherein said catalytic domain of said first endolysin comprises SEQ ID NO:5 or a fragment thereof having antimicrobial activity against E. faecalis.

17. The chimeric polypeptide of any of claims 1 to 13, wherein said catalytic domain of said first endolysin comprises a first peptide having at least 80% sequence identity to a second peptide of the same size, wherein said first peptide has antimicrobial activity against E. faecalis and said second peptide has the amino acid sequence of SEQ ID NO:5 or a fragment thereof.

18. The chimeric polypeptide of claim 17, wherein said first peptide has at least 85% sequence identity to said second peptide.

19. The chimeric polypeptide of claim 17, wherein said first peptide has at least 90% sequence identity to said second peptide.

20. The chimeric polypeptide of claim 17. wherein said first peptide has at least 95% sequence identity to said second peptide.

21. The chimeric polypeptide of claim 1, wherein said catalytic domain of said second endolysin comprises SEQ ID NO: 7 of a fragment thereof having antimicrobial activity against E. faecalis.

22. The chimeric polypeptide of claim 21, wherein said catalytic domain of said second endolysin comprises a third peptide having at least 80% sequence identity to a fourth peptide of the same size, wherein said third peptide has antimicrobial activity against E. faecalis and said fourth peptide has the amino acid sequence of SEQ ID NO:7 or a fragment thereof

23. The chimeric polypeptide of claim 22, wherein said third peptide has at least 85% sequence identity to said fourth peptide.

24. The chimeric polypeptide of claim 22, wherein said third peptide has at least 90% sequence identity to said fourth peptide.

25. The chimeric polypeptide of claim 22, wherein said third peptide has at least 95% sequence identity to said fourth peptide.

26. The chimeric polypeptide of claim 1 , wherein said chimeric polypeptide comprises SEQ ID NO: 9 or a fragment thereof having antimicrobial activity against E. faecalis.

27. A chimeric first polypeptide having at least 80% sequence identity to a second polypeptide of the same size wherein said first polypeptide has antimicrobial activity against E. faecalis and said second polypeptide has the amino acid sequence of SEQ ID NO:9 or a fragment thereof.

28. The chimeric polypeptide of claim 27 wherein said first polypeptide has at least 85% sequence identity to said second polypeptide.

29. The chimeric polypeptide of claim 27 wherein said first polypeptide has at least 90% sequence identity to said second polypeptide.

30. The chimeric polypeptide of claim 27 wherein said first polypeptide has at least 95% sequence identity to said second polypeptide.

3 1. An isolated lysin peptide from phage F 168/08, said lysin peptide comprising the amino acid sequence of SEQ ID NO:7 or a fragment thereof having antimicrobial activity against

E. faecalis.

32. An isolated fifth lysin peptide having a least 80% sequence identity to a sixth lysin peptide of the same size, wherein said fifth lysin peptide has antimicrobial activity against E. faecalis and said sixth lysin peptide has the amino acid sequence of SEQ ID NO:7 or a fragment thereof.

33. The isolated fifth lysin peptide of claim 32, wherein said fifth lysin peptide has at least 85% sequence identity to said sixth lysin domain.

34. The isolated fifth lysin peptide of claim 32, wherein said fifht lysin peptide has at least 90%) sequence identity to said sixth lysin peptide.

35. The isolated fifth lysin peptide of claim 32, wherein said fifth lysin peptide has at least 95% sequence identity to said sixth lysin peptide.

36. A nucleic acid comprising a nucleotide sequence encoding the chimeric polypeptide or lysin protein of any one of claims 1 -35.

37. The nucleic acid of claim 36 comprising the nucleotide sequence corresponding to SEQ ID NO: 8 or a fragment thereof.

38. The nucleic acid of claim 36 comprising the nucleotide sequence

corresponding to SEQ ID NO: 10 or a fragment thereof.

39. A vector comprising the nucleic acid of claim 36.

40. The vector of claim 39 that is an expression vector.

41. A host cell comprising the vector of claim 40.

42. A pharmaceutical composition comprising and a pharmaceutically acceptable carrier and the chimeric polypeptide or lysin peptide of any one of claims 1 -35.

43. A method for treating a bacterial infection in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition of claim 42.

44. The method of claim 43, wherein said bacterial infection is an infection by a Gram-positive bacteria.

45. The method of claim 44, wherein said Gram-positive bacteria is an

Enterococcus, Enter ococcus faecalis, Enterococcus faecium, Staphylococcus aureus.

Staphylococcus haemolyticus, Staphylococcus epidermidis. Bacilus subtilis, Bacilus licheniformis, Streptococcus grupo. Micrococcus luteus, and/or Escherichia coli.

46. The method of claim 45, wherein said Gram-positive bacteria is an

Enterococcus.

47. The method of claim 46, wherein said Gram-positive bacteria is E. faecalis.

48. A method of screening peptides for antibiotic activity, said method comprising screening sequences of contiguous amino acids at least 6 residues in length from SEQ ID NO:7, or 9 for antimicrobial activity against liquid cultures of E. faecalis.

49. The method of claim 48 wherein said sequences of contiguous amino acids are at least 10 residues in length.

50. A method for recombinantly producing the lysin peptide or chimeric polypeptide of any one of claims 1 -35. said method comprising: (i) constructing a nucleic acid encoding said peptide or polypeptide; (ii) culturing in a medium a host cell comprising said nucleic acid, under conditions suitable for the expression of said peptide or polypeptide; and (iii) recovering said peptide or polypeptide from said medium.