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

bHeterogeneous 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 ␤ 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.

Genome Sequence-Based Discriminator for Vancomycin-Intermediate Staphylococcus aureus

“…It has also been shown that mutations to other regulatory genes, including rpoD selected here, can lead to the VISA phenotype in the Mu3 lineage (35). The selection of the cell wall synthesis enzyme pbp4, here implicated in the VISA phenotype, has been confirmed in other studies (35,36) and is related to its role in peptidoglycan cross-linking.…”

“…For example, members of two-component regulatory systems selected here, graR, vraS, and walR, have been implicated in the VISA phenotype via their effects on cell wall composition and capsule expression (4,(32)(33)(34). It has also been shown that mutations to other regulatory genes, including rpoD selected here, can lead to the VISA phenotype in the Mu3 lineage (35). The selection of the cell wall synthesis enzyme pbp4, here implicated in the VISA phenotype, has been confirmed in other studies (35,36) and is related to its role in peptidoglycan cross-linking.…”

Mechanisms of vancomycin resistance in Staphylococcus aureus

“…Comparison of such susceptible and resistant isolates can then be used to identify the altered genetic determinant in the VISA strain. This approach has identified a variable number and kinds of mutations in different VISA isolates (18,28,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40).…”

Performance of vancomycin and teicoplanin disk diffusion test in isogenic vancomycin non-susceptible Staphylococcus aureus

“…The most frequently affected gene was cmk -encoding cytidylate kinase. Reduction of cytidylase kinase activity in cmk mutants encouraged the hVISA-to-VISA conversion by thickening the cell wall and reducing the cell growth rate [29].…”