Restoration of sensitivity of a diverse set of drug-resistant Staphylococcus clinical strains by bactericidal protein P128.

PURPOSE: P128, a phage-derived lysin, exerts antibacterial activity on staphylococci by cleaving the pentaglycine-bridge of peptidoglycan. We sought to determine whether the presence of P128 could re-sensitize drug-resistant bacteria to antibiotics by virtue of its cell wall degrading property. METHODOLOGY: P128 was tested in combination with standard-of-care (SoC) drugs by chequerboard assays on planktonic cells and biofilms of strains individually resistant to these drugs. The bactericidal effect of P128 and drug combinations on planktonic cells and biofilms was measured by c.f.u. reduction assays. A mouse model of MRSA bacteraemia was used to test the efficacy of P128 and oxacillin in combination. RESULTS: A combination of sub-MIC P128 (0.025-0.20 µg ml-1) and 0.5 µg ml-1 of oxacillin resulted in inhibition of bacterial growth in four MRSA strains. Similar results were seen with all the other drugs tested, wherein sub-MIC of P128 re-sensitized S. aureus and CoNS strains to SoC drugs. The chequerboard assays on strains of S. aureus and CoNS showed that combinations of P128 and antibiotics consistently inhibited bacterial growth on biofilms. Data from scanning electron microscopy and c.f.u. reduction assays on drug-resistant S. aureus and CoNS demonstrated that sub-MICs of P128 and SoC antibiotics could kill biofilm-embedded bacteria. In vivo, a combination of sub-therapeutic doses of P128 and oxacillin could help protect animals from fatal bacteraemia. CONCLUSION: The ability of P128 to re-sensitize bacteria to SoC drugs suggests that combinations of P128 and SoC antibiotics can potentially be developed to treat infections caused by drug-resistant strains of staphylococci.

Phage-derived lysins as potential agents for eradicating biofilms and persisters.

Bacterial biofilms are highly resistant to the action of antibiotics. Presence of persisters, phenotypically resistant populations of bacterial cells, is thought to contribute toward recalcitrance of biofilms. The phage-derived lysins, by virtue of their ability to cleave the peptidoglycan of bacterial cells in an enzymatic manner, have the unique ability to kill dormant cells. Several lysins have shown potent antibiofilm activity in vitro. The fact that lysins have shown better efficacy than conventional drugs in animal models of endocarditis and other infections involving biofilms suggests that the lysins can potentially be developed against difficult-to-treat bacterial infections.

Efficient Killing of Planktonic and Biofilm-Embedded Coagulase-Negative Staphylococci by Bactericidal Protein P128.

Coagulase-negative staphylococci (CoNS) are the major causative agents of foreign-body-related infections, including catheter-related bloodstream infections. Because of the involvement of biofilms, foreign-body-related infections are difficult to treat. P128, a chimeric recombinant phage-derived ectolysin, has been shown to possess bactericidal activity on strains of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). We tested the killing potential of P128 on three clinically significant species of CoNS, S. epidermidis, S. haemolyticus, and S. lugdunensis, under a variety of physiological conditions representing growing and nongrowing states. The MIC90 and minimum bactericidal concentration at which 90% of strains tested are killed (MBC90) of P128 on 62 clinical strains of CoNS were found to be 16 and 32 µg/ml (0.58 and 1.16 µM), respectively, demonstrating the bactericidal nature of P128 on CoNS strains. Serum showed a potentiating effect on P128 inhibition, as indicated by 4- to 32-fold lower MIC values observed in serum. P128 caused a rapid loss of viability in all CoNS strains tested. Persisters of CoNS that were enriched in the presence of vancomycin or daptomycin were killed by P128 at 1× the MIC in a rapid manner. Low concentrations of P128 caused a 2- to 5-log reduction in CFU in stationary-phase or poorly metabolizing CoNS cultures. P128 at low concentrations eliminated CoNS biofilms in microtiter plates and on the surface of catheters. Combinations of P128 and standard-of-care (SoC) antibiotics were highly synergistic in inhibiting growth in preformed biofilms. Potent activity on planktonic cells, persisters, and biofilms of CoNS suggests that P128 is a promising candidate for the clinical development of treatments for foreign-body-related and other CoNS infections.

Antibiofilm Activity and Synergistic Inhibition of Staphylococcus aureus Biofilms by Bactericidal Protein P128 in Combination with Antibiotics.

P128 is an antistaphylococcal protein, comprising a cell wall-degrading enzymatic region and a Staphylococcus-specific binding region, which possesses specific and potent bactericidal activity against sensitive and drug-resistant strains of Staphylococcus aureus To explore P128's ability to kill S. aureus in a range of environments relevant to clinical infection, we investigated the anti-S. aureus activity of P128 alone and in combination with standard-of-care antibiotics on planktonic and biofilm-embedded cells. P128 was found to have potent antibiofilm activity on preformed S. aureus biofilms as detected by CFU reduction and a colorimetric minimum biofilm inhibitory concentration (MBIC) assay. Scanning electron microscopic images of biofilms formed on the surfaces of microtiter plates and on catheters showed that P128 at low concentrations could destroy the biofilm structure and lyse the cells. When it was tested in combination with antibiotics which are known to be poor inhibitors of S. aureus in biofilms, such as vancomycin, gentamicin, ciprofloxacin, linezolid, and daptomycin, P128 showed highly synergistic antibiofilm activity that resulted in much reduced MBIC values for P128 and the individual antibiotics. The synergistic effect was seen for both sensitive and resistant isolates of S. aureus Additionally, in an in vitro mixed-biofilm model mimicking the wound infection environment, P128 was able to prevent biofilm formation by virtue of its anti-Staphylococcus activity. The potent S. aureus biofilm-inhibiting activity of P128 both alone and in combination with antibiotics is an encouraging sign for the development of P128 for treatment of complicated S. aureus infections involving biofilms.

Simulated hatchery system to assess bacteriophage efficacy against Vibrio harveyi.

Vibriosis caused by luminous Vibrio harveyi commonly contributes to poor survival in shrimp hatcheries and aquaculture ponds. Lytic bacteriophages pathogenic for V. harveyi are currently being investigated as an alternative to antibiotics to prevent vibriosis. Here, 8 bacteriophages were isolated from oysters and clams using V. harveyi strains as baiting hosts. Among these bacteriophages, 1 strain (VHP6b) identified as broadly pathogenic for 27 V. harveyi strains examined was further characterized by electron microscopy and genome sequence analysis. Phage VHP6b possessed a tail and morphology consistent with it being a member of the family Siphoviridae, and its genome and proteome were most closely related to the Vibrio phages SSP02 and MAR10. An integrase gene essential for lysogeny was not evident. The ability of bacteriophage VHP6b to protect shrimp postlarvae against vibriosis caused by V. harveyi strain VH6 was demonstrated in a model system designed to simulate typical hatchery conditions. Bacteriophage treatment improved survival of postlarvae by 40 to 60% under these conditions, so therapies based on this or other bacteriophages may be useful in shrimp hatcheries.

Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan.

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other ß-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

Efficacy of anti-staphylococcal protein P128 for the treatment of canine pyoderma: potential applications.

In this study, we demonstrate the antibacterial activity of P128 on Staphylococcus isolates responsible for canine pyoderma. Eighty seven swabs were collected from dogs suffering from pyoderma and subjected to antibiotic sensitivity test and 46 Staphylococcus strains were isolated and characterized. In-vitro antimicrobial susceptibility testing with P128 was done by Minimum Inhibitory Concentration (MIC) method as per CLSI guidelines. All the Staphylococci isolated from the dogs with pyoderma, although showed resistance to various antibiotics tested, were lysed by P128. Clinical efficacy of P128 was examined in 17 dogs with pyoderma by application of the P128 hydrogel twice daily for 8 days and the results indicated complete healing of all the lesions of all the dogs under treatment. Under the conditions of this study, P128 was found to be a potent convenient proteinaceous drug for the treatment of staphylococcal pyoderma in dogs.

Antistaphylococcal activity of bacteriophage derived chimeric protein P128.

BACKGROUND: Bacterial drug resistance is one of the most significant challenges to human health today. In particular, effective antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA) are urgently needed. A causal relationship between nasal commensal S. aureus and infection has been reported. Accordingly, elimination of nasal S. aureus reduces the risk of infection. Enzymes that degrade bacterial cell walls show promise as antibacterial agents. Bacteriophage-encoded bacterial cell wall-degrading enzymes exhibit intrinsic bactericidal activity. P128 is a chimeric protein that combines the lethal activity of the phage tail-associated muralytic enzyme of Phage K and the staphylococcal cell wall targeting-domain (SH3b) of lysostaphin.Here we report results of in vitro studies evaluating the susceptibility of staphylococcal strains to this novel protein. RESULTS: Using the broth microdilution method adapted for lysostaphin, we found that P128 is effective against S. aureus clinical strains including MRSA, methicillin-sensitive S. aureus (MSSA), and a mupirocin-resistant S. aureus. Minimum bactericidal concentrations and minimum inhibitory concentrations of P128 (1-64 µg/mL) were similar across the 32 S. aureus strains tested, demonstrating its bactericidal nature.In time-kill assays, P128 reduced colony-forming units by 99.99% within 1 h and inhibited growth up to 24 h.In an assay simulating topical application of P128 to skin or other biological surfaces, P128 hydrogel was efficacious when layered on cells seeded on solid media. P128 hydrogel was lethal to Staphylococci recovered from nares of healthy people and treated without any processing or culturing steps, indicating its in situ efficacy. This methodology used for in vitro assessment of P128 as an agent for eradicating nasal carriage is unique. CONCLUSIONS: The novel chimeric protein P128 is a staphylococcal cell wall-degrading enzyme under development for clearance of S. aureus nasal colonization and MRSA infection. The protein is active against globally prevalent antibiotic-resistant clinical isolates and other clinically significant staphylococcal species including S. epidermidis. The P128 hydrogel formulation was bactericidal against Staphylococci including S. aureus recovered from the nares of 31 healthy people, demonstrating its in situ efficacy.