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Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) clinical strain Mu3 spontaneously generates VISA strains at an extremely high frequency (≥1 × 10 −6 ). The generated VISA strains usually grow more slowly than does the parent hVISA strain, but they form colonies on vancomycin-containing agar plates before 48 h of incubation. However, we noticed a curious group of VISA strains, designated “slow VISA” (sVISA), whose colonies appear only after 72 h of incubation. They have extremely prolonged doubling times but have vancomycin MICs of 8 to ∼24 mg/liter when determined after 72 to ∼144 h of incubation. We established strain Mu3-6R-P (6R-P), which has a vancomycin MIC of 16 mg/liter (at 72 h), as a representative sVISA strain. Its cell wall was thickened and autolytic activity was decreased compared to the respective qualities of the parent hVISA strain Mu3. Whole-genome sequencing of 6R-P revealed only one mutation, encoded by rpoB (R512P), which replaced the 512th arginine of the RNA polymerase β-subunit with proline. Its VISA phenotype was unstable, and the strain frequently reverted to hVISA with concomitant losses of pinpoint colony morphology and cell wall thickness and reduced autolytic activity. Sequencing of the rpoB genes of the phenotypic revertant strains revealed mutations affecting the 512th codon, where the proline of 6R-P was replaced with leucine, serine, or histidine. Slow VISA generated in the tissues of an infected patient serves as a temporary shelter for hVISA to survive vancomycin therapy. The sVISA strain spontaneously returns to hVISA when the threat of vancomycin is lifted. The rpoB (R512P) mutation may be regarded as a regulatory mutation that switches the reversible phenotype of sVISA on and off.

Three types of phenotypic expression of β-lactam resistance have been reported in methicillin-resistant Staphylococcus aureus (MRSA): heterogeneous, homogeneous, and Eagle-type resistance. Heterogeneous-to-homogeneous conversion of β-lactam resistance is postulated to be caused by a chromosomal mutation ( chr* ) in addition to the expression of the mecA gene. Eagle-type resistance is a unique phenotype of chr* occurring in pre-MRSA strain N315 whose mecA gene expression is strongly repressed by an intact mecI gene. We here report that certain mutations of the rpoB gene, encoding the RNA polymerase β subunit, belong to chr* . We studied homogeneous MRSA (homo-MRSA) strain N315ΔIP-H5 (abbreviated as ΔIP-H5), which was obtained from hetero-MRSA strain N315ΔIP by selection with 8 mg/liter imipenem. Whole-genome sequencing of ΔIP-H5 revealed the presence of a unique mutation in the rpoB gene, rpoB (N967I), causing the amino acid replacement of Asn by Ile at position 967 of RpoB. The effect of the rpoB (N967I) mutation was confirmed by constructing a revertant H5 rpoB (I967N) strain as well as an N315-derived mutant, N315 rpoB (N967I). H5 rpoB (I967N) regained the hetero-resistance phenotype, and the N315 rpoB (N967I) strain showed an Eagle-type phenotype similar to that of the typical Eagle-type MRSA strain N315h4. Furthermore, subsequent whole-genome sequencing revealed that N315h4 also had a missense mutation of rpoB (R644H). Introduction of the rpoB (N967I) mutation was accompanied by decreased autolysis, prolonged doubling time, and tolerance to bactericidal concentrations of methicillin. We consider that rpoB mutations are the major cause for heterogeneous-to-homogeneous phenotypic conversion of β-lactam resistance in MRSA strain N315 and its derived strains.

We describe here the genetic analysis of a vancomycin-susceptible Staphylococcus aureus (VSSA) strain, Mu50Ω, a strain related to vancomycin-intermediate S. aureus (VISA) strain Mu50. Using a combination of Mu50Ω whole-genome sequencing and genome engineering, we observed a stepwise evolution of vancomycin resistance from VSSA to VISA after the mutated vraS and graR genes of Mu50 were engineered into Mu50Ω.

In order to better understand the mechanism of daptomycin resistance, we generated a daptomycinnonsusceptible derivative strain, strain 103d1 (MIC 3.0 g/ml), by in vitro exposure of methicillinresistant Staphylococcus aureus strain N315IP (MIC 0.5 g/ml) to daptomycin. We also obtained a daptomycin-susceptible phenotypic revertant strain, strain 103d1-10 (MIC 1.0 g/ml), by passaging 103d1 in drug-free medium for 10 days. The resultant triple-isogenic strains were analyzed for their phenotypes and gene expression by microarray analysis. No significant differences in the membrane fluidities of 103d1 and 103d1-10 compared to the membrane fluidity of N315IP were observed. Resistant strain 103d1 had the highest membrane potential, followed by strains 103d1-10 and N315IP. The vancomycin and teicoplanin MICs also increased. Teichoic acid genes (tagA, tagG), mprF encoding lysyl-phosphatidylglycerol, and cls encoding cardiolipin synthase were downregulated in 103d1 and 103d1-10. The vraF and vraG genes, which encode ATP binding cassette transporter proteins, were upregulated in 103d1. The vraSR two-component regulatory system was upregulated, and electron microscopy revealed that the cell wall of 103d1 was significantly thicker than that of the parental strain. Taken together, daptomycin exposure selected a daptomycin-nonsusceptible strain with a phenotype similar to that of heterogeneous vancomycin-intermediate S. aureus and a transcription profile that partially overlapped that of heterogeneous vancomycin-intermediate S. aureus. After the isolation of the first methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin in 1997, the need for a drug with a new target increased (20, 21). Due to the multiple-drug resistance profile of most S. aureus strains isolated nowadays, there are few therapeutic options available. Daptomycin is a calcium-dependent cyclic lipopeptide approved by the U.S. Food and Drug Administration in 2003 and, afterwards, by the European Agency for Evaluation of Medicinal Products for the treatment of complicated skin and soft tissue infections caused by susceptible bacteria. The U.S. Food and Drug Administration also

Multistep genetic alteration is required for methicillin-resistant Staphylococcus aureus (MRSA) to achieve the level of vancomycin resistance of vancomycin-intermediate S. aureus (VISA). In the progression of vancomycin resistance, strains with heterogeneous vancomycin resistance, designated hetero-VISA, are observed. In studying the whole-genome sequencing of the representative hetero-VISA strain Mu3 and comparing it with that of closely related MRSA strains Mu50 (VISA) and N315 (vancomycin-susceptible S. aureus [VSSA]), we identified a mutation in the response regulator of the graSR two-component regulatory system. Introduction of mutated graR , designated graR *, but not intact graR , designated graRn , could convert the hetero-VISA phenotype of Mu3 into a VISA phenotype which was comparable to that of Mu50. The same procedure did not appreciably increase the vancomycin resistance of VSSA strain N315, indicating that graR * expression was effective only in the physiological milieu of hetero-VISA cell to achieve a VISA phenotype. Interestingly, the overexpression of graR * increased the daptomycin MICs in both Mu3 and N315 and decreased the oxacillin MIC in N315.

As an aggressive pathogen, Staphylococcus aureus poses a significant public health threat and is becoming increasingly resistant to currently available antibiotics, including vancomycin, the drug of last resort for gram-positive bacterial infections. S. aureus with intermediate levels of resistance to vancomycin (vancomycin-intermediate S. aureus [VISA]) was first identified in 1996. The resistance mechanism of VISA, however, has not yet been clarified. We have previously shown that cell wall thickening is a common feature of VISA, and we have proposed that a thickened cell wall is a phenotypic determinant for vancomycin resistance in VISA (L. Cui, X. Ma, K. Sato, et al., J. Clin. Microbiol. 41: 5-14, 2003). Here we show the occurrence of an anomalous diffusion of vancomycin through the VISA cell wall, which is caused by clogging of the cell wall with vancomycin itself. A series of experiments demonstrates that the thickened cell wall of VISA could protect ongoing peptidoglycan biosynthesis in the cytoplasmic membrane from vancomycin inhibition, allowing the cells to continue producing nascent cell wall peptidoglycan and thus making the cells resistant to vancomycin. We conclude that the cooperative effect of the clogging and cell wall thickening enables VISA to prevent vancomycin from reaching its true target in the cytoplasmic membrane, exhibiting a new class of antibiotic resistance in gram-positive pathogens.

The glycopeptide vancomycin has been used successfully for the treatment of serious infections caused by multidrug-resistant Staphylococcus aureus for several decades, and some isolates were reported to be susceptible only to this antibiotic (30, 36). However, with the worldwide emergence of glycopeptide-intermediate S. aureus (GISA [or vancomycin-intermediate S. aureus; VISA]) strains with vancomycin MICs of 8 or 16 mg/liter (5, 17), the likelihood of this resistance reaching global magnitude causes great concern about the possibility of losing the last treatment option for multidrug-resistant staphylococcus infection. Several factors have been reported to be involved in the mechanism of glycopeptide resistance in VISA, including thickened cell wall (7, 11), accumulation of surplus cell wall material (48), reduced peptidoglycan cross-linking (4, 12), inactivation of penicillin-binding protein D (PBP4) (9, 47), a decrease in the level of the muropeptide amidation (4, 7), and/or other cell wall alterations, such as increased glycan chain length (23). The phenomenon of thickened cell wall was discovered in our early study of the first VISA strain, Mu50 (11). Subsequent investigation of the isogenic cells with different cell wall thickness prepared from a single colony of Mu50 cells demonstrated that cell wall thickening is the major factor contributing to glycopeptide resistance in Mu50 (7). Recently we found that 16 VISA clinical isolates collected from seven countries all showed a distinct thickened cell wall compared to glycopeptide-susceptible S. aureus strains (6, 21, 41). An immanent link between cell wall thickness and the level of glycopeptide resistance was also demonstrated in a comparison study using triple sets of 16 VISA strains, their glycopeptide-susceptible derivatives (vancomycin-resistant S. aureus [VRSA]-P) produced by passage on drug-free medium, and phonotically glycopeptide-resistant revertants (VRSA-PR) selected from VRSA-P on vancomycin plates (5, 6). Contrary to the phenotypic mechanism, the genetic mechanism of VISA is far from being clear. Different VISA strains have been studied by many researchers, and altered expression of genes such as pbp2 (46), pbpD (9, 47), sigB (2, 11), ddh (3, 34), tcaA (33), and vraSR (26) were reported. Several sets of up- and down-regulated genes associated with vancomycin resistance, including regulators, were also reported (26, 35, 54). However, until now none of the genes was altered without exception in VISA strains and none could be attributed to VISA strains specifically. Thus there is a need for additional knowledge on the genetic mechanism(s) of glycopeptide resistance acquisition in VISA and for defining genetic alteration(s) resulting in phenotypic change, such as cell wall thickening. Recently, in an effort to efficiently discover genes involved in the mechanism of glycopeptide resistance in S. aureus, a DNA microarray which consists of PCR products covering the whole genome of N315 was used to identify open reading frames (ORFs) that are expressed differently between VISA and VSSA strains. In this report, we describe our findings from transcriptional profiling studies on genes that are differentially expressed as well as genetic studies on genes that are increased in transcription in glycopeptide-resistant strains.

Staphylococcus aureus Mu50, which has reduced susceptibility to vancomycin, has a remarkably thickened cell wall with an increased proportion of glutamine nonamidated muropeptides. In addition, Mu50 had enhanced glutamine synthetase and l -glutamine d -fructose-6-phosphate aminotransferase activities, which are involved in the cell-wall peptidoglycan synthesis pathway. Furthermore, significantly increased levels of incorporation of 14 C-labeled d -glucose into the cell wall was observed in Mu50. Unlike a femC mutant S. aureus strain, increased levels of production of nonamidated muropeptides in Mu50 was not caused by lower levels of glutamine synthetase activity but was considered to be due to the glutamine depletion caused by increased glucose utilization by the cell to biosynthesize increased amounts of peptidoglycan. After the cells were allowed to synthesize cell wall in the absence or presence of glucose and glutamine, cells with different cell-wall thicknesses and with cell walls with different levels of cross-linking were prepared, and susceptibility testing of these cells demonstrated a strong correlation between the cell-wall thickness and the degree of vancomycin resistance. Affinity trapping of vancomycin molecules by the cell wall and clogging of the outer layers of peptidoglycan by bound vancomycin molecules were considered to be the mechanism of vancomycin resistance of Mu50. The reduced cross-linking and the increased affinity of binding to vancomycin of the Mu50 cell wall presumably caused by the increased proportion of nonamidated muropeptides may also contribute to the resistance to some extent.

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.

Vancomycin (VAN)-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) isolates are considered to have emerged from VAN-susceptible S. aureus (VSSA) by spontaneous mutation during VAN exposure. We previously reported that laboratory mutant H14, obtained from VSSA strain ΔIP by exposure to imipenem (IPM), showed overexpression of the vraSR two-component system and a typical hVISA phenotype. In the present study, to elucidate the mechanism of VSSA conversion to hVISA, we further characterized strain H14 by determining its whole-genome sequence, morphology, cell wall synthetic activity, and gene expression. Genome sequencing revealed that H14 harbored a mutated vraS (designated vraS H14 ) that caused an amino acid substitution (S 329 →L). This mutation is different from the VraS mutation (N 5 →I) identified in representative clinical hVISA strain Mu3. However, H14 exhibited a phenotype similar to that of Mu3, including heterogeneous resistance to VAN, enhanced cell wall synthetic activity, and vraSR overexpression. Replacement of the vraS gene of ΔIP with the mutated vraS H14 gene confirmed that the S 329 →L substitution was responsible for both the upregulation of vraSR and conversion to the hVISA phenotype. This conversion was also achieved by using the vraS gene of Mu3, which carries a mutation (N 5 →I), but not with the native vraS gene of strain N315. Finally, we carried out a study to analyze the appearance of hVISA from VSSA by exposure of ΔIP to selective concentrations of VAN and beta-lactam antibiotics. A total of 8 and 5 hVISA isolates were detected among 50 isolates selected with VAN and IPM, respectively. Among the 13 hVISA mutants, mutation in vraSR was detected only in mutant strain H14, suggesting that additional mutational mechanisms can be responsible for evolution to the hVISA phenotype. We conclude that exposure not only to VAN but also to beta-lactams may select for reduced glycopeptide susceptibility in S. aureus .

We present here findings of a strong positive correlation between reduced daptomycin susceptibility and vancomycin resistance in vancomycin-intermediate Staphylococcus aureus (VISA). This correlation is related to cell wall thickening, suggesting that, similar to the case with vancomycin resistance in VISA, the physical barrier of a thickened cell wall may contribute to daptomycin resistance in S. aureus .