In Vitro Activity of the Bacteriophage Endolysin HY-133 against Staphylococcus aureus Small-Colony Variants and Their Corresponding Wild Types

Abstract

Nasal carriage of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) represents both a source and a risk factor for subsequent infections. However, existing MRSA decolonization strategies and antibiotic treatment options are hampered by the duration of administration and particularly by the emergence of resistance. Moreover, beyond classical resistance mechanisms, functional resistance as the formation of the small-colony variant (SCV) phenotype may also impair the course and treatment of S. aureus infections. For the recombinant bacteriophage endolysin HY-133, rapid bactericidal and highly selective in vitro activities against MSSA and MRSA has been shown. In order to assess the in vitro efficacy of HY-133 against the SCV phenotype, minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were evaluated on clinical SCVs, their isogenic wild types, as well as on genetically derived and gentamicin-selected SCVs. For all strains and growth phases, HY-133 MIC and MBC ranged between 0.12 and 1 mg/L. Time-kill studies revealed a fast-acting bactericidal activity of HY-133 resulting in a ≥3 - log₁₀ decrease in CFU/mL within 1 h compared to oxacillin, which required 4⁻24 h. Since the mode of action of HY-133 was independent of growth phase, resistance pattern, and phenotype, it is a promising candidate for future S. aureus decolonization strategies comprising rapid activity against phenotypic variants exhibiting functional resistance.

Keywords: HY-133; Staphylococcus aureus; endolysin; functional resistance; nasal decolonization; oxacillin; small-colony variants; susceptibility.

Figure 1

Time-kill curves of HY-133 shown by plots of mean values for the log₁₀ of the numbers of CFU/mL versus time for two representative clinical Staphylococcus aureus WTs (a,c) and corresponding SCVs (b,d) tested against HY-133. The threshold implicates a ≥3 − log₁₀ decrease in CFU/mL. Time-kill curves for each strain were performed in triplicate (mean ± standard deviation). Asterisks denote statistical difference of the respective concentration of HY-133 used (defined by matching colors) with respect to the untreated growth control at 1 h; p ≤ 0.001 by one-way ANOVA.

Figure 2

Time-kill curves of HY-133 shown by plots of mean values for the log₁₀ of the numbers of CFU/mL versus time for the highly cytotoxic and clinically virulent S. aureus WT strain 6850 (a) and corresponding SCVs JB1 (b) and IIb13 (c) tested against HY-133. The threshold implicates a ≥3 − log₁₀ decrease in CFU/mL. Time-kill curves for each strain were performed in triplicate (mean ± standard deviation). Asterisks denote statistical difference of the respective concentration of HY-133 used (defined by matching colors) with respect to the untreated growth control at 1 h; p ≤ 0.001 by one-way ANOVA.

Figure 3

Time-kill curves of oxacillin shown by plots of mean values for the log₁₀ of the numbers of CFU/mL versus time for two representative clinical S. aureus WTs (a,c) and corresponding SCVs (b,d) tested against oxacillin. The threshold implicates a ≥3 − log₁₀ decrease in CFU/mL. Time-kill curves for each strain were performed in triplicate (mean ± standard deviation). Asterisks denote statistical difference of the respective concentration of oxacillin used (defined by matching colors) with respect to the untreated growth control at 1 h; p ≤ 0.001 by one-way ANOVA.