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To further understand the mechanism of intermediate-level glycopeptide resistance, resulting from multiple endogenous mutations, in both laboratory-derived and clinically isolated Staphylococcus aureus.
Laboratory-derived S. aureus strains were generated under selection using a variety of cell-wall-active antibiotics. Complete sequences of 27 genes, including 17 two-component histidine kinase sensors, were then compared with those of their susceptible parent strain. Further genetic analysis was performed on 125 clinical S. aureus isolates and 42 geographically diverse isolates of vancomycin-intermediate S. aureus (VISA).
Selective pressure using imipenem resulted in single point mutations leading to amino acid substitutions in two genes: vraS, encoding a two-component histidine kinase sensor; and SA1702 (also called yvqF, located immediately upstream of vraS), encoding a conserved hypothetical protein. The accumulation of the mutation in two distinct proteins—MsrR, a peptide methionine sulphoxide reductase regulator, and TcaA, a teicoplanin-resistance-associated protein—correlated with further increases in the glycopeptide MIC. The prevalence of YvqF/VraSR mutants among 125 clinical isolates along with the corresponding teicoplanin MICs was as follows: 0% (0/39), ≤1 mg/L; 48.6% (17/35), 2 mg/L; 72.7% (24/33), 4 mg/L; 93.8% (15/16), 8 mg/L; and 100% (2/2), 16 mg/L. Genetic analysis of 42 VISA isolates also identified the predominant amino acid substitutions in YvqF/VraS: 9 isolates (21.4%) revealed mutations in YvqF, followed by 7 isolates with mutations in VraS (16.7%).
Our findings provide novel insights into the high prevalence and genetic diversity of YvqF/VraSR mutants among clinical S. aureus isolates with reduced susceptibility to teicoplanin.
Methicillin-resistant Staphylococcus aureus (MRSA), which emerged as a major nosocomial pathogen, is now spreading rapidly through the wider community. Moreover, treatment of suspected S. aureus infections is becoming increasingly more complicated. The emergence of glycopeptide resistance in S. aureus is, in particular, considered to be a serious threat around the world, since current treatment of serious infections caused by MRSA relies mainly upon the administration of glycopeptide antibiotics (e.g. vancomycin and teicoplanin). Although highly vancomycin-resistant MRSA (VRSA), resulting from the acquisition of enterococcal vanA operon, has been described,1 such strains are now extremely rare. In contrast, a failure in vancomycin treatment due to S. aureus with reduced susceptibility—so-called vancomycin-intermediate S. aureus (VISA)—is being increasingly identified.2–4 The mechanism of resistance observed in VISA strains is considered to be endogenous, resulting from multiple mutational and metabolic changes.5–7 The vancomycin-intermediate resistance phenotype has been linked to several abnormal physiological properties, including a thickened cell wall, leading to the accumulation of free d-alanyl-d-alanine termini, which act as false targets for glycopeptide and decrease the diffusion rate to its lipid II target.8 The corresponding genetic changes are, however, far from clear. A major problem in understanding the genetic mechanisms has been the lack of a universal resistance marker typical for all VISA strains. In recent years, several genetic alterations in two-component regulatory systems (TCRS) have been reported to be highly related to the glycopeptide resistance phenotype, such as point mutations in vraSR9 and graSR,10 and a defective agr function.11 However, these genetic changes have only been found in a limited number of clinical and laboratory isolates of S. aureus. Furthermore, no mutations were found in vraSR among some pairs of vancomycin-susceptible and -resistant clinical isolates.12 Hence, there are likely to be as yet unidentified loci and pathways linked to the glycopeptide resistance phenotype.
In this study, we focused on TCRS, encoding a histidine kinase sensor and a response regulator, which are widely employed by bacteria to monitor environmental stimuli. TCRS utilize the phosphotransfer cascade to alter specific gene expression profiles13 and have been linked to the resistance phenotype of cell-wall-active agents, such as resistance to glycopeptide in Enterococcus faecium (VanSR),14 tolerance of vancomycin in Streptococcus pneumoniae (VncSR)15 and resistance to bacitracin in Bacillus subtilis (LiaSR).16 It is postulated that genetic alterations in TCRS play an important role in coordinating a response that enhances the resistance phenotype to cell-wall-active agents by sensing cell wall damage and activating the transcription of many enzymes and transporters. S. aureus N315 genome contains 16 pairs of TCRS and an additional pair on a genomic island staphylococcal cassette chromosome mec (SCCmec).17
To better understand the mechanism of glycopeptide resistance, we first generated laboratory-derived S. aureus strains with increasing MICs of glycopeptide by selection using cell-wall-active antibiotics, such as β-lactam and glycopeptide. The complete sequences of 27 genes, including 17 histidine kinase sensors and other genome loci previously suspected to contribute to glycopeptide resistance, were then analysed and compared with those of the glycopeptide-susceptible parent strain. Our analysis gives novel insights into the prevalence of mutations in these loci for >120 clinical S. aureus isolates collected in Japan. Complete sequences for the vraSR operon, including yvqF, were also determined for 42 geographically diverse clinical VISA isolates with different genetic backgrounds.
All strains used in this study are listed in Table 1. S. aureus LR5 strain18 was used as the glycopeptide-susceptible parental strain for serial-passage experiments. Clinical isolates of S. aureus (n=125), with different teicoplanin susceptibilities, collected in Japan during the period 1999–2005 were taken for this study; 104 isolates were MRSA and 21 isolates were methicillin-susceptible S. aureus (MSSA). S. aureus isolates that have either an oxacillin MIC>2 mg/L or harbour the mecA gene were considered to be MRSA. Forty-one geographically diverse clinical VISA isolates were obtained through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) Program. VISA strain Mu50 (ATCC 700699), obtained from the American Type Culture Collection, was also included.
MICs were determined by the agar dilution method according to CLSI guidelines.19 The following antimicrobial agents of known potency were used: arbekacin (Meiji Seika Kaisha, Ltd, Tokyo, Japan); vancomycin (Sigma–Aldrich, St Louis, MO, USA); teicoplanin (Astellas Pharma Inc., Tokyo, Japan); imipenem (Banyu Pharmaceutical Co., Ltd, Tokyo, Japan); oxacillin (Sigma–Aldrich); and levofloxacin (Sequoia Research Products Ltd, Pangbourne, UK).
A series of isogenic strains with increasing glycopeptide resistance levels was generated from a susceptible S. aureus LR5 strain by plating 10-fold dilutions of an overnight culture on brain heart infusion (BHI) agar (BBL Becton Dickinson, Sparks, MD, USA) containing imipenem at 2×MIC (8 mg/L). After an overnight incubation at 35°C, single colonies (first-step mutants) were recovered from the plates and their MICs for glycopeptides were compared with the susceptible parent by the agar dilution method, as described above. Three of the first-step mutants with increasing teicoplanin resistance (MSC15555, MSC15557 and MSC15558) were then used to repeat the selection on BHI agar containing teicoplanin (4 and 8 mg/L). Several independent colonies (second-step mutants) appearing on the plates containing teicoplanin were recovered. From each step, single colonies appearing at the highest concentration were purified and subcultured twice on BHI agar without a selective agent, to ensure the stability of the phenotype. These strains were then kept for further use.
To identify mutations present in S. aureus LR5 derivatives, primers were designed to amplify 27 open reading frames containing 17 two-component histidine kinase sensors, the entire vraSR operon, the msrR region and the tcaRAB region (Table 2). The reference sequences were those of S. aureus subsp. aureus N315 (GenBank accession no. BA000018) and S. aureus subsp. aureus N315 plasmid pN315 (GenBank accession no. AP003139). Bacterial pellets were treated with lysostaphin and lysozyme, and genomic DNA was isolated with a DNeasy tissue kit (Qiagen Inc., Valencia, CA, USA). PCR amplification from genomic DNA was carried out with Ex Taq polymerase (Takara Shuzo Co., Ltd, Kyoto, Japan). The cycling reaction was performed with a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). The presence of a mutation was determined by sequence analysis of the amplified DNA fragments using a BigDye Terminator v1.1 cycle sequencing kit and an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems). The complete amino acid sequence was deduced from the nucleotide sequence in the corresponding gene locus.
In order to determine whether mutations found in S. aureus LR5 derivatives are also detected in the same loci in clinical isolates, the genes coding for yvqF, vraS, vraR, msrR and tcaA were also PCR amplified and sequenced from 125 S. aureus isolates, with different susceptibilities to teicoplanin (collected in Japan). The genetic relatedness of 125 S. aureus isolates was assessed by agr group analysis. Genes coding for the agr locus were PCR amplified and sequenced by the sense primer (5′-ATGCACATGGTGCACATGC-3′) and the antisense primer (5′-ATTGACATAATCATGACGGAAC-3′). The agr group was determined by comparing the obtained sequence with those of S. aureus subsp. aureus USA300-FPR3757 (GenBank accession no. CP000255, agr group I), N315 (agr group II), MW2 (GenBank accession no. BA000033, agr group III) and S. aureus agr locus, complete coding sequence (GenBank accession no. AF288215, agr group IV).
We performed complete sequences for three genes (yvqF, vraS and vraR) using 42 geographically diverse clinical VISA isolates from different genetic backgrounds recovered from nine countries (described in Table 1).
The complete sequences of the 27 genes listed in Table 2, including vraSR operon (orf1, yvqF, vraS and vraR), msrR and tcaA, were determined for the 20 derivatives with increased glycopeptide resistance generated from susceptible S. aureus LR5. Genetic changes that correlate with increasing glycopeptide MICs were identified by sequence comparisons between serial-passage-derived strains and their susceptible parental S. aureus LR5 strain (Table 3). It is worth noting that five first-step mutants selected by imipenem (MSC15553–MSC15557) revealed a point mutation resulting in an Arg91His, Thr125Ile or Pro174Ser substitution in YvqF, which is the conserved hypothetical protein located in the vraSR operon. The remaining first-step mutants (MSC15558 and MSC15559) revealed a point mutation resulting in Ser329Leu in VraS. Determination of MICs for glycopeptides revealed slightly different resistance levels among these first-step mutants. This finding may reflect different effects of diverse amino acid substitutions on glycopeptide MICs. These first-step mutants showed 2- to 8-fold increases in their teicoplanin MICs compared with the MIC for their parental S. aureus LR5 strain, while only an ~2-fold increase in the vancomycin MICs was observed. Interestingly, mutations in the vraSR operon occurred frequently at a lethal concentration (2×MIC) of imipenem. At this concentration, colonies with decreased susceptibilities to teicoplanin were obtained with a frequency of approximately ≥10−7. Three such strains (MSC15555, MSC15557 and MSC15558) were chosen for use in further selection on BHI agar containing teicoplanin at the lethal concentration.
Two second-step mutants (MSC15560 and MSC15561, having MICs of teicoplanin and vancomycin of 8 and 1 mg/L, respectively) contained additional amino acid substitutions in MsrR: Pro292Ser for MSC15560 and Arg230Cys for MSC15561, along with Pro174Ser in YvqF. In contrast, MSC15562 (MICs of teicoplanin and vancomycin of 8 and 1 mg/L, respectively) contained an additional nucleotide substitution of A211T, causing a nonsense mutation in TcaA, possibly resulting in a truncated protein at amino acid position 70. Thus, second-step mutants harbour a mutation in MsrR or TcaA, which was probably responsible for their enhanced resistance to teicoplanin. The susceptibilities to teicoplanin of the mutants were 2-fold lower for those carrying mutations in MsrR and 4-fold lower for those carrying mutations in TcaA than that of their parental first-step mutants. Three out of six second-step mutants (MSC15563, MSC15564 and MSC15565) had no alteration in these regions and the other genes sequenced in this study, indicating that tcaA or msrR does not play a universal role in glycopeptide resistance.
These laboratory-derived strains also showed different susceptibilities toward β-lactams (imipenem and oxacillin), whereas little change was observed in their susceptibility to other antibiotic groups, such as arbekacin (aminoglycoside) and levofloxacin (quinolone), indicating these mutations do not affect the antibiotic susceptibility profile toward non-cell-wall-active antibiotics.
Our findings prompted us to check whether any of the loci mutated in S. aureus LR5 derivatives are also present in clinical isolates. One hundred and twenty-five S. aureus isolates collected in Japan, with different susceptibilities to teicoplanin, were examined for the presence of mutations in the genes coding for yvqF, vraS, vraR, msrR and tcaA (Table 4). Potentially important amino acid substitutions were identified by comparing the obtained sequence with those of S. aureus N315 and highly susceptible clinical isolates (MICs of teicoplanin≤1 mg/L).
The 125 isolates were classified into 27 patterns according to mutations in YvqF, VraS, VraR, MsrR and TcaA. Forty-four isolates possessed mutations in VraS, which was the major pattern and was followed by 13 isolates with mutations in YvqF. Interestingly, the mutation patterns of YvqF and VraS were diverse among the clinical isolates: 6 distinct amino acid changes in YvqF and 17 distinct changes in VraS. Overlapping mutations of YvqF and VraS were not found in any of the isolates, which is consistent with the results from laboratory-derived strains. Eleven out of 12 isolates with the Ile5Asn mutation in VraS were also found to contain a point mutation leading to a Glu146Lys substitution in MsrR. Though rare, mutations in either VraR or TcaA were also detected (two isolates in VraR and a single isolate in TcaA). Sequence analysis of the truncated tcaA locus revealed that the insertion sequence shares 99% similarity to that of the transposase gene in ISEfm2, identified in Enterococcus faecium E59 (GenBank accession no. AY887085).
One hundred and eighteen out of 125 isolates (94.4%) belonged to agr group II, just as in the case of the New York/Japan clone, the major MRSA spreading in hospitals throughout Japan. The remaining seven (5.6%) isolates belonged to agr group I. Twenty out of 21 MSSA isolates also belonged to agr group II. Eighteen out of 21 MSSA isolates had elevated teicoplanin MICs (4–16 mg/L). Among these 18 isolates displaying reduced susceptibility to teicoplanin, 15 had mutations in YvqF or VraS.
None of the 39 isolates with teicoplanin MICs of ≤0.12–1 mg/L showed any changes in these regions, strongly suggesting the potential role of these amino acid substitutions in the expression of the teicoplanin resistance phenotype in S. aureus. Based on these results, the prevalence of mutants with their corresponding teicoplanin MICs was as follows: 0% (0/3), ≤0.25 mg/L; 0% (0/6), 0.5 mg/L; 0% (0/30), 1 mg/L; 48.6% (17/35), 2 mg/L; 72.7% (24/33), 4 mg/L; 93.8% (15/16), 8 mg/L; and 100% (2/2), 16 mg/L.
Several substitutions in VraR (Glu59Asp and Ser164Pro) and in TcaA (Leu218Pro, Tyr237His, Thr262Ser, Arg283His and Gly312Asp) were also identified; however, these were frequently found in highly susceptible clinical isolates (data not shown) and a reference strain, USA300-FPR3757. These variations seem to be owing to the distinct origin of the gene and are less likely to affect the susceptibility to glycopeptide antibiotics. Consequently, these isolates will not be described further in this report.
The vraSR operon was mutated in most clinical isolates displaying reduced susceptibility to teicoplanin. Thus, we performed complete sequence analysis for this locus in 42 geographically diverse clinical VISA isolates from different genetic backgrounds (Table 5). Nine out of 42 isolates (21.4%) revealed mutations in YvqF, which was the major pattern and followed by 7 isolates with mutations in VraS (16.7%). A mutation in VraR was detected in one isolate. Except for mutations in YvqF (Trp119Arg and Pro126Ser) and in VraS (Ile5Asn and Ala260Val), the mutation patterns in YvqF, VraS and VraR were different from those identified in clinical S. aureus isolates collected in Japan, as described in Table 4. A strong link was not found between YvqF/VraSR mutations and resistance to glycopeptide among geographically diverse 42 VISA isolates. Teicoplanin MICs tended to be identical or higher than those of vancomycin. In contrast, no significant relationship was found between MICs of glycopeptides and those of other antibiotic groups, such as imipenem, oxacillin and arbekacin.
The lack of a universal resistance marker common to all VISA strains currently limits our understanding the genetic mechanism of glycopeptide resistance. Thus, our ability to detect and eliminate the development of glycopeptide-resistant strains is severely restricted. The most remarkable finding obtained in the present study was the high prevalence of YvqF/VraSR mutants among the clinical S. aureus isolates with reduced susceptibility to teicoplanin (Table 4). Clearly, the present study was not designed to identify all of the mutations that correlate with increases in glycopeptide MIC. The mutations identified in this study may, however, act as useful diagnostic markers to detect risk factors associated with glycopeptide resistance, particularly teicoplanin, in S. aureus.
Mutations in several genetic loci other than TCRS, such as sigB,20 trfAB21 and tcaA,22 are known to contribute to glycopeptide resistance. The isogenic series of S. aureus LR5 strain provided us a unique opportunity to study genetic changes involved in the development of the glycopeptide resistance phenotype. We showed that the selective pressure of growth at a lethal concentration of imipenem resulted in a single point mutation leading to amino acid substitutions in two proteins: VraS, a two-component histidine kinase sensor; and YvqF, a conserved hypothetical protein. Exposure to teicoplanin resulted in accumulation of mutations in two distinct proteins—MsrR, a peptide methionine sulphoxide reductase regulator, and TcaA, a teicoplanin-resistance-associated protein—which correlated with a further increase in glycopeptide MIC.
Although YvqF is predicted to be a membrane protein, its biochemical functions and the impact of the observed mutations on its functions still need to be determined. Nonetheless, YvqF is thought to be involved in cell wall metabolism on the basis of three separate lines of evidence: (i) the yvqF gene is located immediately upstream of vraS, and co-transcribed with vraS and vraR;23 (ii) transcription of yvqF is regulated by the VraSR system;24 and (iii) up-regulation of YvqF or its homologue in the presence of cell-wall-active antibiotics (i.e. vancomycin and oxacillin) has been identified in both S. aureus25 and B. subtilis.26 Surprisingly, overlapping YvqF and VraS mutations were not found in any of the clinical isolates or laboratory-derived strains generated from S. aureus LR5 in this study. These findings suggest that mutations in YvqF and VraS are mutually exclusive. Thus, our results imply that these events are genetically redundant and that the simultaneous occurrence of both mutations does not confer any further advantage. Alternatively, the presence of both mutations may, in fact, be disadvantageous to the cell.
MsrR shares a high level of sequence identity and similarity with Psr, the PBP5 synthesis repressor of Enterococcus faecalis and Enterococcus hirae, and with LytR of B. subtilis.27 A correlation between amino acid substitutions in MsrR and glycopeptide resistance has not been previously reported, and the role of MsrR mutations in resistance is unclear. However, Glu146Lys in MsrR was also found in VISA strain Mu50 (GenBank accession no. BA000017). MsrR was highly expressed in glycopeptide-resistant strains compared with glycopeptide-susceptible strains.28 Moreover, a slight decrease in glycopeptide susceptibility was observed when MsrR was overexpressed in S. aureus N315.28 We predict that alterations in MsrR that result in decreased susceptibility to glycopeptide antibiotics do not inactivate protein function, because disruption of msrR leads to increased teicoplanin susceptibility.27 These observations may at least partly explain the contribution of MsrR mutations to glycopeptide resistance.
In the study of isogenic S. aureus isolates recovered from a single patient undergoing chemotherapy with antibiotics (including vancomycin and imipenem), a mutation in the vraSR operon was detected in the early step in the evolution of vancomycin resistance.29 Our data shows that a single point mutation in YvqF/VraS affects glycopeptide resistance at several levels, although its impact on resistance in a glycopeptide-susceptible background is only minor. Nevertheless, the emergence of a physiologically stable first-step YvqF/VraSR mutant is of concern, because the accumulation of additional mutations could lead to the formation of VISA.
The appearance of resistant isolates may, in part, be owing to the continued widespread use of glycopeptide. However, it is important to note that mutations in YvqF/VraSR occurred frequently at the lethal concentration of imipenem (Table 3). β-Lactam antibiotics are thought to be implicated in the emergence of hetero-VISA, containing VISA cells within its small subpopulation that express a heterogeneous type of resistance to vancomycin.30 Our results imply that the use of β-lactam antibiotics to treat MRSA infections might be one of the risk factors for the emergence of hetero-VISA. Indeed, this mechanism may sometimes involve mutation in YvqF/VraSR.
Of the 86 clinical isolates with teicoplanin MICs of ≥2 mg/L, Ile5Asn and Ile46Met in VraS were the most prevalent mutations [i.e. 14.0% (12/86) for each] (see Table 4). We previously reported the spread of Ile5Asn mutants in a Cancer Institute Hospital (Tokyo, Japan).31 Except for the Mu50 strain, however, Ile5Asn was not detected in any of the geographically diverse VISA isolates (Table 5). Furthermore, mutation patterns in YvqF/VraSR selected by cell-wall-active antibiotics, other than imipenem, remain to be proven. Further understanding of selective pressures in a clinical setting may help in establishing proper treatment protocols, thereby avoiding the development of antibiotic-resistant strains. Although more remains to be learned concerning the genetic basis of VISA formation, we believe that our findings will help further understanding of the mechanism of glycopeptide resistance in S. aureus.
No specific funding was received for this study.
All authors are employees of Meiji Seika Kaisha, Ltd, and have no significant declarations to make with respect to external funding, ownership of company stock or shares and reimbursement for preparing this article.
We thank the investigators who kindly provided S. aureus strains used in this study: Keiichi Hiramatsu for S. aureus LR5 strain; and the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) Program for 41 clinical VISA strains supported under NIAID/NIH Contract No. N01-AI-95359.