# Patent

## Title
Antimicrobial compositions comprising a hyaluronic acid binding peptide and a β-lactam antibiotic

## Abstract

Compositions and methods for the treatment of antibiotic resistant Staphylococcus aureus infections are provided comprising β-lactam antibiotics, as well as a hyaluronic acid binding peptide. The antibiotics include cephalosporins as well as the ηoη-β lactam antibiotic vancomycin. The methods provide administering the composition to the subject in an amount effective to reduce or eliminate the infection.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. national stage of International Application No. PCT/CA2013/050346, filed May 3, 2013, which claims the benefit of and priority to U.S. Provisional Application no. 61/642,721, filed on May 4, 2012 under the title ANTIMICROBIAL COMPOSITIONS. The content of each of the above patent applications is hereby expressly incorporated by reference into the detailed description hereof.


DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: EMER_049_01US_SubSeqList.txt, date recorded: Mar. 27, 2015, file size 3 kilobytes).
FIELD OF THE INVENTION
The invention relates to new compositions which comprise selective antibiotics and a peptide. The invention further relates to a method of potentiating the antimicrobial effectiveness of antibiotics for the treatment of bacterial infections in a vertebrate subject. Methods are provided for administering the composition to the vertebrate subject in an amount effective to reduce or eliminate infection or further limit opportunities for antibiotic resistant phenotypes. Methods for the treatment or prevention of Staphylococcus aureus infection in an organism are provided.
BACKGROUND OF THE INVENTION
Staphylococcus aureus (S. aureus) is a ubiquitous gram positive bacterium that can colonize the nares and skin of humans without causing disease. Approximately one third of the human population is colonized with S. aureus making it difficult to avoid transmittance. The bacteria can cause a wide variety of disease from mild skin infections to more serious diseases such as bacteremia and endocarditis. The patient populations most at risk are dialysis patients, patients with ventriculoperitoneal shunts, patients at risk of infective endocarditis, patients who are immunocompromised, and residents of nursing homes. In healthcare settings it is the main pathogen responsible for infections of the skin and soft tissues, as well as for those associated with medical procedures and indwelling devices such as catheters. Since catheter- and device-related infections remain the most significant cause of morbidity, prolonged length of stay and increased cost in affected patients, S. aureus infections are of concern. S. aureus has developed resistance to multiple antibiotics and has a methicillin-resistant variant (MRSA) which is becoming widespread in the community and nosocomial environments. This is leading to increased incidences of infection in both the hospital and community settings. With reduced treatment options available, alternative approaches are required.
S. aureus is an important cause of serious infections in both hospitals and community. Unfortunately, this pathogen has the ability to quickly respond to each new antibiotic and has been particularly efficient at developing resistance mechanisms. In 1942, two years after the introduction of penicillin for medical use, the first penicillin-resistant S. aureus isolate was observed in a hospital. Since 1960, around 80% of all S. aureus strains are resistant to penicillin. In 1961, two years after the introduction of methicillin, a penicillinase-resistant penicillin, S. aureus, developed methicillin-resistance due to the acquisition of the mecA gene. During the last several decades, various methicillin-resistant S. aureus (MRSA) clones disseminated worldwide become a global health threat and a significant challenge for healthcare systems. Since the widespread emergency of MRSA, vancomycin has represented the cornerstone of therapy for MRSA infections. Over the last decade, strains that are not susceptible to vancomycin have occurred, showing either intermediate resistance (VISA) or, worse, full resistance to this antibiotic (VRSA).
Currently, the rapid emergence of bacteria resistant to commonly used antibiotics has become a serious problem and one of the major challenges for the healthcare systems worldwide. Antibiotic resistant infections are associated with higher treatment cost, longer hospital stay and a 1.3 to 2-fold increase in mortality. The resistant bacteria also spread to community and become broader infection-control problems, since some community-associated resistant strains are more virulent due to the production of virulent factors like toxins.
Despite the urgent need for effective agents to overcome the bacterial resistance, the antibiotic drug discovery and development has slowed considerably in recent years. Traditional approaches and the newer genomic mining approaches have not yielded novel classes of antibacterial compounds. An alternative strategy is to improve analogues of existing classes of antibacterial drugs by modifying the action sites or combining with other compounds to improve the potency and minimize the resistance.
Methicillin resistant bacteria typically exhibit resistance to all, or at least most, β-lactam antibiotics, including penicillins, cephalosporins and carbapenems. β-lactam antibiotics target the transpeptidase activity of penicillin-binding proteins (PBPs) involved in cell wall biosynthesis. The peptidoglycan layer in the bacterial cell wall is a crystallattice structure formed from linear chains of two alternating amino sugars, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Each MurNac is attached to a short amino acid chain, containing L-alanine, D-glutamic acid, L-lysine, and D-alanyl-D-alanine in the case of S. aureus. Cross-linking between amino acids in different linear amino sugar occurs with the help of the enzyme transpeptidases and results in a 3-dimensional structure that is strong and rigid. β-lactams contain the highly reactive CO—N bond in the β-lactam ring, which lie in exactly the same position as the CO—N bond in D-alanyl-D-alanine, resulting in nearly identical conformation of the terminal portion of the peptidoglycan peptide chain, which is the target of transpeptidation. The binding of β-lactams to transpeptidase enzymes (also known as penicillin-binding proteins, PBPs) results in acylation of a serine residue in the active site of PBP, this irreversible reaction inactivates the enzyme and prevents the final cross linking (transpeptidation) of the nascent peptidoglycan layer, disrupting cell wall synthesis.
Methicillin sensitive S. aureus (MSSA) express four naïve penicillin-binding enzymes (PBP1, PBP2, PBP3, and PBP4), and their activities are specifically prevented by the covalent binding of β-lactam antibiotics to their active sites. The MRSA isolates acquired the mecA gene, coding for a novel 78 KDa penicillin-binding protein 2a (PBP2a). The crystal structure of PBP2a reveals it to have a closed active site, and the interactions of PBP2a with peptidoglycan at an allosteric site trigger a conformational change that leads to accessibility to the active site. PBP2a is not inhibited in the presence of β-lactams due to the blockage of access to the active site, and is able to take over the peptidoglycan biosynthesis from the naïve PBPs to perform the critical cell wall cross-linking reaction.
Vancomycin is a glycopeptide antibiotic that is able to form hydrogen bond interactions with the terminal D-alanyl-D-alanine moieties of the peptidoglycan (murein) monomer. The binding of vancomycin to the D-Ala-D-Ala prevents cell wall synthesis in two ways. It can completely or partially inhibit the peptidoglycan polymerization if it binds to murein monomers located in the cytoplasmic membrane. It also can target the D-Ala-D-Ala residues in the completed peptidoglycan layers or on the nascent peptidoglycan chain to prevent the cross-linking of the backbone polymers.
VISA and VRSA have emerged almost exclusively from MRSA, with few exceptions involving strains with hetero-resistance. Vancomycin resistance does not develop step-wise and VRSA does not progress from VISA, since VISA and VRSA have completely different resistance mechanisms.
The intermediate resistance in VISA has been associated to the presence of a thickened cell wall. The wall is rich in peptidoglycan chains that are not cross-linked and display free terminal D-Ala-D-Ala residues, which act as decoy targets, blocking vancomycin in the external layer of the cell wall and diverting the antibiotic from reaching its true target-peptidoglycan precursors at cytoplasmic membrane level. The cell wall is clogged by the trapped vancomycin and this further block the antibiotic penetration. No characteristic genetic trait has been tightly associated with VISA resistance.
Different from VISA, VRSA strains have acquired the complete genetic apparatus for glycopeptides resistance from vancomycin-resistant enterococci (VRE). VRSA strains acquired the vanA operon that confers high level resistance to glycopeptides, vancomycin and teicoplanin. The vanA operon contains an assembly of genes that encode the synthesis of modified peptidoglycan precursors containing a terminal D-Ala-D-Lac instead of D-Ala-D-Ala and the elimination of the susceptible wild-type targets. The D-alanyl-D-lactate variation results in the loss of one hydrogen-bonding interaction (4, as opposed to 5 for D-alanyl-D-alanine) possible between vancomycin and the peptide, which results in a 1000-fold decrease in affinity. The resistance mechanism is under the regulation of a two-component signal transduction system (gene vanS and vanR), which activates only in the presence of vancomycin.
Therefore, there is an unmet need for effective treatment and/or prevention of S. aureus associated infections that are resistance to current antibiotics, including MRSA, VISA, and VRSA.
SUMMARY OF THE INVENTION
The applicant has discovered that the antibiotic effect of certain antibiotics is synergistically potentiated when they are coadministered with hyaluronic acid binding peptides, specifically PEP35.
According to one aspect of the invention is provided a method for the prevention or treatment of antibiotic resistant S. Aureus infection or reinfection, comprising administering a therapeutically effective amount of a beta-lactam antibiotic and a hyaluronic acid binding peptide.
According to a further aspect of the present invention is the use of a hyaluronic acid binding peptide in the potentiation of a beta-lactam antibiotic against antibiotic resistant S. Aureus. 
According to yet a further aspect of the present invention is the use of a hyaluronic acid binding peptide and a beta-lactam antibiotic for the treatment or prevention of antibiotic resistant S. Aureus infection or reinfection.
According to yet a further aspect of the present invention is provided a composition comprising a beta-lactam antibiotic and a hyaluronic acid binding peptide.
In certain embodiments, the S. aureus is a methicillin resistant variant (MRSA), a vancomycin resistant variant (VRSA), or a vancomycin intermediate resistant variant (VISA).
In certain embodiments, the beta-lactam antibiotic and the hyaluronic acid binding peptide are administered simultaneously.
In certain embodiments, the beta-lactam antibiotic and the hyaluronic acid binding peptide are administered concurrently.
In certain embodiments, the beta-lactam antibiotic and the hyaluronic acid binding peptide are administered separately, for example, within 15 minutes of each other.
In certain embodiments, the beta-lactam antibiotic is a cephalosporin.
In certain embodiments, the cephalosporin is a first generation cephalosporin, for example, cefacetrile, Cefadroxil, Cephalexin, Cefaloglycin, Cefalonium, Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, Cefazaflur, Cefazedone, Cefazolin, Cefradine, Cefroxadine, or Ceftezole.
In certain embodiments, the cephalosporin is a second generation cephalosporin, for example, Cefaclor, Cefonicid, Cefprozil, Cefuroxime, Cefuzonam, Cefmetazole, Cefotetan, Cefoxitin Carbacephems or Cephamycins.
In certain embodiments, the cephalosporin is a third generation cephalosporin, for example, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefixime, Cefmenoxime, Cefodizime, Cefotaxime, Cefovecin, Cefpimizole, Cefpodoxime, Cefteram, Ceftibuten, Ceftiofur, Ceftiolene, Ceftizoxime, Ceftriaxone, Cefoperazone, Ceftazidime, or Oxacephems.
In certain embodiments, the cephalosporin is a fourth generation cephalosporin, for example, Cefclidine, Cefepime, Cefluprenam, Cefoselis, Cefozopran, Cefpirome, Cefquinome, or Oxacephems.
In certain embodiments, the cephalosporin is a fifth generation cephalosporin, for example, Ceftobiprole, medocaril, Ceftaroline, Cefaloram, Cefaparole, Cefcanel, Cefedrolor, Cefempidone, Cefetrizole, Cefivitril, Cefmatilen, Cefmepidium, Cefoxazole, Cefrotil, Cefsumide, Ceftaroline, Ceftioxide, or Cefuracetime.
In certain embodiments, the antibiotic is selected from methicillin, cefazolin, ceftriaxone, vancomycin, meropenem, and piperacillin/tazobactam.
In certain embodiments, the antibiotic is vancomycin.
In certain embodiments, the hyaluronic acid binding peptide is a PEP35 peptide, for example, a mouse PEP35 having an amino acid sequence represented by SEQ ID NO.: 1, or a human PEP35 having an amino acid sequence represented by SEQ ID NO.: 2, or a hybrid PEP35 having an amino acid sequence represented by SEQ ID NO.: 3, or a scrambled PEP35 having an amino acid sequence represented by SEQ ID NO.: 4.
BRIEF DESCRIPTION OF THE DRAWINGS
NONE


DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.
The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
“Vertebrate,” “mammal,” “subject,” “mammalian subject,” or “patient” are used interchangeably and refer to all vertebrates, e.g., mammals and non-mammals, such as mice, sheep, dogs, cows, avian species, ducks, geese, pigs, chickens, amphibians, and reptiles, for example, mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, cows, horses, goats, dogs, and primates.
The present invention generally relates to compositions and methods for the prevention or treatment of bacterial infection by S. aureus, in a vertebrate. Methods for inducing an immune response to S. aureus infection are provided. The methods provide administering an antibody or agent to subject in need thereof in an amount effective to reduce, eliminate, or prevent S. aureus bacterial infection or bacterial carriage.
“Bacterial carriage” is the process by which bacteria can thrive in a normal subject without causing the subject to get sick. Bacterial carriage is a very complex interaction of the environment, the host and the pathogen. Various factors dictate asymptomatic carriage versus disease. Therefore an aspect of the invention includes treating or preventing bacterial carriage.
“Treating” or “treatment” refers to either (i) the prevention of infection or reinfection, e.g., prophylaxis, or (ii) the reduction or elimination of symptoms of the disease of interest, e.g., therapy. “Treating” or “treatment” can refer to the administration of a composition comprising a polypeptide of interest, e.g., S. aureus alpha-hemolysin antigens or antibodies raised against these antigens. Treating a subject with the composition can prevent or reduce the risk of infection and/or induce an immune response to the polypeptide of interest. Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
“Preventing” or “prevention” refers to prophylactic administration or vaccination with polypeptide or antibody compositions.
“Therapeutically-effective amount” or “an amount effective to reduce or eliminate bacterial infection” or “an effective amount” refers to an amount of polypeptide or antibiotic that is sufficient to prevent S. aureus bacterial infection or to alleviate (e.g., mitigate, decrease, reduce) at least one of the symptoms associated with S. aureus bacterial infection or to induce an immune response to S. aureus alpha-hemolysin protein. It is not necessary that the administration of the composition eliminate the symptoms of S. aureus bacterial infection, as long as the benefits of administration of compound outweigh the detriments. Likewise, the terms “treat” and “treating” in reference to S. aureus bacterial infection, as used herein, are not intended to mean that the subject is necessarily cured of infection or that all clinical signs thereof are eliminated, only that some alleviation or improvement in the condition of the subject is effected by administration of the composition.
The term “amino acid” as used herein includes the twenty alpha-amino acids found in mammalian proteins, including both the L-isomeric and D-isomeric forms. The term also includes alternate amino acid residues, such as hydroxyproline, α-aminoisobutyric acid, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, 4-aminobutyric acid, and so forth, which can also be included in the peptide sequence in a completely analogous way. The D forms of the encoded amino acids and of alternate amino acids can, of course, also be employed. The manner of determining relative rate constants, of conducting syntheses, and of conducting selection and analysis is entirely analogous to that described below for the naturally-occurring amino acids. Accordingly, the results in terms of the number of rate constants required, the number of representative peptides in the mixture, etc., are also directly applicable to peptides that include as one, or more, or all residues, these nonencoded amino acids.
The following standard one letter and three letter abbreviations for the amino acid residues may be used throughout the specification: A, Ala-alanine; R, Arg-Arginine; N, Asn-Asparagine; D, Asp-Aspartic acid; C, Cys-Cysteine; Q, Gln-Glutamine; E, Glu-Glutamic acid; G, Gly-Glycine; H, His-Histidine; I, Ile-Isoleucine; L, Leu-Leucine; K, Lys-Lysine; M, Met-Methionine; F, Phe-Phenyalanine; P, Pro-Proline; S, Ser-Serine; T, Thr-Threonine; W, Trp-Tryptophan; Y, Tyr-Tyrosine; and V, Val-Valine.
PEP35 is an anti-infective antimicrobial agent, previously disclosed in U.S. Pat. No. 8,044,022. The sequence of PEP35 is based on the two hyaluronic acid binding domains (HABDs) of the murine HA-binding protein RHAMM (receptor hyaluronic acid-mediated motility). RHAMM, a cell surface receptor.
The HABDs of RHAMM contain B[X7]B motifs (where B is a basic amino acid arginine or lysine), which are shared with other hyaladherins such as CD44, and are believed to interact electrostatically with the negatively charged glucuronic acids of hyaluronic acid. PEP35 contains multiple B[X7]B motifs separated by a tri-valine linker.
PEP35 is a peptide comprised of 27 amino acids with a pI ̃12.1 and molecular weight of  ̃3240. It contains multiple positive charges at physiological pH, due to the presence of eight lysine residues as well as two arginines and one histidine residue in addition to an unmodified N-terminus. The C-terminal end is amidated to improve peptide half-life. The remaining residues on the peptide chain are hydrophobic resulting in an amphipathic peptide. Based on CD analysis, the peptide is capable of alpha-helix formation in polar environments. The sequence of PEP35 is: LKQKIKHVVKLKVVVKLRSQLVKRKQN (SEQ ID NO.:1).
Several variants of the PEP35 sequence were synthesized and tested. The equivalent peptide structure hPEP35 was based on the human RHAMM domain 1 and 2 linked by a tri-valine sequence. Conservative substitutions to the human peptide variant of PEP35 were also synthesized and tested (hybPEP35-1). Although all of these peptides are very similar in size and charge, surprising differences in efficacy were noted in various assays.
Peptide sequences are found below:














Peptides
Sequence









PEP35
LKQKIKHVVKLKVVVKLRSQLVKRKQN




(SEQ ID NO.: 1)



 



hPEP35
LKQKIKHVVKLKVVVKLRCQLAKKKQS




(SEQ ID NO.: 2)



 



hybPEP35-1
LKQKIKHVVKLKVVVKLRSQLAKKKQS




(SEQ ID NO.: 3)



 



Scrambled PEP35 
KKKKKLQLQLNLIKKKVQVSVVVVRRH



(PEP #5)
(SEQ ID NO.: 4)



 



PEP52
GAHWQFNALTVRGGGS 




(SEQ ID NO.: 5)










The sequence of PEP35 is based on the two hyaluronic acid binding domains (HABDs) of the mouse HA-binding protein RHAMM (receptor hyaluronic acid-mediated motility).















PEP35
Sequence
BX7Bs
Mass
pI
Charge







mouse
LKQKIKHVVKLKVVV
4
3239
12.1
10+ 0− =


10+
KLRSQLVKRKQN 







(SEQ ID NO.: 1)






 


human
LKQKIKHVVKLKVVV
4
3172
10.9
10+ 0− =


10+
KLRCQLAKKKQS 







(SEQ ID NO.: 2)






 


hybrid 1
LKQKIKHVVKLKVVV
4
3156
11.5
10+ 0− =


10+
KLRSQLAKKKQS







(SEQ ID NO.: 3)









The scrambled PEP35 (PEP#5, SEQ ID NO.:4) contains the same 27 a.a. as PEP35 (SEQ ID NO.:1) does, but reordered to not contain any BX7B motifs. It does, however, contain other BXB motifs, including: X1, X2, X3, X8 and X9. PEP35 has the potential to form a helix based on CD data. PEP35 has at least 6 uninterrupted hydrophobic residues on the wheel in a face of helix; while scrambled PEP35 has 5 uninterrupted hydrophobic residues in a face. This implies that PEP35 and scrambled PEP35 potentially have some degree of similar activities.
PEP52 (SEQ ID NO.: 5) is a HA inhibitor peptide designed by Mark E. Mummert (J. Exp. Med., vol 192, 2000). A 12-mer peptide (GAHWQFNALTVR (SEQ ID NO.: 6)) was developed by using phage display technology, and showed specific binding to soluble, immobilized, and cell-associated forms of HA. Mummert designed the synthetic peptide to include the spacer sequence G-G-G-S(SEQ ID NO.: 7) at the COOH terminus to mimic the original configuration of the peptide moieties fused to the pIII proteins and displayed on phage surfaces.
Cephalosporins are a class of β-lactam antibiotics originally derived from the fungi Acremonium, and are generally known in the art. They are structurally and pharmacologically related to the penicillins. Like the penicillins, cephalosporins have a beta-lactam ring structure that interferes with synthesis of the bacterial cell wall and so are bactericidal.
The cephalosporin nucleus can be modified to gain different properties. Cephalosporins are sometimes grouped into “generations” by their antimicrobial properties. The first cephalosporins were designated first generation, whereas later, more extended spectrum cephalosporins were classified as second-generation cephalosporins. Each newer generation of cephalosporins has significantly greater Gram-negative antimicrobial properties than the preceding generation, in most cases with decreased activity against Gram-positive organisms. Fourth-generation cephalosporins, however, have true broad spectrum activity.
Although first-generation cephalosporins are moderate spectrum agents, with a spectrum of activity of bacteria that includes penicillinase-producing, methicillin-susceptible staphylococci and streptococci, they are not the drugs of choice for such infections. They also have activity against some Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis, but have no activity against Bacteroides fragilis, enterococci, methicillin-resistant staphylococci, Pseudomonas, Acinetobacter, Enterobacter, indole-positive Proteus, or Serratia. First-generation cephalosporins include; Cefacetrile (cephacetrile), Cefadroxil (cefadroxyl; Duricef), Cephalexin (cephalexin; Keflex), Cefaloglycin (cephaloglycin), Cefalonium (cephalonium), Cefaloridine (cephaloradine), Cefalotin (cephalothin; Keflin), Cefapirin (cephapirin; Cefadryl), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (cephazolin; Ancef, Kefzol), Cefradine (cephradine; Velosef), Cefroxadine, Ceftezole.
The second-generation cephalosporins have a greater Gram-negative spectrum while retaining some activity against Gram-positive cocci. They are also more resistant to beta-lactamase. Second-generation cephalosporins include Cefaclor (Ceclor, Distaclor, Keflor, Raniclor), Cefonicid (Monocid), Cefprozil (cefproxil; Cefzil), Cefuroxime (Zinnat, Zinacef, Ceftin, Biofuroksym), Cefuzonam, Cefmetazole, Cefotetan, Cefoxitin Carbacephems (loracarbef, Lorabid) and Cephamycins.
Third-generation cephalosporins have a broad spectrum of activity and further increased activity against Gram-negative organisms. Some members of this group (in particular, those available in an oral formulation, and those with anti-pseudomonal activity) have decreased activity against Gram-positive organisms. Third-generation cephalosporins include Cefcapene, Cefdaloxime, Cefdinir (Omnicef, Kefnir), Cefditoren, Cefetamet, Cefixime (Suprax), Cefmenoxime, Cefodizime, Cefotaxime (Claforan), Cefovecin (Convenia), Cefpimizole, Cefpodoxime (Vantin, PECEF), Cefteram, Ceftibuten (Cedax), Ceftiofur, Ceftiolene, Ceftizoxime (Cefizox), Ceftriaxone (Rocephin), Cefoperazone (Cefobid), Ceftazidime (Fortum, Fortaz), Oxacephems (latamoxef, moxalactam).
Fourth-generation cephalosporins are extended-spectrum agents with similar activity against Gram-positive organisms as first-generation cephalosporins. They also have a greater resistance to beta-lactamases than the third-generation cephalosporins. Fourth-generation cephalosporins include Cefclidine, Cefepime (Maxipime), Cefluprenam, Cefoselis, Cefozopran, Cefpirome (Cefrom), Cefquinome, Oxacephems (flomoxef).
Cephalosporins that are described as “fifth generation” or yet to be classified include Ceftobiprole (and the soluble prodrug medocaril), Ceftaroline, Cefaloram, Cefaparole, Cefcanel, Cefedrolor, Cefempidone, Cefetrizole, Cefivitril, Cefmatilen, Cefmepidium, Cefoxazole, Cefrotil, Cefsumide, Ceftaroline, Ceftioxide, Cefuracetime.
EXAMPLES
Example 1
Minimum MIC of Four Peptides
Activity of the four peptides PEP35, scrambled PEP35, hybPEP35-1 and PEP-52 (SEQ ID NOs.: 1, 4, 3, and 5, respectively) were assessed against MRSA (both community-acquired, eg. USA400/CMRSA10, and healthcare-acquired eg USA100/800), obtained from the CANWARD study [(Canadian Journal of Infectious Diseases and Medical Microbiology 2009; 20 (Suppl A): 3-71)]. VISA (strains 92, 95 and 96), VRSA (strains 105 and 106) and E. coli (strain 69717) isolates were also tested. One MSSA was used as a control. All MICs were assessed using CLSI guidelines. Also assessed was the activity of cefazolin, ceftriazone, meropenem, and a piperacillin/tazobactam combination antibiotic. Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after incubation.
Antimicrobial Susceptibility Testing: A custom designed panel of antimicrobials was created. The panel included: PEP35, penicillin, piperacillin/tazobactam, cefazolin, ceftriaxone, meropenem, clarithromycin, clindamycin, gentamicin, amikacin, vancomycin, ciprofloxacin, levofloxacin, moxifloxacin, linezolid, trimethoprim/sulfamethoxazole and doxycycline. These agents were obtained as laboratory grade powders from their respective manufacturers. Stock solutions were prepared and dilutions made as described by the Clinical Laboratory Standards Institute (2005 CLSI). Following two subcultures from frozen stock the MICs of the antimicrobial agents for the isolates were determined by the CLSI 2005 approved broth macrodilution method. Briefly, test tubes containing doubling antibiotic dilutions (2 ml per tube) of cation adjusted Mueller-Hinton broth (eg. for Enterobacteriaceae) with or without lysed horse blood (2-5% V/V) [eg. for S. pneumoniae] were inoculated to achieve a final concentration of approximately 5×105 CFU/ml and incubated in ambient air for 24 hours prior to reading. Colony counts were performed periodically to confirm inocula. Quality control was performed every two weeks using a variety of ATCC QC organisms including; S. aureus 29213, E. faecalis 29212, E. coli 25922 and P. aeruginosa 27853. For all antimicrobials as well as PEP35, CLSI criteria were used. PEP35 was solubilized in 10 mM sodium acetate. Results were tabulated in Table 1. PEP35 and scrambled PEP35 exhibited similar activity, versus MRSA, VISA and VRSA and E. coli. HybPEP35-1 and PEP52 exhibited no activity versus MRSA, VISA, VRSA and E. coli. 
Example 2
Checkerboard Assays
Checkerboard assays were carried out as described by Bonapace et al.
Diagnostic Microbiology and Infectious Diseases 2002; 44:363-366. In brief, checkerboard assays were performed in 96 well microtitre trays using an 8-by-8 well configuration. Dilutions of each antimicrobial were performed with concentrations ranging from 0.03×MIC to 4×MIC. 96-well custom designed microtitre plates contained 100 ul/well of cation adjusted Mueller-Hinton broth (eg. for Enterobacteriaceae) with or without lysed horse blood (2-5% V/V) [eg. for S. pneumoniae] and were inoculated to achieve a final concentration of approximately 5×105 CFU/ml and incubated in ambient air for 24 hours prior to reading. Colony counts were performed periodically to confirm inocula. Quality control was performed every two weeks using a variety of ATCC QC organisms including; S. pneumoniae 49619, S. aureus 29213, E. faecalis 29212, E. coli 25922 and P. aeruginosa 27853. The checkerboard assays were carried out with four peptides (PEP35, scrambled PEP35, HybPEP35-1 and PEP-52) versus MRSA, VISA, VRSA and E. coli with MSSA (as a control). All peptides were assessed for synergy with β-lactams and vancomycin. For all antimicrobials as well as the peptides, CLSI criteria were used.
The fractional inhibitory concentration index (FIC index) was used to interpret synergy, antagonism or additivity/indifference. FIC index=FIC of drug A+FIC of drug B where:
The FIC of drug A and drug B was calculated as follows, FIC drug A=MIC of drug A in combination/MIC of drug A alone and FIC of drug B=MIC of drug B in combination/MIC of drug B alone. Synergy was defined as FIC index ≦0.5, additivity/indifference defined as a FIC between 0.5 and 4 and antagonism defined as an FIC index >4. Results were tabulated in Tables 2-4. This data suggests that both PEP35 and scrambled PEP35 showed synergistic effect with cefazolin and ceftriaxone, versus CA-MRSA, HA-MRSA, VISA and VRSA, and with meropenem versus CA-MRSA, HA-MRSA, and VISA. The data also suggest that PEP35 and scrambled PEP35 were not synergistic with β-lactams (except at a very low level, with ceftriaxone), against E. Coli. Finally, the data suggest that Pep52 exhibited no synergistic activity.
Example 3
MICs of PEP35 with Cephalosporins
Cefazolin, ceftriaxone and vancomycin MICs were performed against MRSA (both community-acquired, eg. USA300/CMRSA10, and healthcare-acquired), VISA (strains 92, 95 and 96) and VRSA (strains 105 and 106) isolates using fixed concentrations of PEP35 (8 ug/ml and 32 ug/ml). One MSSA was used as a control. All MICs were assessed using CLSI guidelines.
Example 4
Checkerboard Assay—Timing
The checkerboard assays were carried out with PEP35 and cefazolin, ceftriaxone or vancomycin versus 2 isolates of MRSA using the methods previously described in example 2. In these checkerboard studies, PEP35 and the cephalosporins were added 15 minutes before the other to determine if pre-exposure to the peptide or antibiotic is required for the synergy. Thus, experiments were performed to expose the MRSA to PEP35, 15 minutes before the addition of the cephalosporin or vancomycin, as well as the opposite (to expose the MRSA to cephalosporins 15 minutes before exposure to PEP35).
The fractional inhibitory concentration index (FIC index) was used to interpret synergy, antagonism or additivity/indifference. FIC index=FIC of drug A+FIC of drug B where the FIC of drug A and drug B was calculated as follows, FIC drug A=MIC of drug A in combination/MIC of drug A alone and FIC of drug B=MIC of drug B in combination/MIC of drug B alone. Synergy was defined as FIC index ≦0.5, additivity/indifference defined as an FIC of between 0.5 and 4 and antagonism defined as an FIC index >4. Results were tabulated in Tables 5-10. Cephalosporin MICs, versus CA-MRSA, HA-MRSA, and VRSA, were reduced in a concentration dependent fashion, in the presence of PEP35. The MIC was not affected by the order in which PEP35 and cephalosporin were added to the assays. The results also showed that PEP35 activity on vancomycin MICs versus VRSA was concentration dependent, but order neutral.
Example 5
Checkerboard Assay—Versus Gram Positive and Gram Negative Bacteria
To assess whether any synergy, antagonism or additivity/indifference occurs when PEP35 is combined with marketed antimicrobials against a variety of Gram-positive and Gram-negative organisms from respiratory, skin/skin structure, urinary and bacteremic pathogens obtained from Canadian hospitalized patients.
The checkerboard assays were carried out as described by Eliopoulos and Moellering (Chapter 9) in Antibiotic in Laboratory Medicine, Lorian Ed. 1996 as well as Bonapace et al. Diagnostic Microbiology and Infectious Diseases 2002; 44:363-366 and Bonapace et al. Diagnostic Microbiology and Infectious Diseases 2000; 38:43-50. In brief, checkerboard assays were performed in 96 well microtitre trays using an 8-by-8 well configuration. Dilutions of each antimicrobial were performed with concentrations ranging from 0.03×MIC to 4×MIC. 96-well custom designed microtitre plates contained 100 ul/well of cation adjusted Mueller-Hinton broth (eg. for Enterobacteriaceae) with or without lysed horse blood (2-5% V/V) [eg. for S. pneumoniae] and were inoculated to achieve a final concentration of approximately 5×105 CFU/ml and incubated in ambient air for 24 hours prior to reading. Colony counts were performed periodically to confirm inocula. Quality control was performed every two weeks using a variety of ATCC QC organisms including; S. pneumoniae 49619, S. aureus 29213, E. faecalis 29212, E. coli 25922 and P. aeruginosa 27853. For all antimicrobials as well as PEP35, CLSI criteria was used.
The fractional inhibitory concentration index (FIC index) was used to interpret synergy, antagonism or additivity/indifference. FIC index=FIC of drug A+FIC of drug B where:
The FIC of drug A and drug B is calculated as follows, FIC drug A=MIC of drug A in combination/MIC of drug A alone and FIC of drug B=MIC of drug B in combination/MIC of drug B alone. Synergy is defined as FIC index ≦0.5, additivity/indifference FIC >0.5-4 and antagonism FIC index >4.
Antimicrobials used included PEP35, cefazolin, ceftriaxone, vancomycin, linezolid, tigecycline and daptomycin versus Gram-positive cocci and PEP35, gentamicin, levofloxacin, piperacillin/tazobactam, meropenem and colistin (Polymyxin E) versus Gram-negative bacilli. These agents were obtained as laboratory grade powders from their respective manufacturers. Stock solutions were prepared and dilutions made as described by the Clinical Laboratory Standards Institute (2005 CLSI).
Organisms included Gram-positive cocci (2 strains of each): Methicillin-susceptible Staphylococcus aureus (MSSA), community-associated (CA) and healthcare-associated (HA) methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate Staphylococcus aureus (VISA), vancomycin-resistant Staphylococcus aureus (VRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, Enterococcus faecium and vancomycin-resistant enterococcus (VRE). Gram-negative bacilli tested (2 strains of each) included: E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, K. oxytoca, Enterobacter cloacae, P. mirabilis and Stenotrophomonas maltophilia. MIC testing, following two subcultures from frozen stock were determined by the CLSI 2005 approved broth microdilution method.
Example 6
MICs of PEP35 with and without Cefazolin or Cephalexin Against MRSA
Cefazolin and cephalexin MICs were performed against MRSA (50 community-acquired (CA-MRSA) and 50 healthcare-acquired (HA-MRSA) isolates) using a fixed concentration of PEP35 (32 ug/ml). One MS SA and ATCC reference strains were used as controls. All MICs were assessed using CLSI guidelines.
Results
Table 1 shows the activity of Cangene peptides versus MRSA, VISA, VRSA and E. coli. These data show that PEP35 and scrambled PEP35 demonstrate similar activity versus MRSA, VISA and VRSA. Unlike PEP35, scrambled PEP35 was active vs E. coli. Other Cangene peptides (HybPEP35-1 or PEP52) were not active versus these organisms.
Table 2 shows the synergy studies with β-lactams and PEP35 versus MRSA, VISA, VRSA and E. coli. These data show that PEP35 is synergistic with cefazolin, ceftriaxone and meropenem versus CA-MRSA, HA-MRSA, VISA and VRSA (not meropenem). No combination of PEP35 and β-lactams were synergistic versus E. coli. 
Table 3 describes synergy studies with β-lactams and Scrambled PEP35 versus MRSA, VISA, VRSA and E. coli. These data show that like PEP35, scrambled PEP35 is synergistic with cefazolin, ceftriaxone and meropenem versus CA-MRSA, HA-MRSA, VISA and VRSA (not meropenem). The combination of scrambled PEP35 and β-lactams were not synergistic (except at low level with ceftriaxone) versus E. coli. 
Table 4 shows the synergy studies with β-lactams and PEP-52 versus MRSA, VISA, VRSA and E. coli. These data show no synergistic activity with PEP_52 and β-lactams.
Table 4b shows the synergy studies with β-lactams and HybPEP35-1 Versus MRSA, VISA, VRSA and E. coli. The data shows synergistic activity with HybPEP35-1 and certain β-lactams, against certain isolates.
Table 5 shows the effect of increasing the concentrations of PEP35 on cephalosporin activity (MICs) versus CA-MRSA and HA-MRSA. These data show that cephalosporin MICs versus CA-MRSA and HA-MRSA are reduced in the presence of PEP35 and that these cephalosporin MICs are reduced in a concentration dependent fashion.
Table 5 also shows the effect of adding PEP35 15 minutes before or after cephalosporins on activity (MICs) versus CA-MRSA and HA-MRSA. These data show that whether PEP35 is added 15 minutes before the cephalosporin or the cephalosporin is added 15 minutes before PEP35 does not in any way influence the MIC.
Table 6 shows the effect of increasing the concentrations of PEP35 on cephalosporin activity (MICs) versus CA-MRSA, HA-MRSA, VISA and VRSA. These data show that cephalosporin MICs versus CA-MRSA, HA-MRSA and VRSA are reduced in the presence of PEP35 and that these cephalosporin MICs are reduced in a concentration dependent fashion. PEP35 reduced the MICs of cephalosporins versus VISA in 2 of 3 strains and this reduction was concentration dependent. Values from independent experiments for each condition shown in table separated by a “/”.
Table 7 shows the effect of adding PEP35 15 Minutes before or after cephalosporins on synergy versus CA-MRSA and HA-MRSA. These data show that whether PEP35 is added 15 minutes before the cephalosporin or the cephalosporin is added 15 minutes before PEP35, does not in any way influence the presence of synergy. Synergy occurs with PEP35 and cephalosporins versus CA-MRSA and HA-MRSA.
Table 8 shows the effect of increasing the concentration of PEP35 on vancomycin activity (MICs) versus VRSA. These data show that PEP35 activity on vancomycin MIC is concentration dependent. Increasing the concentration of PEP35 lowers the vancomycin MIC. Table 8 also shows the effect of adding PEP35 15 Minutes before or after vancomycin on activity (MICs) versus VRSA. These data show that whether PEP35 is added 15 minutes before the vancomycin or the vancomycin is added 15 minutes before PEP35, does not in any way influence the vancomycin MIC.
Table 9 shows the effect of adding PEP35 15 minutes before or after vancomycin on synergy versus VRSA. These data show that whether PEP35 is added 15 minutes before the vancomycin or the vancomycin is added 15 minutes before PEP35, does not in any way influence synergy. PEP35 and vancomycin are synergistic versus VRSA.
Table 10 shows the effect of increasing PEP35 concentration on activity (MICs) with and without vancomycin versus MRSA, VISA and VRSA. These data show that PEP35 activity on vancomycin MIC is concentration dependent for VRSA and that VRSA MICs decrease significantly with PEP35. No effect on MIC occurred with PEP35 and vancomycin versus MRSA or VISA. Values from independent experiments for each condition shown in table separated by a “/”.
Table 11 shows the synergy studies with PEP35 versus Gram-positive cocci, in combination with cefazolin, ceftriaxone, daptomycin, linezolid, tigecycline and vancomycin did not demonstrate antagonism against any streptococci, staphylococci and enterococci tested. Versus Gram-positive cocci, PEP35 interacted with cefazolin, ceftriaxone, daptomycin, linezolid, tigecycline and vancomycin in an additive fashion against all streptococci, staphylococci and enterococci tested with the exception of CA-MRSA and HA-MRSA. Versus CA-MRSA and HA-MRSA, PEP35 (MIC alone, range 512-1024 ug/ml) with both beta-lactams tested (cefazolin and ceftriaxone, [MIC alone, range 16-256 ug/ml]) interacted in a synergistic fashion (FIC index≦0.5).
Table 12 shows the synergy studies with PEP35 Versus Gram-negative bacilli, in combination with gentamicin, levofloxacin, piperacillin-tazobactam, meropenem, and colistin (polymyxin E). PEP35 did not demonstrate antagonism or synergy against any Gram-negative bacilli tested. Versus Gram-negative bacilli, PEP35 in combination with gentamicin, levofloxacin, piperacillin-tazobactam, meropenem, and colistin (polymyxin E) interacted in an additive fashion with all Gram-negative bacilli tested.
Table 13 shows the synergy studies with HybPEP35-1 versus gram positive cocci in combination with cefazolin, ceftriaxone, meropenem and pipercillin-tazobactam. The data shows synergy in certain isolates between HybPEP35-1 and β-lactams and meropenem. Strong synergy was evident versus CA-MRSA, weak synergy was found with one HA-MRSA and stronger synergy with another HA-MRSA strain. Synergy ranged from mild to strong for VISA and VRSA. Values from independent experiments for each condition shown in table separated by a “/”.
Table 14 shows the in vitro activity of PEP35 with 50 CA-MRSA strains with and without Cefazolin or Cephalexin. Cefazolin alone MICs ranged from 2-256 ug/ml with 45 of the 50 strains being resistant to cefazolin. Combining PEP35 with cefazolin resulted in MIC reductions of 2-128 fold, with 32-64 fold reductions being most common. All CA-MRSA MICs with combination of PEP35 with cefazolin were ≦4 ug/ml, which is considered susceptible (≦8 ug/ml) as per CLSI breakpoints.
Cephalexin alone MICs ranged from 64-512 ug/ml with 50 of the 50 strains being resistant to cephalexin. Combining PEP35 with cephalexin resulted in MIC reductions of 4-32 fold, with 4-8 fold reductions being the most common. 16% of CA-MRSA MICs with combination of PEP35 with cephalexin were ≦8 ug/ml, which is considered susceptible as per CLSI breakpoints.
Table 15 shows the in vitro activity of PEP35 with 50 HA-MRSA strains with and without Cefazolin or Cephalexin. Cefazolin alone MICs ranged from 2-512 ug/ml with 47 of the 50 strains being resistant to cefazolin. Combining PEP35 with cefazolin resulted in MIC reductions of 0-256 fold, with 4-32 fold reductions being most common. 64% of HA-MRSA MICs with combination of PEP35 with cefazolin were ≦8 ug/ml, which is considered susceptible (≦8 ug/ml) as per CLSI breakpoints.
Cephalexin alone MICs ranged from 128-512 ug/ml with 50 of the 50 strains being resistant to cephalexin. Combining PEP35 with cephalexin resulted in MIC reductions of 0-32 fold, with 2-4 fold reductions being the most common. 4% of HA-MRSA MICs with combination of PEP35 with cephalexin were ≦8 ug/ml, which is considered susceptible as per CLSI breakpoints.
As would be evident to a person of skill in the art, often, when faced with a clinical situation, it is not known which strain is being combated. As such, a combination exhibiting synergy against some strains, and effectiveness (but not necessarily synergy) against others, is, in all cases, a useful and powerful weapon against infection, regardless of whether it offers synergy against the specific strain, since the specific strain is sometimes not known, and the treatment will, in many cases, work better than the alternative treatment.







TABLE 1





MIC Activity of Cangene Peptides Versus MRSA, VISA, VRSA and E. coli



























Piperacillin/

Scrambled




ISOLATE
Cefazolin 
Ceftriaxone
Meropenem
Tazobactam
PEP35
PEP35
HybPEP35-1
PEP-52





MSSA 79333
1
8
0.12
≦1
128
64
256
>512/?≦128


CA-MRSA 69615 (USA400)
64
256
0.5
32
512
256
>2048/512
>512/1024


CA-MRSA 70857 (USA400)
32
128
2
32
256
256
>2048/512
>512/1024


HA-MRSA 77090
128
512
4
16
256
512
>2048/512
>512/1024


(USA100/800)










HA-MRSA 70798
512
2048
64
4
512
256
>2048/1024
>512/>1024


(USA100/800)










VRSA 105
256
2048
32
64
128
64
256/128
>512/>1024


VRSA 106
256
1024
16
64
128
128
256
>512/>1024


VISA 92
128
1024
16
64
1024
2048
>2048/>2048
>512/1024


VISA 95
128
512
16
64
1024
2048
>2048/2048
>512/1024


VISA 96
64
256
8
64
512
1024
1024
>512/1024











Scrambled








Piperacillin/

PEP 35
hybPEP35-1
PEP-52


ISOLATE
Cefazolin
Ceftriaxone 
Meropenem
Tazobactam
PEP 35
(28-00639)
(RC-0127)
(28-00900)





E.coli 69716
2
   ≦1 (0.03)
0.03
2
128
8
64
>512/>1024


E.co1i 70752
128
   >64 (1024)
0.03
8
128
8
32
















TABLE 2







Synergy Studies With β-lactams and PEP35 Versus MRSA, VISA, VRSA and E. coli












Cefazolin
Ceftriaxone
Meropenem
Piperacillin/Tazobactam


ISOLATE
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)

















MSSA 79333
0.56
(0.37-1.015)
0.61
(0.37-1.03)
0.52
(0.37-0.62)



CA-MRSA 69615
0.24
(0.06-0.53)
0.30
(0.12-0.56)
0.49
(0.24-1.008)


(USA400)


HA-MRSA 70798
0.27
(0.09-0.53)
0.24
(0.075-0.53)
0.30
(0.12-0.53)


(USA100/800)


VRSA 106
0.39
(0.12-1.015)
0.26
(0.09-0.53)
0.91
(0.62-1.12)


VISA 96
0.24
(0.06-0.53)
0.28
(0.09-0.56)
0.41
(0.24-0.62)


E. coli 69716
1.1
(1.0-1.25)
1.04
(0.56-2.015)
1.21
(1.03-1.5)
0.87 (0.56-1.12)


E. coli 70752






0.98 (0.62-1.25)
















TABLE 3







Synergy Studies With β-lactams and Scrambled PEP35 Versus MRSA, VISA, VRSA and E. coli












Cefazolin
Ceftriaxone
Meropenem
Piperacillin/Tazobactam


ISOLATE
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)


















MSSA 79333
0.57
(0.50-0.56)
0.98
(0.62-1.25)
1.11
(1.0-1.25)




CA-MRSA 69615
0.24
(0.06-0.53)
0.31
(0.15-0.56)
0.70
(0.37-1.03)


(USA400)


HA-MRSA 70798
0.35
(0.018-0.56)
0.32
(0.015-0.56)
0.38
(0.31-0.53)


(USA100/800)


VRSA 106
0.52
(0.28-1.06)
0.39
(0.28-0.56)
0.91
(0.62-1.12)


VISA 96
0.30
(0.12-0.53)
0.26
(0.09-0.53)
0.45
(0.31-0.56)


E. coli 69716
1.1
(1.0-1.25)
0.41
(0.26-0.56)
1.21
(1.03-1.5)
0.59
(0.37-1.015)


E. coli 70752






0.67
(0.5-1.06)
















TABLE 4







Synergy Studies With β-lactams and PEP-52 Versus MRSA, VISA, VRSA and E. coli












Cefazolin
Ceftriaxone
Meropenem*
Piperacillin/Tazobactam


ISOLATE
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)





MSSA 79333
>1*
>1*
>1*



CA-MRSA 69615
  >0.5*
  >0.5*
>1*


(USA400)


HA-MRSA 70798
>1*
>1*
>1*


(USA100/800)


VRSA 106
>1*
>1*
>1*


VISA 96
>1*
>1*
>1*


E. coli 69716
>1*
>1*
>1*
>1*


E. coli 70752



>1*





*PEP52 MIC > 1024













TABLE 4b







Synergy Studies With β-lactams and HybPEP35-l Versus MRSA, VISA, VRSA and E. coli












Cefazolin
Ceftriaxone
Meropenem*
Pippercillin/Tazobactam


ISOLATE
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)


















MSSA 79333
0.57
(0.50-0.56)
0.98
(0.62-1.25)
1.11
(1.0-1.25)




CA-MRSA 69615
0.24
(0.06-0.53)
0.31
(0.15-0.56)
0.70
(0.37-1.03)


(USA400)


HA-MRSA 70798
0.35
(0.018-0.56)
0.32
(0.015-0.56)
0.38
(0.31-0.53)


(USA100/800)


VRSA 106
0.52
(0.28-1.06)
0.39
(0.28-0.56)
0.91
(0.62-1.12)


VISA 96
0.30
(0.12-0.53)
0.26
(0.09-0.53)
0.45
(0.31-0.56)


E. coli 69716
1.1
(1.0-1.25)
0.41
(0.26-0.56)
1.21
(1.03-1.5)
0.59
(0.37-1.015)


E. coli 70752






0.67
(0.5-1.06)





*frozen HybPEP35-1 stock used













TABLE 5







Effect of Increasing Concentration of PEP35 or Adding PEP35 15 Minutes Before or After Cephalosporins on


Activity (MICs) Versus CA-MRSA and HA-MRSA

















CEFAZOLIN 
CEFAZOLIN

CEFTRIAXONE 
CEFTRIAXONE





& PEP35
& PEP35

& PEP35
& PEP35


ORGANISM
PEP35
CEFAZOLIN
(8 ug/mL)
(32 ug/mL)
CEFTRIAXONE
(8 ug/mL)
(32 ug/mL)

















CA-MRSA 69615 (USA400)
256
64
16
≦2
256
64
16


CA-MRSA 69615 (USA400)a

64
32
≦2
512
128
16


CA-MRSA 69615 (USA400)b

64
32
≦2
256
128
16


HA-MRSA 77090 (USA100/800)
256
128
4
≦2
1024
64
16


HA-MRSA 77090 (USA100/800)a

128
8
≦2
512
64
16


HA-MRSA 77090 (USA100/800)b

128
8
≦2
512
64
16





aCFZ or CTR added 15 minutes post PEP35 & organism exposure;


bPEP35 added 15 minutes post CFZ or CTR & organism exposure













TABLE 6







Effect of Increasing Concentration of PEP35 on Cephalosporin Activity (MICs) Versus CA-MRSA, HA-MRSA


VISA and VRSA

















CEFAZOLIN
CEFAZOLIN

CEFTRIAXONE 
CEFTRIAXONE





& PEP35
& PEP35

& PEP35
& PEP35


ISOLATE
PEP35
CEFAZOLIN
(8 ug/mL)
(32 ug/mL)
CEFTRIAXONE
(8 ug/mL)
(32 ug/mL)

















MSSA 79333
256/256/128
0.5/0.5
0.5/0.5
0.5/0.25
4/4
4/2
2/2


CA-MRSA 69615 
512/512/256/512/512
64/32/8/32
8/≦2/0.5/2
≦2/≦2/1/1
128/64/256
32/16/32
16/16/16


(USA400)









HA-MRSA 77090 
512/512/256/256/512
128/64/64
4/≦2/2
≦2/≦2/1
512/128/512
32/16/64
16/16/32


(USA100/800)









VRSA 105
128/128/64/128/64
128/128/128/
64/64/8/128
4/≦2/2/4
2048/1024/>512/
512/1024/256/
32/8/8/16




256


>512
>512



VRSA 106
128/128/128
256/128
128/128
64/64
1024/1024
512/512
256/256


VISA 92
2048/2048/2048
128/128
256/128
256/128
1024/1024
1024/1024
1024/512


VISA 95
1024/1024/512/2048/
128/64/64
64/32/64
64/16/32
1024/512/512/
512/256/256/512 
512/64/64/256



1024



512




VISA 96
512/512/512
32/32
16/16
8/4
128/128
64/64
32/16
















TABLE 7







Effect of Adding PEP35 15 Minutes Before or After Cephalosporins on Synergy versus CA-MRSA and HA-MRSA














PEP35 & CFZ
PEP35 & CFZa
PEP35b & CFZ
PEP35 & CTR
PEP35 & CTRc
PEP35d & CTR


ORGANISM
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)
















CA-MRSA 69615 (USA400)
0.29 (0.12-0.53)
0.29 (0.12-0.53)
0.21 (0.045-0.508)
0.26 (0.09-0.53)
0.26 (0.09-0.53)
0.24 (0.06-0.53)



0.21 (0.045-0.515)
0.21 (0.045-0.515)

0.26 (0.09-0.53)
0.26 (0.09-0.53)



CA-MRSA 70857 (USA400)
0.21 (0.45-0.515)
0.26 (0.09-0.53)
0.23 (0.075-0.53)
0.30 (0.12-0.53)
0.34 (0.18-0.53)
0.26 (0.09-0.53)


HA-MRSA 77090 (USA100/800)
0.21 (0.045-0.515)
0.19 (0.03-0.515)
0.21 (0.045-0.53)
0.23 (0.06-0.515) 
0.23 (0.06-0.515)
0.21 (0.06-0.53)



0.21 (0.045-0.53)
0.18 (0.03-0.515)

0.26 (0.9-0.53)
0.29 (0.12-0.53) 



HA-MRSA 70798 (USA 100/800)
0.56 (0.028-1.015)
0.56 (0.28-1.015)
0.47 (0.24-1.008) 
0.33 (0.18-0.515)
0.40 (0.28-0.56) 
0.44 (0.31-0.56)





aCFZ added 15 minutes post PEP35 & organism exposure


bPEP35 added 15 minutes post CFZ & organism exposure


cCTR added 15 minutes post PEP35 & organism exposure


dPEP35 added 15 minutes post CTR & organism exposure













TABLE 8







Effect of Increasing Concentration of PEP35


or Adding PEP35 15 Minutes Before or After


Vancomycin on Activity (MICs) Versus VRSA














VANCOMYCIN &
VANCOMYCIN &


ORGANISM
PEP35
VAN
PEP35 (8 ug/mL)
PEP35 (32 ug/mL)





VRSA 105
128
512
128
8


VRSA 105a


256
8


VRSA 105b


256
8


VRSA 106
128
512
128
4


VRSA 106a


256
4


VRSA 106b


256
4





aVAN added 15 minutes post PEP35 & organism exposure


bPEP35 added 15 minutes post VAN & organism exposure













TABLE 9







Effect of Adding PEP35 15 Minutes Before or


After Vancomycin on Synergy Versus VRSA











VAN & PEP35
VAN & PEP35a
VAN & PEP35b


ORGANISM
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)
















VRSA 105
0.27
(0.09-0.53)
0.27
(0.09-0.53)
0.36
(0.135-1.03)


VRSA 106
0.35
(0.09-1.015)
0.35
(0.09-1.015)
0.35
(0.09-1.015)





aVAN added 15 minutes post PEP35 & organism exposure


bPEP35 added 15 minutes post VAN & organism exposure













TABLE 10







Effect of Increasing PEP35 Concentration on Activity (MICs)


With and Without Vancomycin Versus MRSA, VISA and VRSA












VANCOMYCIN &
VANCOMYCIN &


ISOLATE
VANCOMYCIN
PEP35 (8 ug/mL)
PEP35 (32 ug/mL)





MSSA 79333
  1/0.5
1/1
1/1


CA-MRSA 69615
1/1
1/1
1/1


(USA400)


HA-MRSA 77090
1/1
1/1
1/1


(USA100/800)


VRSA 105
512/512/512
16/256/128
4/4/8


VRSA 106
512/512/512
32/32/128
4/4/4


VISA 92
4/4
4/4
4/4


VISA 95
2/4
2/2
2/2


VISA 96
4/4
4/4
4/4
















TABLE 11







Synergy studies with PEP35 against Gram-positive cocci with various antibiotics
















Cefazolin
Ceftriaxone
Daptomycin
Linezolid
Tigecycline
Vancomycin




Avg ΣFIC 
Avg ΣFIC 
Avg ΣFIC 
Avg ΣFIC 
Avg ΣFIC 
Avg ΣFIC 




(range)
(range)
(range)
(range)
(range)
(range)





Stock.
Organism






















S. pneumoniae-Pen S n = 2 (MHB)




















70400
Streptococcus
0.68 (0.515-1.06)
0.99 (0.62-1.06)
1.21 (1.03-1.5)
0.63 (0.5-1.06)
0.91 (0.62-1.12)
0.91 (0.56-1.25)



pneumoniae








71263
Streptococcus
0.99 (0.62-1.25)
0.91 (0.62-1.12)
1.39 (1.03-2.5)
0.92 (0.62-1.12)
0.92 (0.56-1.25)
1.05 (0.75-1.25)



pneumoniae



















S. pneumoniae-Pen I n = 2 (MHB)




















72302
Streptococcus
 1.1 (1.0-1.25)
1.05 (0.75-1.25)
0.99 (0.62-1.25)
0.86 (0.56-1.12)
0.68 (0.53-1.03)
1.26 (1.06-1.5)



pneumoniae








78663
Streptococcus
 1.1 (1.0-1.25)
 1.1 (1.0-1.25)
1.19 (1.03-1.5)
1.05 (0.75-1.25)
 1.0 (0.56-1.12)
 1.1 (1.0-1.25)



pneumoniae



















S. pneumoniae-Pen R n = 2 (MHB)




















78519
Streptococcus
0.97 (0.75-1.12)
1.05 (0.75-1.25)
1.19 (1.03-1.5)
0.92 (0.75-1.12)
0.98 (0.75-1.12)
 1.2 (1.015-1.5)



pneumoniae








76065
Streptococcus
0.70 (0.53-1.06)
1.21 (1.03-1.5)
1.09 (0.56-1.5)
0.74 (0.53-1.12)
0.91 (0.62-1.12)
0.86 (0.53-1.25)



pneumoniae



















MSSA-n = 2 (MH)




















79333
Staphylococcus
1.04 (0.75-1.25)
0.86 (0.56-1.12)
1.19 (1.03-1.5)
1.05 (0.75-1.25)
0.99 (0.62-1.5)
1.11 (1.0-1.25)



aureus (2008)








83424
Staphylococcus
1.02 (0.75-1.25)
0.80 (0.5-1.12)
1.58 (1.015-2.5)
0.74 (0.515-1.06)
0.85 (0.5-1.12)
1.19 (1.03-1.5)



aureus (2008)



















CA-MRSA n = 2 (MH)




















69615
Staphylococcus
0.27 (0.09-0.56)
0.30 (0.12-0.53)
1.18 (1.015-1.5)
0.80 (0.515-1.12)
0.93 (0.62-1.12)
1.19 (1.03-1.5)



aureus, MRSA
0.24 (0.06-0.53)
0.33 (0.18-0.53)






70857
Staphylococcus
 0.3 (0.12-0.56)
0.51 (0.18-1.015)
1.18 (1.015-1.5)
0.89 (0.5-1.12)
0.74 (0.5-1.06)
1.05 (0.75-1.25)



aureus, MRSA
0.26 (0.09-0.53)


















HA-MRSA-n = 2 (MH)




















77090
Staphylococcus
0.30 (0.12-0.56)
0.27 (0.09-0.53)
1.27 (1.015-2.03)
0.74 (0.53-1.06)
0.83 (0.5-1.12)
0.95 (0.56-1.25)



aureus, MRSA
0.24 (0.075-0.53)
0.27 (0.09-0.56)






70798
Staphylococcus
0.30 (0.15-0.53)
 0.4 (0.265-0.56)
0.99 (0.53-1.5)
0.70 (0.5-1.06)
0.77 (0.5-1.06)
1.19 (1.03-1.5)



aureus, MRSA
0.54 (0.24-1.03)
0.39 (0.15-1.015)





R&D STK#
Organism
















VISA-n = 3 (MH)




















92
S. aureus Van 
0.33 (0.18-0.53)
0.52 (0.28-1.06)
1.54 (1.015-2.5)
0.99 (0.75-1.12)
0.56 (0.31-1.06)
 1.1 (1.0-1.25)



Intermediate
0.45 (0.18-1.015)







95
S. aureus Van 
0.49 (0.31-0.62)
0.28 (0.09-0.56)
1.23 (1.06-1.5)
0.98 (0.62-1.25)
0.46 (0.37-0.56)
 1.1 (1.03-1.25)



Intermediate
0.37 (0.18-0.56)
0.37 (0.18-0.56)


0.43 (0.31-0.56)



96
S. aureus Van 
0.34 (0.18-0.56)
0.31 (0.15-0.53)
1.21 (1.03-1.5)
0.77 (0.56-1.06)
0.91 (0.62-1.12)
0.88 (0.53-0.125)



Intermediate
0.53 (0.31-1.03)
0.47 (0.24-1.03)

















VRSA-n = 2 (MH)




















105
S. aureus Van 
0.63 (0.5-1.015)
0.45 (0.24-0.56)
0.52 (0.37-0.62)
0.80 (0.5-1.06)
0.93 (0.62-1.12)
 0.3 (0.15-0.53)



Resistant

0.54 (0.18-1.06)



0.49 (0.24-1.06)


106
S. aureus Van 
0.47 (0.265-1.06)
0.34 (0.135-0.56)
0.91 (0.56-1.25)
0.83 (0.5-1.12)
 1.2 (0.56-2.12)
0.27 (0.09-0.53)



Resistant
0.59 (0.31-1.03)
0.73 (0.5-1.06)



0.40 (0.15-1.06)





Stock.
Organism
















MRSE-n = 2 (MH)




















79617
Staphylococcus
0.88 (0.53-1.25)
0.88 (0.53-1.25)
1.19 (1.03-1.5)
1.10 (0.56-1.5)
1.19 (1.03-1.5)
0.99 (0.62-1.25)



epidermidis (2008)








83100
Staphylococcus
0.52 (0.37-0.62)
1.05 (0.75-1.25)
 1.1 (1.0-1.25)
0.73 (0.515-1.12)
1.21 (1.03-1.5)
0.60 (0.31.12) rpt



epidermidis (2008)



















S. agalactiae-n = 2 (MHB)




















80870
Streptococcus
0.92 (0.56-1.25)
0.99 (0.53-1.25)
0.83 (0.53-1.12)
 1.1 (1.0-1.25)
1.10 (1.0-1.25)
1.05 (0.75-1.25)



agalactiae (2008)








81262
Streptococcus
1.10 (1-1.25)
1.05 (0.75-1.25)
1.69 (1.06-2.5)
0.91 (0.53-1.25)
1.05 (0.75-1.25)
 1.1 (1.0-1.25)



agalactiae (2008)



















S. pyogenes-n =2 (MHB)




















77765
Streptococcus
1.04 (0.75-1.25)
1.19 (0.03-1.5)
1.19 (1.03-1.5)
1.05 (0.75-1.25)
 1.1 (1.0-1.25)
1.09 (1.0-1.25)



pyogenes (2008)








79531
Streptococcus
1.19 (1.03-1.5)
1.19 (1.03-1.5)
1.19 (1.03-1.5)
1.19 (1.03-1.5)
 1.1 (1.0-1.25)
1.27 (1.015-2.5)



pyogenes (2008)



















E. faecalis n =2 (MH)




















78145
Enterococcus faecalis
0.93 (0.62-1.06)
0.72 (0.53-1.12)
1.39 (1.03-2.25)
0.88 (0.62-1.06)
 0.9 (0.5-1.12)
1.19 (1.03-15)


70468
Enterococcus faecalis
0.94 (0.56-1.25)
 1.1 (1-1.25)
 1.5 (1-2.25)
0.99 (0.62-1.25)
0.82 (0.5-1.06)
 1.1 (1-1.25)













E. Faecium-n = 2 (MH)




















78024
Enterococcus faecium
1.16 (1.015-1.5)
>1*
1.19 (1.03-1.5)
 1.1 (1-1.25)
1.19 (1.03-1.5)
1.19 (1.03-1.5)


78352
Enterococcus faecium
1.10 (1-1.25)
0.99 (0.62-1.25)
1.10 (0.75-1.5)
0.80 (0.5-1.06)
0.99 (0.75-1.12)
1.59 (1.06-2.5)













VRE-n =2 (MH)




















70769
Enterococcus faecium
>1*
>1*
0.82 (0.28-1.25)
1.09 (1-1.25)
 1.2 (1.03-1.05)
0.92 (0.506-1.25)


77872
Enterococcus faecium
>1*
>1*
1.34 (1.03-1.5)
1.19 (1.03-1.5)
1.39 (1.03-2.06)
1.19 (1.03-1.5)
















TABLE 12







Synergy studies with PEP35 against Gram-negative cocci with various antibiotics















Gentamicin
Levofloxacin
Piperacillin/Tazobactam
Meropenem
Colistin


Stock.
Organism
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)
Avg ΣFIC (range)















E.coli n =2 (MH)


















69716
Escherichia coli
 1.4 (1.06-2.12)
0.93 (0.56-1.12)
0.63 (0.5-1.03)
0.86 (0.53-1.25)
0.71 (0.31-1.06)


70752
Escherichia coli
1.05 (0.75-1.25)
1.13 (0.75-1.12)
0.63 (0.24-1.03)
1.21 (1.03-1.5)
0.81 (0.31-1.25)












P. aeruginosa-n =2 (MH)


















70849
Pseudomonas aeruginosa
1.11 (1.03-1.25)
1.21 (1.03-1.5)
0.92 (0.62-1.12)
1.21 (1.03-1.5)
0.70 (0.28-1.12)


71841
Pseudomonas aeruginosa
1.05 (0.75-1.25)
1.13 (1.0-1.25)
0.88 (0.56-1.12)
1.10 (1.0-1.25)
0.89 (0.53-1.25)












K. pneumoniae-n =2 (MH)


















77081
Klebsiella pneumoniae
0.89 (0.56-1.25)
1.14 (1.06-1.25)
1.05 (0.75-1.25)
1.21 (1.03-1.5)
0.61 (0.18-1.06)


70508
Klebsiella pneumoniae
1.13 (1.0-1.25)
0.97 (0.75-1.12)
0.93 (0.62-1.12)
1.19 (1.03-1.5)
0.67 (0.31-1.06)












K. oxytoca-n =2 (MH)


















76874
Klebsiella oxytoca
1.10 (0.75-2.06)
0.94 (0.62-1.12)
0.83 (0.53-1.12)
0.80 (0.53-1.12)
0.83 (0.31-2.03)


75316
Klebsiella oxytoca
1.24 (0.75-2.12)
0.88 (0.53-1.25)
0.93 (0.62-1.12)
 1.1 (1.0-1.25)
0.41 (0.24-0.56)








0.62 (0.18-1.06)








0.65 (0.18-1.12)












E. cloacae-n =2 (MH)


















80194
Enterobacter cloacae (2008)
 1.4 (0.5-2.12)
1.13 (1.0-1.25)
0.79 (0.5-1.06)
1.32 (1.03-2.12)
0.70 (0.31-1.12)


83128
Enterobacter cloacae (2008) 
1.05 (0.75-1.25)
0.98 (0.75-1.12)
0.71 (0.37-1.06)
1.05 (0.75-1.25)
0.77 (0.5-1.06)












P. mirabilis-n =2 (MH)


















80267
Proteus mirabilis (2008)
>1*
>1*
>0.5*
>1*
>1**


81569
Proteus mirabilis (2008)
>1*
>1*
>0.5*
>1*
>1**












S. maltophilia-n =2 (MH)


















73062
Stenotrophomonas
 1.4 (0.62-2.25)
1.21 (1.03-1.5)
0.98 (0.62-1.25)
0.77 (0.5-1.03)
0.88 (0.53-1.25)



maltophilia







77424
Stenotrophomonas
1.99 (1.0-4.25)
0.94 (0.62-1.12)
1.04 (0.75-1.25)
1.05 (0.75-1.25)
0.32 (0.18-0.53)



maltophilia
1.98 (1.06-4.25) 



0.92 (0.37-1.5)








0.71 (0.37-1.06)




*PEP35 MIC > 4096
*PEP35 MIC > 4096
*PEP MIC > 4096
*PEP MIC > 4096
*PEP MIC > 4096








** PEP35 MIC > 4096








COL MIC > 4096
















TABLE 13







Effect of Combining HybPEP35-l and β-lactams on Activity (MICs) Versus CA-MRSA, HA-MRSA VISA and VRSA

























Pipercillin-





Cefazolin

Ceftriaxone

Meropenem

Tazobactam





& hybPEP35
Ceftri-
& hybPEP35

& hybPEP35
Piperacillin-
& hybPEP35


ISOLATE
hybPEP35
Cefazolin
(32 ug/mL)
axone
(32 ug/mL)
Meropenem
(32 ug/mL)
Tazobactam
(32 ug/mL)





MSSA 79333
128/128
0.5/0.5
   0.5/≦0.25
4/4
2/2
0.25/0.12
0.12/0.06




CA-MRSA 69615
1024/1024
64/64
2/2
512/256
16/16
8/4
2/2


(USA400)


CA-MRSA 70857
1024/1024
32/32
2/2
256/128
16/16
4/4
2/2


(USA400)


HA-MRSA 77090
1024/512 
256/256
 8/16
1024/1024
64/64
32/32
 8/16


(USA100/800)


HA-MRSA 77098
≧2048/2048 
512/512
256/256
4096/4096
2048/2048
64/64
64/64


(USA100/800)


VRSA 105
128/128
128/256
8/8
1024/1024
64/64
16/32
4/4


VRSA 106
128/256
256/256
 64/128
2048/2048
512/512
16/16
16/16


VISA 96
1024/2048
64/32
4/8
256/256
32/32
8/8
4/4


E. coli 69716
128/64 






4/2
≦0.06/0.12 


E. coli 70752
64/64






4/4
≦0.06/≦0.06
















TABLE 14







In Vitro Activity of Pep35 with and without Cefazolin or Cephalexin against CA-MRSA















Cefazolin & PEP35

Cephalexin & PEP35





32 ug/mL

32 ug/mL


















Fold 


Fold 


Organism
PEP35
Cefazolin
MIC *
reduction
Cephalexin
MIC *
reduction

















CA-MRSA
512
64
1
64
256
16
16


(CMRSA7/USA400/MW2)









CA-MRSA
512
128
2
64
512
64
8


(CMRSA7/USA400/MW2)









CA-MRSA
512
128
2
64
512
64
8


(CMRSA7/USA400/MW2)









CA-MRSA
512
128
2
64
512
64
8


(CMRSA7/USA400/MW2)









CA-MRSA
512
8
1
8
128
8
16


(CMRSA7/USA400/MW2)









CA-MRSA
512
64
2
32
256
64
4


(CMRSA7/USA400/MW2)









CA-MRSA
256
8
1
8
128
8
16


(CMRSA7/USA400/MW2)









CA-MRSA
512
256
2
128
256
64
4


(CMRSA7/USA400/MW2)









CA-MRSA
512
64
2
32
256
64
4


(CMRSA7/USA400/MW2)









CA-MRSA
512
128
2
64
512
64
8


(CMRSA7/USA400/MW2)









CA-MRSA
512
64
2
32
256
64
4


(CMRSA7/USA400/MW2)









CA-MRSA
512
64
1
64
256
32
8


(CMRSA7/USA400/MW2)









CA-MRSA
512
64
2
32
256
64
4


(CMRSA7/USA400/MW2)









CA-MRSA
512
64
2
32
256
32
8


(CMRSA7/USA400/MW2)









CA-MRSA (CMRSA10/USA300)
512
64
2
32
256
32
8


CA-MRSA (CMRSA10/USA300)
512
128
2
64
512
64
8


CA-MRSA (CMRSA10/USA300)
512
64
2
32
256
32
8


CA-MRSA (CMRSA10/USA300)
512
128
2
64
256
64
4


CA-MRSA (CMRSA10/USA300)
256
128
2
64
512
64
8


CA-MRSA (CMRSA10/USA300)
512
128
2
64
256
64
4


CA-MRSA (CMRSA10/USA300)
1024
64
1
64
256
32
8


CA-MRSA (CMRSA10/USA300) 
256
128
2
64
512
128
4


CA-MRSA (CMRSA10/USA300)
1024
128
2
64
512
128
4


CA-MRSA (CMRSA10/USA300)
1024
32
2
16
256
64
4


CA-MRSA (CMRSA10/USA300) 
512
64
2
32
256
32
8


CA-MRSA (CMRSA10/USA300) 
512
128
2
64
512
64
8


CA-MRSA (CMRSA10/USA300) 
512
64
1
64
256
16
16


CA-MRSA (CMRSA10/USA300) 
512
128
2
64
512
32
16


CA-MRSA (CMRSA10/USA300)
1024
128
2
64
256
64
4


CA-MRSA (CMRSA10/USA300) 
512
64
2
32
256
64
4


CA-MRSA (CMRSA10/USA300) 
512
4
1
4
128
8
16


CA-MRSA (CMRSA10/USA300) 
512
64
4
16
256
64
4


CA-MRSA (CMRSA10/USA300) 
512
32
2
16
256
32
8


CA-MRSA (CMRSA10/USA300) 
512
64
2
32
256
32
8


CA-MRSA (CMRSA10/USA300) 
512
4
2
2
64
8
8


CA-MRSA (CMRSA10/USA300) 
512
64
2
32
256
8
32


CA-MRSA (CMRSA10/USA300) 
512
128
4
32
512
128
4


CA-MRSA (CMRSA10/USA300) 
512
64
1
64
256
8
32


CA-MRSA (CMRSA10/USA300) 
512
64
2
32
256
64
4


CA-MRSA (CMRSA10/USA300) 
512
64
2
32
256
32
8


CA-MRSA (CMRSA10/USA300) 
512
64
1
64
256
8
32


CA-MRSA (CMRSA10/USA300) 
512
64
2
32
256
32
8


CA-MRSA (CMRSA10/USA300) 
512
64
2
32
256
32
8


CA-MRSA (CMRSA10/USA300) 
512
2
1
2
64
4
16


CA-MRSA (CMRSA10/USA300) 
256
128
4
32
256
128
2


CA-MRSA (CMRSA10/USA300)
1024
64
2
32
256
32
8


CA-MRSA (CMRSA10/USA300) 
512
64
2
32
256
32
8


CA-MRSA (CMRSA10/USA300) 
256
64
1
64
256
16
16


CA-MRSA (CMRSA10/USA300)
512
64
2
32
256
32
8


CA-MRSA (CMRSA10/USA300)
512
128
4
32
256
128
2


S. aureus
512
0.5/0.5
0.5
0.5
4/8
2
2


E. coli
64
2/2
1
2
8/8
8
8





* MIC ≦ 8 ug/ml is considered susceptible to treatment


* MIC ≧ 32 ug/ml is considered resistant to treatment













TABLE 15







In Vitro Activity of Pep35 with and without Cefazolin or Cephalexin against HA-MRSA















Cefazolin & 

Cephalexin & 





PEP35 32 ug/mL

PEP35 32 ug/mL


















Fold 


Fold


Organism
PEP35 
Cefazolin
MIC *
reduction
Cephalexin
MIC *
reduction

















HA-MRSA (CMRSA1/USA600) 
256
128
8
16
256
128
2


HA-MRSA
256
8
1
8
128
16
8


(CMRSA2/USA100/800/NY) 









HA-MRSA
512
256
4
64
512
128
4


(CMRSA2/USA100/800/NY)









HA-MRSA
512
512
256
2
>512
512
≧2


(CMRSA2/USA100/800/NY)









HA-MRSA
512
128
2
64
256
32
8


(CMRSA2/USA100/800/NY)









HA-MRSA
512
512
128
4
512
512
0


(CMRSA2/USA100/800/NY)









HA-MRSA
512
128
2
64
512
64
8


(CMRSA2/USA100/800/NY)









HA-MRSA
1024
512
256
2
>512
512
≧2


(CMRSA2/USA100/800/NY)









HA-MRSA
512
256
2
128
512
64
8


(CMRSA2/USA100/800/NY)









HA-MRSA
1024
512
128
4
512
512
0


(CMRSA2/USA100/800/NY)









HA-MRSA
512
256
2
128
512
16
32


(CMRSA2/USA100/800/NY)









HA-MRSA
256
256
64
4
512
256
2


(CMRSA2/USA100/800/NY)









HA-MRSA
128
512
8
64
>512
256
≧4


(CMRSA2/USA100/800/NY)









HA-MRSA
512
256
4
64
256
256
0


(CMRSA2/USA100/800/NY)









HA-MRSA
256
256
1
256
256
16
16


(CMRSA2/USA100/800/NY)









HA-MRSA
512
128
2
64
512
64
8


(CMRSA2/USA100/800/NY)









HA-MRSA
128
32
1
32
256
16
16


(CMRSA2/USA100/800/NY)









HA-MRSA
512
256
8
32
512
128
4


(CMRSA2/USA100/800/NY)









HA-MRSA
512
32
1
32
256
8
32


(CMRSA2/USA100/800/NY)









HA-MRSA
512
32
2
16
256
64
4


(CMRSA2/USA100/800/NY)









HA-MRSA
256
512
128
4
512
256
2


(CMRSA2/USA100/800/NY)









HA-MRSA
512
128
2
64
512
128
1


(CMRSA2/USA100/800/NY)









HA-MRSA
512
256
4
64
512
128
1


(CMRSA2/USA100/800/NY)









HA-MRSA
1024
32
2
16
256
64
1


(CMRSA2/USA100/800/NY)









HA-MRSA
256
256
64
4
512
256
2


(CMRSA2/USA100/800/NY)









HA-MRSA
1024
256
64
4
512
256
2


(CMRSA2/USA100/800/NY)









HA-MRSA
1024
512
256
2
>512
512
≧2


(CMRSA2/USA100/800/NY)









HA-MRSA
1024
4
1
4
128
32
4


(CMRSA2/USA100/800/NY)









HA-MRSA
512
256
128
2
512
256
2


(CMRSA2/USA100/800/NY)









HA-MRSA
256
256
16
16
512
256
2


(CMRSA2/USA100/800/NY)









HA-MRSA
512
128
4
32
512
128
4


(CMRSA2/USA100/800/NY)









HA-MRSA
512
256
8
32
512
128
4


(CMRSA2/USA100/800/NY)









HA-MRSA
512
128
4
32
256
64
4


(CMRSA2/USA100/800/NY)









HA-MRSA
256
256
16
16
512
256
2


(CMRSA2/USA100/800/NY)









HA-MRSA
512
32
2
16
256
32
8


(CMRSA2/USA100/800/NY)









HA-MRSA
512
512
256
20
>512
512
≧2


(CMRSA2/USA100/800/NY)









HA-MRSA
512
128
4
32
512
128
4


(CMRSA2/USA100/800/NY)









HA-MRSA
512
32
8
4
512
128
4


(CMRSA2/USA100/800/NY)









HA-MRSA
512
64
2
8
256
64
4


(CMRSA2/USA100/800/NY)









HA-MRSA
256
256
8
32
512
128
4


(CMRSA2/USA100/800/NY)









HA-MRSA
512
128
4
32
512
128
4


(CMRSA2/USA100/800/NY)









HA-MRSA
512
64
2
32
256
64
4


(CMRSA2/USA100/800/NY)









HA-MRSA (CMRSA6)
512
256
64
4
256
256
0


HA-MRSA (CMRSA6)
512
512
256
2
512
256
2


HA-MRSA (CMRSA3/6)
256
256
64
64
256
256
0


HA-MRSA (CMRSA3/6)
512
256
256
0
512
512
0


HA-MRSA (CMRSA5/USA500) 
512
2
1
2
256
8
32


HA-MRSA (CMRSA8)
1024
64
4
16
256
128
2


HA-MRSA (CMRSA4/USA200) 
512
256
128
2
512
256
2


HA-MRSA (CMRSA4/USA200)
512
128
2
64
256
128
2


S. aureus
1024
0.5/0.5
0.5
0.5
4/4
2
4


E. coli
64
2/2
1
2
8/8
4
8





* MIC ≦ 8 ug/ml is considered susceptible to treatment


* MIC ≧ 32 ug/ml is considered resistant to treatment

## Claims

1. A method for the prevention or treatment of antibiotic resistant S. aureus infection or reinfection, comprising administering a therapeutically effective amount of a beta-lactam antibiotic and a hyaluronic acid binding peptide comprising the amino acid sequence SEQ ID NO: 2.

2. The method of claim 1 wherein the S. aureus is a methicillin resistant variant (MRSA), a vancomycin resistant variant (VRSA), or a vancomycin intermediate resistant variant (VISA).

3. The method of claim 1 wherein the beta-lactam antibiotic and the hyaluronic acid binding peptide are administered within 15 minutes of each other.

4. The method of claim 1 wherein the beta-lactam antibiotic is a cephalosporin.

5. The method of claim 1 wherein the cephalosporin is a first generation cephalosporin.

6. The method of claim 1 wherein the cephalosporin is a second, third, fourth or fifth generation cephalosporin.

7. The method of claim 4 wherein the cephalosporin is selected from the group consisting of: cefacetrile, Cefadroxil, Cephalexin, Cefaloglycin, Cefalonium, Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, Cefazaflur, Cefazedone, Cefazolin, Cefradine, Cefroxadine, Ceftezole, Cefaclor, Cefonicid, Cefprozil, Cefuroxime, Cefuzonam, Cefmetazole, Cefotetan, Cefoxitin, Carbacephems, Cephamycins, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefixime, Cefmenoxime, Cefodizime Cefotaxime, Cefovecin, Cefpimizole, Cefpodoxime Cefteram, Ceftibuten, Ceftiofur, Ceftiolene, Ceftizoxime, Ceftriaxone, Cefoperazone, Ceftazidime, Oxacephems, Cefclidine, Cefepime, Cefluprenam, Cefoselis, Cefozopran, Cefpirome, Cefquinome, Oxacephems, Ceftobiprole, medocaril, Ceftaroline, Cefaloram, Cefaparole, Cefcanel, Cefedrolor, Cefempidone, Cefetrizole, Cefivitril, Cefmatilen, Cefmepidium, Cefoxazole, Cefrotil, Cefsumide, Ceftaroline, Ceftioxide, and Cefuracetime.

8. The method of claim 1 wherein the antibiotic is selected from the group consisting of methicillin, vancomycin, meropenem, and piperacillin/tazobactam.

9. The method of claim 1 wherein the hyaluronic acid binding peptide consists of the amino acid sequence SEQ ID NO: 2.

10. A method of potentiating a beta-lactam antibiotic against antibiotic resistant S. aureus comprising administering a hyaluronic acid binding peptide, comprising the amino acid sequence SEQ ID NO: 2, with the beta-lactam antibiotic.

11. The method of claim 10 wherein the antibiotic resistant S. aureus is a methicillin resistant variant (MRSA), a vancomycin resistant variant (VRSA) or a vancomycin intermediate resistant variant (VISA).

12. The method of claim 10 wherein the beta-lactam antibiotic and the hyaluronic acid binding peptide are administered simultaneously or concurrently.

13. The method of claim 12 wherein the beta-lactam antibiotic and the hyaluronic acid binding peptide are for administration within 15 minutes of each other.

14. The method of claim 12 wherein the beta-lactam antibiotic is a cephalosporin.

15. The method of claim 14 wherein the cephalosporin is a first generation cephalosporin.

16. The method of claim 14 wherein the cephalosporin is a second, third, fourth or fifth generation cephalosporin.

17. The method of claim 14 wherein the cephalosporin is selected from the group consisting of: cefacetrile, Cefadroxil, Cephalexin, Cefaloglycin, Cefalonium, Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, Cefazaflur, Cefazedone, Cefazolin, Cefradine, Cefroxadine, Ceftezole, Cefaclor, Cefonicid, Cefprozil, Cefuroxime, Cefuzonam, Cefmetazole, Cefotetan, Cefoxitin, Carbacephems, Cephamycins, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefixime, Cefmenoxime, Cefodizime, Cefotaxime, Cefovecin, Cefpimizole, Cefpodoxime, Cefteram, Ceftibuten, Ceftiofur, Ceftiolene, Ceftizoxime, Ceftriaxone, Cefoperazone, Ceftazidime, Oxacephems, Cefclidine, Cefepime, Cefluprenam, Cefoselis, Cefozopran, Cefpirome, Cefquinome, Oxacephems, Ceftobiprole, medocaril, Ceftaroline, Cefaloram, Cefaparole, Cefcanel, Cefedrolor, Cefempidone, Cefetrizole, Cefivitril, Cefmatilen, Cefmepidium, Cefoxazole, Cefrotil, Cefsumide, Ceftaroline, Ceftioxide, and Cefuracetime.

18. The method of claim 12 wherein the antibiotic is selected from the group consisting of methicillin, vancomycin, meropenem, and piperacillin/tazobactam.

19. The method of claim 12 wherein the hyaluronic acid binding peptide consists of the amino acid sequence SEQ ID NO: 2.

20. A composition comprising a beta-lactam antibiotic and a hyaluronic acid binding peptide comprising the amino acid sequence SEQ ID NO: 2.

21. The composition of claim 20 wherein the beta-lactam antibiotic is a cephalosporin.

22. The composition of claim 21 wherein the cephalosporin is a first generation cephalosporin.

23. The composition of claim 21 wherein the cephalosporin is a second, third, fourth or fifth generation cephalosporin.

24. The composition of claim 21 wherein the cephalosporin is selected from the group consisting of: cefacetrile, Cefadroxil, Cephalexin, Cefaloglycin, Cefalonium, Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, Cefazaflur, Cefazedone, Cefazolin, Cefradine, Cefroxadine, Ceftezole, Cofactor, Cefonicid, Cefprozil, Cefuroxime, Cefuzonam, Cefmetazole, Cefotetan, Cefoxitin, Carbacephems, Cephamycins, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefixime, Cefmenoxime, Cefodizime, Cefotaxime, Cefovecin, Cefpimizole, Cefpodoxime, Cefteram, Ceftibuten, Ceftiofur, Ceftiolene, Ceftizoxime, Ceftriaxone, Cefoperazone, Ceftazidime, Oxacephems, Cefclidine, Cefepime, Cefluprenam, Cefoselis, Cefozopran, Cefpirome, Cefquinome, Oxacephems, Ceftobiprole, medocaril, Ceftaroline, Cefaloram, Cefaparole, Cefcanel, Cefedrolor, Cefempidone, Cefetrizole, Cefivitril, Cefmatilen, Cefmepidium, Cefoxazole, Cefrotil, Cefsumide, Ceftaroline, Ceftioxide, and Cefuracetime.

25. The composition of claim 20 wherein the antibiotic is selected from the group consisting of methicillin, vancomycin, meropenem, and piperacillin/tazobactam.

26. The composition of claim 20 wherein the hyaluronic acid binding peptide consists of the amino acid sequence SEQ ID NO: 2.

27. The method of claim 2, wherein the combination of the hyaluronic acid binding protein with the antibiotic reduces the minimum inhibitory concentration of the MRSA strain.

28. The method of claim 2, wherein the combination of the hyaluronic acid binding protein with the antibiotic reduces the minimum inhibitory concentration of the MRSA strain 2-256 fold.

29. The method of claim 2, wherein the combination of the hyaluronic acid binding protein with the antibiotic reverts the minimum inhibitory concentration of the MRSA strain to susceptible (≦8 ug/mL).