Lung-Targeting Lysostaphin Microspheres for Methicillin-Resistant Staphylococcus aureus Pneumonia Treatment and Prevention.

Multifunctional antimicrobial strategies are urgently needed to treat methicillin-resistant Staphylococcus aureus (MRSA) caused pneumonia due to its increasing resistance, enhanced virulence, and high pathogenicity. Here, we report that lysostaphin, a bacteriolytic enzyme, encapsulated within poly(lactic-co-glycolic acid) microspheres (LyIR@MS) specially treats planktonic MRSA bacteria, mature biofilms, and related pneumonia. Optimized LyIR@MS with suitable diameters could deliver a sufficient amount of lysostaphin to the lung without a decrease in survival rate after intravenous injection. Furthermore, the degradable properties of the carrier make it safe for targeted release of lysostaphin to eliminate MRSA, repressing the expression of virulence genes and improving the sensitivity of biofilms to host neutrophils. In the MRSA pneumonia mouse model, treatment or prophylaxis with LyIR@MS significantly improved survival rate and relieved inflammatory injury without introducing adverse events. These findings suggest the clinical translational potential of LyIR@MS for the treatment of MRSA-infected lung diseases.

The bactericidal effect of lysostaphin coupled with liposomal vancomycin as a dual combating system applied directly on methicillin-resistant Staphylococcus aureus infected skin wounds in mice.

BACKGROUND AND AIM: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of surgical infection, and its resistance to numerous conventional antibiotics makes treatment difficult. Although vancomycin is often an effective agent for the initial therapy of MRSA, clinical failure sometimes occurs. Therefore, there is an urgent need to develop better therapies. Here, we prepared some vancomycin-loaded nanoliposomes coupled with anti-staphylococcal protein (lysostaphin) and evaluated their in vitro and in vivo efficacy as a topical MRSA therapy. METHODS: Vancomycin was encapsulated in liposomes, and the coupling of lysostaphin with the surface of liposomes was carried out through cyanuric functional groups. The bactericidal efficacies and a full characterization were evaluated. To define different nanoliposomal-bacterium interactions and their bactericidal effect, flow cytometry was employed. Finally, in vivo, the topical antibacterial activity of each formulation was measured against surgical wound MRSA infection in a mouse model. RESULTS: High encapsulation and conjugation efficiency were achieved for all formulations. All the formulations showed a significant reduction in bacterial counts (p<0.05). The targeted liposomes more effectively suppress bacterial infection in vitro and in vivo relative to equivalent doses of untargeted vancomycin liposome. The flow cytometry results confirmed liposome-bacterium interactions, which increased during the incubation time. The maximum binding rate and the bactericidal effect were significantly higher in targeted liposomes (p<0.05) compared with control liposomes. CONCLUSION: Our data suggest a novel nano-vehicle (lysostaphin-conjugated coupled liposomal vancomycin) which could be used as a great topical antimicrobial construct for treatment of MRSA skin infections.

Reduced vancomycin susceptibility and increased macrophage survival in Staphylococcus aureus strains sequentially isolated from a bacteraemic patient during a short course of antibiotic therapy.

PURPOSE: The purpose of the present study was to determine the relatedness of Staphylococcus aureus strains successively isolated over a 7-day period from a single bacteraemic patient undergoing antibiotic treatment with vancomycin. METHODS: The S. aureus strains had been isolated and sequenced previously. Antibiotic susceptibility testing, population analysis profiling, and lysostaphin sensitivity and phagocytic killing assays were used to characterize these clonal isolates. RESULTS: The seven isolates (MEH1-MEH7) were determined to belong to a common multilocus sequence type (MLST) and spa type. Within the third and fifth day of vancomycin treatment, mutations were observed in the vraS and rpsU genes, respectively. Population analysis profiles revealed that the initial isolate (MEH1) was vancomycin-susceptible S. aureus (VSSA), while those isolated on day 7 were mostly heteroresistant vancomycin-intermediate S. aureus (hVISA). Supporting these findings, MEH7 was also observed to be slower in growth, to have an increase in cell wall width and to have reduced sensitivity to lysostaphin, all characteristics of VISA and hVISA strains. In addition, MEH7, although phagocytosed at numbers comparable to the initial isolate, MEH1, survived in higher numbers in RAW 264.7 macrophages. Macrophages infected with MEH7 also released more TNF-α and IFN-1ß. CONCLUSION: We report an increasing resistance to vancomycin coupled with daptomycin that occurred within approximately 3 days of receiving vancomycin and steadily increased until the infection was cleared with an alternative antibiotic therapy. This study reiterates the need for rapid, efficient and accurate detection of hVISA and VISA infections, especially in high-bacterial load, metastatic infections like bacteraemia.

Treatment of methicillin-resistant Staphylococcus aureus in neonatal mice: lysostaphin versus vancomycin.

S. aureus is a significant cause of late-onset sepsis in neonates. Increasing antibiotic resistance, however, requires additional treatment options. Lysostaphin, an endopeptidase, has that potential. The objective of this study is to compare lysostaphin versus vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) in a neonatal mouse model. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against MRSA strain USA300 were determined using standard methods. To determine pharmacokinetics, neonatal pups received either vancomycin or lysostaphin intraperitoneal and serum samples were obtained. To evaluate efficacy, pups were infected s.c. and littermates randomized to receive either saline, vancomycin, or lysostaphin intraperitoneal. Pups were observed for survival and growth. Quantitative blood cultures were obtained 24 h after infection. The MIC/MBC for vancomycin and lysostaphin were 0.71/1.19 microg/mL and <0.008/0.015 microg/mL, respectively. Mean lysostaphin concentrations ranged from 2.34 to 8.92 microg/mL. Mean vancomycin concentrations ranged from 1.72 to 11.2 microg/mL. Lysostaphin improved survival compared with placebo (p < 0.00001) and vancomycin (p < 0.03). There was no significant difference in growth among the groups. All treatment regimens resulted in less bacteremia compared with placebo (p < 0.0001). Lysostaphin appears to be more effective than vancomycin in treating MRSA in a neonatal model.

In vitro activity of lysostaphin, mupirocin, and tea tree oil against clinical methicillin-resistant Staphylococcus aureus.

Colonization of methicillin-resistant Staphylococcus aureus (MRSA) commonly leads to infection by the same strain. We examined the activity of lysostaphin, mupirocin, and tea tree oil against clinical MRSA (n = 98) isolates. MIC(50) (range) were as follows: lysostaphin, 0.125 mg/L (0.125-0.25); mupirocin, 0.5 mg/L (0.19-1024); tea tree oil, 1024 mg/L (512-2048). High- and low-level mupirocin resistance was noted in 9.2% of our MRSA isolates. Time kill results indicate MRSA activity at 24 h was lysostaphin = gentamicin = vancomycin (P mupirocin > tea tree oil (P >or= .05). Checkerboard testing indicated a synergistic relationship between lysostaphin and mupirocin in combination with gentamicin. Antagonism was observed with the combination of vancomycin and tea tree oil; time kill studies confirmed this result. Decolonization options are limited and resistance to mupirocin exists. Lysostaphin and tea tree oil may offer additional therapeutic options for the decolonization of MRSA where current treatment alternatives are limited.

Lysostaphin as a potential therapeutic agent for staphylococcal biofilm eradication.

The aim was to study the activity of lysostaphin in monotherapy or in combination with oxacillin, towards biofilms built by clinical and reference S. aureus and S. epidermidis strains in the wells of microplate, in the chambers of a LabTekII chamber slide or on the polyethylene catheter. MICs of oxacillin and lysostaphin for planktonic bacteria were determined according to the standards of NCCLS. BIC (Biofilm Inhibitory Concentration) was estimated by the MTT assay. The integrity of biofilm treated with antimicrobials was also examined: by staining with FITC and laser scanning fluorescence confocal microscopy and visually by TTC reduction assay. Despite the fact that susceptibility of planktonic cultures of 25 staphylococcal strains to lysostaphin action was various, we have demonstrated the effectiveness of lysostaphin in the treatment of biofilm, built not only on the flat surface of the microplates but also on catheter's surface. The synergistic effect of subBIC lysostaphin+oxacillin was observed for MSSA and MRSA biofilms but not for 1474/01 hVISA strain. Also BICOXA for S. epidermidis RP12 and A4c strains, but not for 6756/99 MRSE biofilms was reduced when lysostaphin was simultaneously used.

Cell wall composition and decreased autolytic activity and lysostaphin susceptibility of glycopeptide-intermediate Staphylococcus aureus.

The cell wall composition and autolytic properties of passage-selected glycopeptide-intermediate Staphylococcus aureus (GISA) isolates and their parent strains were studied in order to investigate the mechanism of decreased vancomycin susceptibility. GISA had relatively modest changes in peptidoglycan composition involving peptidoglycan interpeptide bridges and somewhat decreased cross-linking compared to that of parent strains. The cell wall phosphorus content of GISA strains was lower than that of susceptible parent strains, indicating somewhat lower wall teichoic acid levels in the GISA strains. Similar to whole cells, isolated crude cell walls retaining autolytic activity of GISA had drastically reduced autolytic activity compared to that of parent strains, and this arose early in the development of the GISA phenotype. This was due to an alteration in the autolytic enzymes of GISA as revealed by normal susceptibility of GISA-purified cell walls to parental strain autolysin extract and lower activity and altered peptidoglycan hydrolase activity profiles in GISA autolysin extracts compared to those of parent strains. Northern blot analysis indicated that expression of atl, the major autolysin gene, was significantly downregulated in a GISA strain compared to that of its parent strain. In contrast to whole cells, which showed decreased lysostaphin susceptibility, purified cell walls of GISA showed increased susceptibility to lysostaphin. We suggest that in our GISA strains, decreased autolytic activity is involved in the tolerance of vancomycin and the activities of endogenous autolysins are important in conferring sensitivity to lysostaphin on whole cells.

Pseudomonas aeruginosa LasA protease in treatment of experimental staphylococcal keratitis.

LasA protease is a staphylolytic endopeptidase secreted by Pseudomonas aeruginosa. We have examined the effectiveness of LasA protease in the treatment of staphylococcal keratitis caused by methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates in a rabbit model. Keratitis was induced by intrastromal injection of the bacteria. The eyes were treated topically, and the efficacy of LasA protease was compared to those of lysostaphin (a staphylolytic protease secreted by Staphylococcus simulans) and vancomycin. When treatment was initiated early (4 h) after infection, practically all of the MSSA- and MRSA-infected corneas were sterilized by LasA protease, and its efficacy in eradicating the bacteria was comparable to those of lysostaphin and vancomycin. By contrast, most of the control corneas were heavily infected, with median values of 4.5 x 10(6) (MSSA) and 5 x 10(5) (MRSA) CFU/cornea (P < 0.001). When treatment was initiated late (10 h) after infection, LasA protease reduced the numbers of CFU in both MSSA- and MRSA-infected corneas by 3 to 4 orders of magnitude compared to the numbers of CFU for the controls (median values, 1,380 and 30 CFU/cornea, respectively, for the treated animals compared to 1.2 x 10(6) and 5 x 10(5) CFU/cornea for the respective controls [P = 0.001]), and it was more effective than vancomycin in eradicating MRSA cells (P = 0.02). In both the early- and the late-treatment protocols, the clinical scores for eyes treated with LasA protease were significantly lower than those for the eyes of the corresponding controls and comparable to those for the lysostaphin- and vancomycin-treated eyes. We conclude that LasA protease is effective in the treatment of experimental S. aureus keratitis in rabbits and may have potential for the treatment of disease in humans.

Determination of phagocytosis of 32P-labeled Staphylococcus aureus by bovine polymorphonuclear leukocytes.

A procedure for the measurement of phagocytosis by bovine polymorphonuclear leukocytes (PMN) of 32P-labeled Staphylococcus aureus was modified so that a larger number of samples could be compared in a single run, and smaller volumes of sample, PMN, and 32P-labeled S aureus could be used. Results were highly reproducible, with a coefficient of variation between duplicate determinations of less than or equal to 2%. Lysostaphin was prepared from the supernatant of S staphylolyticus and was compared with a commercially available preparation. Effects of lysostaphin on PMN and influence of incubation media on release of 32P from 32P-labeled S aureus by lysostaphin were examined.

Cell-wall proteins of Staphylococcus aureus : a kinetic study of release by lysostaphin.

The kinetics of cell-wall hydrolysis of Staphylococcus aureus (strain Cowan I) by lysostaphin was studied by turbidity, quantitative determination of hexosamines, phosphorus, total proteins, protein A and by electron microscopy. In our experimental conditions, the lytic activity of lysostaphin was very fast and no characteristic cell-wall structures could be seen after five minutes. The release of hexosamines, phosphorus and protein A was synchronous and could be recorded even though turbidity had become stable. At the end of the hydrolysis, the totality of these components was solubilized. Total proteins represented about 14% of the cell-wall dry weight. During hydrolysis protein A was the only protein solubilized.