Comprehensive identification of mutations responsible for heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA)-to-VISA conversion in laboratory-generated VISA strains derived from hVISA clinical strain Mu3

Abstract

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10(-6) or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes, rpoB, rpoC, walK, pbp4, and pp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) was cmk, which encodes cytidylate kinase, followed closely by rpoB (5 out of 32), encoding the β subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number of cmk mutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity in cmk mutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate.

Fig 1

Population analysis of hVISA parent strains. The number of colonies on BHI agar plates containing various concentrations of vancomycin was counted after 48 h of incubation at 37°C.

Fig 2

Mapping of the cmk mutations of the VISA strains. Substituted amino acid residues of strain Mu3 derivatives and clinical isolates are shown with the strain name in open and gray boxes, respectively. Nine α-helixes and 8 β-sheets assigned previously (22) are shown in gray and dark gray, respectively. The parallel β-sheet consisting of strands β1-β2 and β6-β8 flanked by α-helixes α1, α6, and α9 make up the CORE domain, which contains a highly conserved phosphate-binding loop (22). NMP, nucleoside monophosphate.

Fig 3

Effects of cmk mutations on the vancomycin-resistant subpopulations of Mu3, Mu3p27, and Mu3fdh2*. (A) Population analysis of the cmk(A20G) mutant and the parent strains. Mu3cmk* and Mu3fdh2*cmk* are Mu3- and Mu3fdh2*-derived mutant strains bearing the cmk(A20G) mutation introduced by gene replacement methods. Mu3V6-7 is a vancomycin-selected VISA strain from Mu3. (B) Population analysis of the cmk(SD*) mutant and parent strains. Mu3 fdh2*cmk(SD*) is a Mu3fdh2*-derived mutant strains with the cmk(SD*) mutation introduced by gene replacement methods. Mu3p27V6-10 is a vancomycin-selected VISA strain from Mu3p27.

Fig 4

Transmission electron microscopy of the cmk mutant strains and their parent strains. (A) cmk(A20G) mutants and their parent strain. (B) cmk(SD*) mutants and their parent strains. Data are presented as the mean and standard deviation of the cell wall thickness for each strain. Note that all cmk mutant cells had thick cell walls compared to those of the parent strain. Magnification, ×30,000.