Fitness cost of VanA-type vancomycin resistance in methicillin-resistant Staphylococcus aureus

Abstract

We have quantified the biological cost of VanA-type glycopeptide resistance due to the acquisition of the resistance operon by methicillin-resistant Staphylococcus aureus (MRSA) from Enterococcus sp. Exponential growths of recipient strain HIP11713, its transconjugant VRSA-1, VRSA-5, and VRSA-6 were compared in the absence or, except for HIP11713, in the presence of vancomycin. Induction of resistance was performed by adding vancomycin in both the preculture and the culture or the culture at only 1/50 the MIC. In the absence of vancomycin, the growth rates of the vancomycin-resistant S. aureus (VRSA) strains were similar to that of susceptible MRSA strain HIP11713. When resistance was induced, and under both conditions, there was a significant reduction of the growth rate of the VRSA strains relative to that of HIP11713 and to those of their noninduced counterparts, corresponding to a ca. 20% to 38% reduction in fitness. Competition experiments between isogenic VRSA-1 and HIP11713 mixed at a 1:1, 1:100, or 100:1 ratio revealed a competitive disadvantage of 0.4% to 3% per 10 generations of the transconjugant versus the recipient. This slight fitness burden can be attributed to the basal level of expression of the van genes in the absence of induction combined with a gene dosage effect due to the presence of the van operon on multicopy plasmids. These data indicate that VanA-type resistance, when induced, is highly costly for the MRSA host, whereas in the absence of induction, its biological cost is minimal. Thus, the potential for the dissemination of VRSA clinical isolates should not be underestimated.

FIG. 1.

Comparison of the growth kinetics of VRSA strains. (A) Strains were grown in BHI broth without vancomycin (−/−), pregrown without vancomycin and subcultured with 1/50 the MIC of vancomycin (−/+), or pregrown and subcultured with 1/50 the MIC of vancomycin (+/+). The gray area represents the exponential interval in which the growth rates were calculated. (B and C) Growth rates of VRSA-1 relative to that of HIP11713 (B) and of VRSA-1, VRSA-5, and VRSA-6 relative to that of the noninduced corresponding VRSA isolate (C). Each value represents the mean of four independent experiments carried out in duplicates. Error bars represent the standard deviations of the means. OD₆₀₀, optical density at 600 nm.

FIG. 2.

Growth competition between VRSA-1 and HIP11713 mixed at an initial ratio of 1:1 (A), 1:100 (B), or 100:1 (C). Log₁₀ CFU counts of the resistant and susceptible populations were plotted against the number of transfers. The selection coefficient (s) was calculated as the slope of the linear regression model ln(CI)/ln(d), where CI is the CI obtained by the CFU ratio of the resistant and susceptible population at t₁ divided by the same ratio at t₀ and where d is the dilution factor. The log₁₀ values represent the means of CFU counts for three cultures in at least two independent experiments.