Comparison of Four Methods for Determining Lysostaphin Susceptibility of Various Strains of Staphylococcus aureus

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Abstract
Lysostaphin is an endopeptidase that cleaves the pentaglycine cross-bridges of the staphylococcal cell wall rapidly lysing the bacteria. Recently, lysostaphin has been examined for its potential to treat infections and to clear Staphylococcus aureus nasal colonization, requiring a reliable method for determining the lysostaphin susceptibility of strains of S. aureus. We compared four methods for determining the lysostaphin susceptibility of 57 strains of methicillin-sensitive S. aureus, methicillin-resistant S. aureus, vancomycin intermediately susceptible S. aureus (VISA), mupirocin-resistant S. aureus, and various defined genetic mutants of S. aureus. Three reference lysostaphin-resistant S. aureus variants were also included in the assays as negative controls. The assays examined included turbidity, MIC, minimum bactericidal concentration (MBC), and disk diffusion assays. All of the strains of S. aureus tested, including a VISA strain which had previously been reported to be lysostaphin resistant, were susceptible to lysostaphin by all four methods. The three reference lysostaphin-resistant variants were resistant by all four methods. The disk diffusion assay was the simplest method to differentiate lysostaphin-susceptible S. aureus strains from lysostaphin-resistant variants, while the MBC assay could be used as a follow-up assay if required. In the disk diffusion assay, all strains of S. aureus tested revealed zones of inhibition of ≥11 mm using a 50-μg lysostaphin disk, while the three reference lysostaphin-resistant S. aureus variants had no zones of inhibition. In MBC assays, concentrations of lysostaphin ranging from 0.16 μg/ml to 2.5 μg/ml were found to cause a 3 log or greater drop from the initial CFU of S. aureus within 30 min for all strains tested.