REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

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FIELD OF THE INVENTION

The present invention relates generally to prevention, control, disruption and treatment of bacterial biofilms with lysin, particularly lysin having capability to kill Staphylococcal bacteria, including drug resistantStaphylococcus aureus, particularly the lysin PlySs2. The invention also relates to compositions and methods for modulation of bacterial biofilm(s) and biofilm formation.

BACKGROUND OF THE INVENTION

The development of drug resistant bacteria is a major problem in medicine as more antibiotics are used for a wide variety of illnesses and other conditions. The use of more antibiotics and the number of bacteria showing resistance has prompted longer treatment times. Furthermore, broad, non-specific antibiotics, some of which have detrimental effects on the patient, are now being used more frequently. A related problem with this increased use is that many antibiotics do not penetrate mucus linings easily.

Gram-positive bacteria are surrounded by a cell wall containing polypeptides and polysaccharide. Gram-positive bacteria include but are not limited to the generaActinomyces, Bacillus, Listeria, Lactococcus, Staphylococcus, Streptococcus, Enterococcus, Mycobacterium, Corynebacterium, andClostridium. Medically relevant species includeStreptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, andEnterococcus faecalis. Bacillusspecies, which are spore-forming, cause anthrax and gastroenteritis. Spore-formingClostridiumspecies are responsible for botulism, tetanus, gas gangrene and pseudomembranous colitis.Corynebacteriumspecies cause diphtheria, andListeriaspecies cause meningitis.

Novel antimicrobial therapy approaches include enzyme-based antibiotics (“enzybiotics”) such as bacteriophage lysins. Phages use these lysins to digest the cell wall of their bacterial hosts, releasing viral progeny through hypotonic lysis. A similar outcome results when purified, recombinant lysins are added externally to Gram-positive bacteria. The high lethal activity of lysins against gram-positive pathogens makes them attractive candidates for development as therapeutics (Fischetti, V. A. (2008) Curr Opinion Microbiol 11:393-400; Nelson, D. L. et al (2001) Proc Natl Acad Sci USA 98:4107-4112). Bacteriophage lysins were initially proposed for eradicating the nasopharyngeal carriage of pathogenic streptococci (Loeffler, J. M. et al (2001) Science 294: 2170-2172; Nelson, D. et al (2001) Proc Natl Acad Sci USA 98:4107-4112). Lysins are part of the lytic mechanism used by double stranded DNA (dsDNA) phage to coordinate host lysis with completion of viral assembly (Wang, I. N. et al (2000) Annu Rev Microbiol 54:799-825). Lysins are peptidoglycan hydrolases that break bonds in the bacterial wall, rapidly hydrolyzing covalent bonds essential for peptidoglycan integrity, causing bacterial lysis and concomitant progeny phage release.

Lysin family members exhibit a modular design in which a catalytic domain is fused to a specificity or binding domain (Lopez, R. et al (1997) Microb Drug Resist 3:199-211). Lysins can be cloned from viral prophage sequences within bacterial genomes and used for treatment (Beres, S. B. et al (2007) PLoS ONE 2(8):1-14). When added externally, lysins are able to access the bonds of a Gram-positive cell wall (Fischetti, V. A. (2008) Curr Opinion Microbiol 11:393-400). Bacteriophage lytic enzymes have been established as useful in the assessment and specific treatment of various types of infection in subjects through various routes of administration. For example, U.S. Pat. No. 5,604,109 (Fischetti et al.) relates to the rapid detection of Group A streptococci in clinical specimens, through the enzymatic digestion by a semi-purified Group C streptococcal phage associated lysin enzyme. This enzyme work became the basis of additional research, leading to methods of treating diseases. Fischetti and Loomis patents (U.S. Pat. Nos. 5,985,271, 6,017,528 and 6,056,955) disclose the use of a lysin enzyme produced by group C streptococcal bacteria infected with a C1 bacteriophage. U.S. Pat. No. 6,248,324 (Fischetti and Loomis) discloses a composition for dermatological infections by the use of a lytic enzyme in a carrier suitable for topical application to dermal tissues. U.S. Pat. No. 6,254,866 (Fischetti and Loomis) discloses a method for treatment of bacterial infections of the digestive tract which comprises administering a lytic enzyme specific for the infecting bacteria. The carrier for delivering at least one lytic enzyme to the digestive tract is selected from the group consisting of suppository enemas, syrups, or enteric coated pills. U.S. Pat. No. 6,264,945 (Fischetti and Loomis) discloses a method and composition for the treatment of bacterial infections by the parenteral introduction (intramuscularly, subcutaneously, or intravenously) of at least one lytic enzyme produced by a bacteria infected with a bacteriophage specific for that bacteria and an appropriate carrier for delivering the lytic enzyme into a patient.

Phage associated lytic enzymes have been identified and cloned from various bacteriophages, each shown to be effective in killing specific bacterial strains. U.S. Pat. Nos. 7,402,309, 7,638,600 and published PCT Application WO2008/018854 provides distinct phage-associated lytic enzymes useful as antibacterial agents for treatment or reduction ofBacillus anthracisinfections. U.S. Pat. No. 7,569,223 describes lytic enzymes forStreptococcus pneumoniae. Lysin useful forEnterococcus(E. faecalisandE. faecium, including vancomycin resistant strains) are described in U.S. Pat. No. 7,582,291. US 2008/0221035 describes mutant Ply GBS lysins highly effective in killing Group B streptococci. A chimeric lysin denoted ClyS, with activity against Staphylococci bacteria, includingStaphylococcus aureus, is detailed in WO 2010/002959. ClyS is specific for Staphylococcal bacteria and is inactive againstStreptococcusand other gram positive bacteria.

Based on their rapid, potent, and specific cell wall-degradation and bactericidal properties, lysins have been suggested as antimicrobial therapeutics to combat Gram-positive pathogens by attacking the exposed peptidoglycan cell walls from outside the cell (Fenton, M et al (2010) Bioengineered Bugs 1:9-16; Nelson, D et al (2001) Proc Natl Acad Sci USA 98:4107-4112). Efficacies of various lysins as a single agents have been demonstrated in rodent models of pharyngitis (Nelson, D et al (2001) Proc Natl Acad Sci USA 98:4107-4112), pneumonia (Witzenrath, M et al (2009) Crit Care Med 37:642-649), otitis media (McCullers, J. A. et al (2007) PLOS pathogens 3:0001-0003), abscesses (Pastagia, M et al Antimicrobial agents and chemotherapy 55:738-744) bacteremia (Loeffler, J. M. et al (2003) Infection and Immunity 71:6199-6204), endocarditis (Entenza, J. M. et al (2005) Antimicrobial agents and chemotherapy 49:4789-4792), and meningitis (Grandgirard, D et al (2008) J Infect Dis 197:1519-1522). In addition, lysins are generally specific for their bacterial host species and do not lyse non-target organisms, including human commensal bacteria which may be beneficial to gastrointestinal homeostasis (Blaser, M. (2011) Nature 476:393-394; Willing, B. P. et al (2011) Nature reviews. Microbiology 9:233-243)

Microorganisms tend to form surface-attached biofilm communities as an important survival strategy in different environments. Biofilms consist of microbial cells and a wide range of self-generated extracellular polymeric substances, including polysaccharides, nucleic acids, and proteins (Flemming H C et al (2007) J Bacteriol 189:7945-7947). Biofilms are found in natural and industrial aquatic environments, tissues, and medical materials and devices (Costerton J W et al (1994) J Bacteriol 176:2137-2142). Biofilms can be formed by a single bacterial strain, although most natural biofilms are formed by multiple bacterial species (Yang L et al (2011) Int J Oral Sci 3:74-81). Applications of antibiotics are often ineffective for biofilm populations due to their unique physiology and physical matrix barrier.

Staphylococci often form biofilms, sessile communities encased in an extracellular matrix that adhere to biomedical implants or damaged and healthy tissue. Infections associated with biofilms are difficult to treat, and it is estimated that sessile bacteria in biofilms are 1,000 to 1,500 times more resistant to antibiotics than their planktonic counterparts. This antibiotic resistance of biofilms often leads to the failure of conventional antibiotic therapy and necessitates the removal of infected devices. Lysostaphin has been shown to killS. aureusin biofilms and also disrupted the extracellular matrix ofS. aureusbiofilms in vitro on plastic and glass surfaces (Wu, J A et al (2003) Antimicrob Agents and Chemoth 47(11):3407-3414). This disruption ofS. aureusbiofilms was specific for lysostaphin-sensitiveS. aureus, and biofilms of lysostaphin-resistantS. aureuswere not affected. High concentrations of oxacillin (400 ag/ml), vancomycin (800 tag/ml), and clindamycin (800 ag/ml) had no effect on the establishedS. aureusbiofilms, even after 24 h. Lysostaphin also disruptedS. epidermidisbiofilms, however, higher concentrations were required. Application of phage lysins for the removal of staphylococcal biofilms have been reported, with mixed results. Bacteriophage lysin SAL-2 was reported to removeS. aureusbiofilms (Son J S et al (2010) Appl Microbiol Biotechnol 86(5):1439-1449), while in the case of two similar phage lysins, phi11 and phi12, while phi11 hydrolyzed staphylococcal biofilms, phi12 was inactive (Sass P and Bierbaum G (2007) Appl Environ Microbiol 73(1):347-352). Various combinations of enzymes have been studied for the removal and disinfection of bacterial biofilms in various systems (Johansen C et al (1997) Appl Environ Microbiol 63:3724-3728). This process, however, requires a minimum of two enzymes or agents, one enzyme or agent for removal of the adherent bacteria of the biofilms and a second enzyme or agent with bactericidal activity.

It is evident from the deficiencies and problems associated with current traditional antibacterial agents that there still exists a need in the art for additional specific bacterial agents and therapeutic modalities and also for broader spectrum agents, particularly without risks of acquired resistance, for the effective and efficient treatment, control and prevention of bacterial biofilms. It is notable that to date, no lysin demonstrating lytic activity against multiple distinct species of pathogenic and clinically relevant gram positive bacteria, which is readily manufacturable and stable, and has no or limited risk of resistance, has been shown to be effective on biofilms. Accordingly, there is a commercial need for new antibacterial approaches, especially those that operate via new modalities or provide new means to kill pathogenic bacteria in biofilms.

The citation of references herein shall not be construed as an admission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods are provided for the prevention, disruption and treatment of bacterial biofilms. In its broadest aspect, the present invention provides use and application of a lysin having broad killing activity against multiple bacteria, particularly Gram-positive bacteria, including particularlyStaphylococcus, Streptococcus, particularlyStreptococcus pyogenes(Group A strep) andStreptococcus agalactiae(Group B strep) bacterial strains, in the prevention, disruption and treatment of biofilms. The lysin and compositions of the invention are useful and applicable in killingEnterococcusandListeriabacterial strains, and in applicable biofilms thereof. The invention provides a method for decolonizing, dispersing and removal of bacterial biofilm utilizing bacteriophage lysin capable of killing bacteria effectively and efficiently in a biofilm. The invention thus contemplates treatment, decolonization, and/or decontamination of bacterial biofilms and the prevention of infections after dispersion of biofilm(s) wherein one or more gram positive bacteria, particularly one or more ofStaphylococcus, Streptococcus, EnterococcusandListeriabacteria, is suspected or present.

In accordance with the present invention, bacteriophage lysin derived fromStreptococcus suisbacteria are utilized in the methods and applications of the invention. The lysin polypeptide(s) of use in the present invention, particularly PlySs2 lysin as provided herein and inFIG.5(SEQ ID NO: 1), are unique in demonstrating broad killing activity against multiple bacteria, particularly gram-positive bacteria, includingStaphylococcus, Streptococcus, EnterococcusandListeriabacterial strains. In one such aspect, the PlySs2 lysin is capable of killingStaphylococcus aureusstrains and bacteria in biofilms, as demonstrated herein. PlySs2 is effective against antibiotic-resistant bacteria, includingStaphylococcus aureussuch as methicillin-resistantStaphylococcus aureus(MRSA), vancomycin resistantStaphylococcus aureus(VRSA), daptomycin-resistantStaphylococcus aureus(DRSA) and linezolid-resistantStaphylococcus aureus(LRSA). PlySs2 is effective against bacteria with altered antibiotic sensitivity such as vancomycin intermediate-sensitivityStaphylococcus aureus(VISA).

In an aspect of the invention, a method is provided of killing gram-positive bacteria in a biofilm comprising the step of contacting the biofilm with a composition comprising an amount of an isolated lysin polypeptide effective to kill gram-positive bacteria in a biofilm, includingS. aureus, the isolated lysin polypeptide comprising the PlySs2 lysin polypeptide or variants thereof effective to kill gram-positive bacteria. Thus, a method is provided of killing gram-positive bacteria in a biofilm comprising the step of contacting the biofilm with a composition comprising an amount of an isolated lysin polypeptide effective to kill the gram-positive bacteria in the biofilm, the isolated lysin polypeptide comprising the amino acid sequence provided inFIG.5or SEQ ID NO: 1 or variants thereof having at least 80% identity, 85% identity, 90% identity, 95% identity or 99% identity to the polypeptide ofFIG.5or SEQ ID NO: 1 and effective to kill the gram-positive bacteria in the biofilm.

In an aspect of the invention, a method is provided of dispersing gram-positive bacteria in a biofilm so as to decontaminate and to release bacteria then susceptible to antibiotics, comprising the step of contacting the biofilm with a composition comprising an amount of an isolated lysin polypeptide effective to disperse gram-positive bacteria in a biofilm, includingS. aureus, the isolated lysin polypeptide comprising the PlySs2 lysin polypeptide, including as set out inFIG.5or SEQ ID NO: 1 or variants thereof effective to kill gram-positive bacteria.

In an aspect of the above methods, the methods are performed in vitro or ex vivo so as to sterilize or decontaminate a solution, material or device, particularly intended for use by or in a human.

The invention provides a method for reducing a population of gram-positive bacteria in a biofilm comprising the step of contacting the biofilm with a composition comprising an amount of an isolated polypeptide effective to kill or release at least a portion of the gram-positive bacteria in the biofilm, the isolated polypeptide comprising the amino acid sequence ofFIG.5(SEQ ID NO: 1) or variants thereof having at least 80% identity to the polypeptide ofFIG.5(SEQ ID NO: 1) and effective to kill the gram-positive bacteria.

The present invention further provides a method for dispersing or treating an antibiotic-resistantStaphylococcus aureusinfection which involves or includes a biofilm in a human comprising the step of administering to a human with an antibiotic-resistantStaphylococcus aureusbiofilm infection, an effective amount of a composition comprising an isolated polypeptide comprising the amino acid sequence ofFIG.5(SEQ ID NO: 1) or variants thereof having at least 80% identity, 85% identity, 90% identity or 95% identity to the polypeptide ofFIG.5(SEQ ID NO: 1) and effective to disperse the biofilm and killStaphylococcus aureustherein and//or released therefrom, whereby the number ofStaphylococcus aureusin the human is reduced and the biofilm and attendant infection is controlled.

A method of the invention also includes a method for preventing, dispersing or treating a gram-positive bacterial biofilm comprising one or more ofStaphylococcusor Streptococcusbacteria in a human comprising the step of administering to a subject having or suspected of having or at risk of a bacterial biofilm, an effective amount of a composition comprising an isolated polypeptide comprising the amino acid sequence ofFIG.5(SEQ ID NO: 1) or variants thereof having at least 80% identity, 85% identity, 90% identity or 95% identity to the polypeptide ofFIG.5(SEQ ID NO: 1) and effective to kill the gram-positive bacteria, whereby the number of gram-positive bacteria in the human is reduced and the biofilm contamination or infection is controlled. In an aspect of the method, biofilm comprising or including one or more of anEnterococcusorListeriabacteria is effectively prevented, dispersed or treated. In a particular aspect of this method, wherein the subject is exposed to or at risk of one of or one or more ofStaphylococcus(such asStaphylococcus aureus),Streptococcus(particularly Group A strep or Group B strep such asStreptococcus pyogenesorStreptococcus agalactiae, respectively)bacteria. An alternative bacteria such asListeria(such asL. monocytogenes) orEnterococcus(such asE. faecalis) bacteria may also be involved and addressed, prevented, dispersed or treated in accordance with the methods and compositions of the invention. The subject may be a human. The subject may be a human adult, child, infant or fetus.

In any such above method or methods, the susceptible, killed, dispersed or treated biofilm bacteria may be selected fromStaphylococcus aureus, Listeria monocytogenes, Staphylococcus simulans, Streptococcus suis, Staphylococcus epidermidis, Streptococcus equi, Streptococcus equizoo,Streptococcus agalactiae(GBS),Streptococcus pyogenes(GAS),Streptococcus sanguinis, Streptococcus gordonii, Streptococcus dysgalactiae, Group GStreptococcus, Group EStreptococcus, Enterococcus faecalisandStreptococcus pneumonia.

In accordance with any of the methods of the invention, the susceptible bacteria or biofilm bacteria may be an antibiotic resistant bacteria. The bacteria may be antibiotic resistant, including methicillin-resistantStaphylococcus aureus(MRSA), vancomycin resistantStaphylococcus aureus(VRSA), daptomycin-resistantStaphylococcus aureus(DRSA), or linezolid-resistantStaphylococcus aureus(LRSA). The bacteria may have altered antibiotic sensitivity, such as for example, vancomycin intermediate-sensitivityStaphylococcus aureus(VISA), The susceptible bacteria may be a clinically relevant or pathogenic bacteria, particularly for humans. In an aspect of the method(s), the lysin polypeptide(s) is effective to killStaphylococcus, Streptococcus, EnterococcusandListeriabacterial strains.

It has been shown that coating medical implants with antimicrobials may effectively prevent the initial adherence of staphylococcal biofilms to the implants. Coating biomedical materials with lysin may also prove successful in preventing early adherence of bacteria, including staphylococci, to the implants, thus averting biofilm formation. The present invention thus also provides methods for reducing or preventing biofilm growth on the surface of devices, implants, separation membranes (for example, pervaporation, dialysis, reverse osmosis, ultrafiltration, and microfiltration membranes) by administering or coating with the lysin of the invention, including PlySs2 lysin.

Alternative active and suitable lysin(s) may be utilized in accordance with the methods and compositions of the present invention, including as the lysin(s) of use and/or as one or more additional effective and useful lysins. In an additional aspect or embodiment of the methods and uses provided herein, the staphylococcal specific lysin ClyS is used herein alone or in combination with the PlySs2 lysin as provided and described herein.

Other objects and advantages will become apparent to those skilled in the art from a review of the following description which proceeds with reference to the following illustrative drawings.

