Heterogeneous Vancomycin-intermediate Staphylococcus Aureus Research Articles

Impact of vancomycin use trend change due to the availability of alternative antibiotics on the prevalence of Staphylococcus aureus with reduced vancomycin susceptibility: a 14-year retrospective study

BackgroundWe investigated the trend change in vancomycin-intermediate Staphylococcus aureus (VISA)/heterogeneous VISA (hVISA) prevalence among methicillin-resistant S. aureus (MRSA) bacteremia strains and antistaphylococcal antibiotic use together with mutation studies of vancomycin resistance-related gene loci to evaluate the impact of changes in antibiotic use after new antistaphylococcal antibiotics became available.MethodsAmong 850 healthcare-associated MRSA isolates from 2006 to 2019 at a tertiary hospital in South Korea, hVISA/VISA was determined by modified PAP/AUC analysis, and the identified hVISA/VISA strains were genotyped. Gene mutations at vraSR, graSR, walKR, and rpoB were studied by full-length sequencing. Antistaphylococcal antibiotic use in 2005–2018 was analyzed.ResultsTwo VISA and 23 hVISA strains were identified. The prevalence rate ratio of hVISA/VISA carrying mutations at the two-component regulatory systems among MRSA was 0.668 (95% CI 0.531–0.841; P = 0.001), and the prevalence rate ratio of hVISA/VISA carrying rpoB gene mutations was 1.293 (95% CI 0.981–1.702; 174 P = 0.068). Annual vancomycin use density analyzed by days of therapy (DOT) per 1,000 patient-days did not decrease significantly, however the annual average length of time analyzed by the number of days vancomycin was administered for each case showed a significantly decreasing trend.ConclusionsDuring the 14-year period when the average length of vancomycin therapy decreased every year with the availability of alternative antibiotics, the prevalence of hVISA/VISA did not decrease significantly. This seems to be because the resistant strains carrying the rpoB mutations increased despite the decrease in the strains carrying the mutations at the two-component regulatory systems.

Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (HVISA) at a Tertiary Hospital in Malaysia

Methicillin-resistant Staphylococcus aureus remains a global problem. The emergence of reduced susceptibility to Vancomycin in MRSA strains, leads to treatment failure and prolonged hospital stay. Therefore, we aimed to determine the strains with reduced susceptibility among MRSA clinical isolates. S. aureus isolates were collected from identified clinical samples. Antibiotic susceptibility was tested using the Kirby-Bauer method. MRSA strains were confirmed using PCR for mecA gene and subjected to the Epsilometer test (Etest®) for determination of Vancomycin minimum inhibitory concentration (MIC). Isolates with intermediate or reduced susceptibility were subjected to broth microdilution (BMD) and further confirmed by population analysis-area under curve (PAP-AUC) testing. Disc diffusion tests showed that all isolates (n = 105; 100%) were susceptible to Rifampicin, followed by Trimethoprim/Sulfamethoxazole. Meanwhile, a high resistance rate was demonstrated against Penicillin (n = 93; 88.6%). Among all isolates, only 26.0% (n = 27) were MRSA. According to the Vancomycin MIC value by Etest®, only two strains (A3, A106) had intermediate susceptibility, and one strain (A30) had reduced susceptibility to Vancomycin (MIC 3 μg/mL). No susceptibility to Vancomycin was identified among strains using the BMD method. According to the PAP-AUC method, it was confirmed that strain A3 was a heterogeneous VISA strain. Vancomycin Etest®, is a reliable screening test for VISA detection. Vancomycin BMD result was not in agreement with the Vancomycin Etest® result. PAP-AUC, the gold standard test used to detect Vancomycin resistance, should be conducted whenever possible for further confirmation and epidemiological record.

High prevalence of a globally disseminated hypervirulent clone, Staphylococcus aureus CC121, with reduced vancomycin susceptibility in community settings in China.

Most vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) are derived from hospital-associated MRSA due to treatment failure; however, the prevalence of hVISA/VISA in community settings remains unclear. Four hundred and seventy-six community-associated isolates were collected between 2010 and 2011 during national surveillance for antimicrobial resistance in 31 county hospitals across China. Drug susceptibility evaluation and mecA detection were performed by using broth microdilution and PCR analysis, respectively. hVISA/VISA were identified by using macro-Etest and a modified population analysis profile (PAP)-AUC method. The genetic features of all hVISA/VISA isolates were genotyped. Among 476 isolates, MRSA and MSSA accounted for 19.7% (n = 94) and 80.3% (n = 382), respectively. Two VISA and 36 hVISA isolates were identified by PAP-AUC testing. The VISA isolates and 29 of the hVISA isolates were MRSA. The proportion of hVISA/VISA was significantly higher in MRSA (30.9%) than in MSSA (1.8%). The hVISA/VISA isolates were assigned to 18 STs classified into seven clonal complexes (CCs). CC121 (n = 12) followed by ST239 (n = 11) was the most prevalent hVISA/VISA clone. All ST239-hVISA/VISA were MRSA, while 12 CC121-hVISA isolates included 6 MSSA and 6 MRSA isolates. SCCmec III was predominant among MRSA-hVISA/VISA isolates. agr I and agr IV were detected in ST239 and CC121, respectively. All except two strains were positive for Panton-Valentine leucocidin genes. To the best of our knowledge, this is the first report of CC121 as a prevalent hVISA clone in community settings, highlighting the necessity of surveillance and stricter infection control measures for this globally disseminated lineage.

Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Uses the VraSR Regulatory System to Modulate Autophagy for Increased Intracellular Survival in Macrophage-Like Cell Line RAW264.7.

The VraSR two-component system is a vancomycin resistance-associated sensor/regulator that is upregulated in vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) strains. VISA/hVISA show reduced susceptibility to vancomycin and an increased ability to evade host immune responses, resulting in enhanced clinical persistence. However, the underlying mechanism remains unclear. Recent studies have reported that S. aureus strains have developed some strategies to survive within the host cell by using autophagy processes. In this study, we confirmed that clinical isolates with high vraR expression showed increased survival in murine macrophage-like RAW264.7 cells. We constructed isogenic vraSR deletion strain Mu3ΔvraSR and vraSR-complemented strain Mu3ΔvraSR-C to ascertain whether S. aureus uses the VraSR system to modulate autophagy for increasing intracellular survival in RAW264.7. Overall, the survival of Mu3ΔvraSR in RAW264.7 cells was reduced at all infection time points compared with that of the Mu3 wild-type strain. Mu3ΔvraSR-infected RAW264.7 cells also showed decreased transcription of autophagy-related genes Becn1 and Atg5, decreased LC3-II turnover and increased p62 degradation, and fewer visible punctate LC3 structures. In addition, we found that inhibition of autophagic flux significantly increased the survival of Mu3ΔvraSR in RAW264.7 cells. Together, these results demonstrate that S. aureus uses the VraSR system to modulate host-cell autophagy processes for increasing its own survival within macrophages. Our study provides novel insights into the impact of VraSR on bacterial infection and will help to further elucidate the relationship between bacteria and the host immune response. Moreover, understanding the autophagic pathway in vraSR associated immunity has potentially important implications for preventing or treating VISA/hVISA infection.

VraSR has an important role in immune evasion of Staphylococcus aureus with low level vancomycin resistance

Vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) are increasingly being reported as associated with treatment failure. Previous studies indicated that VISA/hVISA resists clearance by the host immune system, thereby allowing persistence within the host. VraSR is a vancomycin-resistance-associated sensor/regulator that is highly expressed in VISA/hVISA strains. Whether VraSR plays an important role in immune escape by VISA/hVISA strains is unclear. Here, we constructed a vraSR deletion mutant strain (ΔvraSR) and complementary strain (CΔvraSR) in Mu3 to investigate the effect of VraSR on S. aureus viability in polymorphonuclear leukocytes (PMNs). The ΔvraSR strain was more susceptible to phagocytosis by PMNs and reduced the ability of S. aureus to survive within PMNs. ΔvraSR showed phenotypic changes, including a thinner cell wall, reduced adhesion, and decreased biofilm-forming ability. Real-time quantitative PCR revealed that the transcript levels of cell wall synthesis-related genes (cap5K, cap5N, nanA, tagA, murD) and adhesion-associated genes (fnbA, fnbB, clfA, ebps, sbi) were significantly decreased in the ΔvraSR strain compared with Mu3. In summary, VraSR promotes the survival of S. aureus in the host, which may be associated with an increase in the thickness of the cell wall, adhesion, and biofilm formation.

Prevalence of Slow-Growth Vancomycin Nonsusceptibility in Methicillin-Resistant Staphylococcus aureus.

ABSTRACTWe previously reported a novel phenotype of vancomycin-intermediate Staphylococcus aureus (VISA), i.e., “slow VISA,” whose colonies appear only after 72 h of incubation. Slow-VISA strains can be difficult to detect because prolonged incubation is required and the phenotype is unstable. To develop a method for detection of slow-VISA isolates, we studied 23 slow-VISA isolates derived from the heterogeneous VISA (hVISA) clinical strain Mu3. We identified single nucleotide polymorphisms (SNPs) in genes involved in various pathways which have been implicated in the stringent response, such as purine/pyrimidine synthesis, cell metabolism, and cell wall peptidoglycan synthesis. We found that mupirocin, which also induces the stringent response, caused stable expression of vancomycin resistance. On the basis of these results, we developed a method for detection of slow-VISA strains by use of 0.032 μg/ml mupirocin (Yuki Katayama, 7 March 2017, patent application PCT/JP2017/008975). Using this method, we detected 53 (15.6%) slow-VISA isolates among clinical methicillin-resistant S. aureus (MRSA) isolates. In contrast, the VISA phenotype was detected in fewer than 1% of isolates. Deep-sequencing analysis showed that slow-VISA clones are present in small numbers among hVISA isolates and proliferate in the presence of vancomycin. This slow-VISA subpopulation may account in part for the recurrence and persistence of MRSA infection.

VraR Binding to the Promoter Region of agr Inhibits Its Function in Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA.

Acquisition of vancomycin resistance in Staphylococcus aureus is often accompanied by a reduction in virulence, but the mechanisms underlying this change remain unclear. The present study was undertaken to investigate this process in a clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain, 10827; an hVISA reference strain, Mu3; and a VISA reference strain, Mu50, along with their respective series of vancomycin-induced resistant strains. In these strains, increasing MICs of vancomycin were associated with increased expression of the vancomycin resistance-associated regulator gene (vraR) and decreased expression of virulence genes (hla, hlb, and coa) and virulence-regulated genes (RNAIII, agrA, and saeR). These results suggested that VraR might have a direct or indirect effect on virulence in S. aureus In electrophoretic mobility shift assays, VraR did not bind to promoter sequences of hla, hlb, and coa genes, but it did bind to the agr promoter region. In DNase I footprinting assays, VraR protected a 15-nucleotide (nt) sequence in the intergenic region between the agr P2 and P3 promoters. These results indicated that when S. aureus is subject to induction by vancomycin, expression of vraR is upregulated, and VraR binding inhibits the function of the Agr quorum-sensing system, causing reductions in the virulence of VISA/hVISA strains. Our results suggested that VraR in S. aureus is involved not only in the regulation of vancomycin resistance but also in the regulation of virulence.

Systematic Review and Meta-Analysis of the Epidemiology of Vancomycin-Intermediate and Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Isolates.

BackgroundVancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) are associated with vancomycin treatment failure, and are becoming an increasing public health problem. Therefore, we undertook this study of 91 published studies and made subgroup comparisons of hVISA/VISA incidence in different study years, locations, and types of clinical samples. We also analyzed the genetic backgrounds of these strains.MethodsA systematic literature review of relevant articles published in PubMed and EMBASE from January 1997 to August 2014 was conducted. We selected and assessed journal articles reporting the prevalence rates of hVISA/VISA.ResultsThe pooled prevalence of hVISA was 6.05% in 99,042 methicillin-resistant S. aureus (MRSA) strains and that of VISA was 3.01% in 68,792 MRSA strains. The prevalence of hVISA was 4.68% before 2006, 5.38% in 2006–2009, and 7.01% in 2010–2014. VISA prevalence was 2.05%, 2.63%, and 7.93%, respectively. In a subgroup analysis of different isolation locations, the prevalence of hVISA strains was 6.81% in Asia and 5.60% in Europe/America, and that of VISA was 3.42% and 2.75%, respectively. The frequencies of hVISA isolated from blood culture samples and from all clinical samples were 9.81% and 4.68%, respectively, and those of VISA were 2.00% and 3.07%, respectively. The most prevalent genotype was staphylococcal cassette chromosome mec (SCCmec) II, which accounted for 48.16% and 37.74% of hVISA and VISA, respectively. Sequence Type (ST) 239 was most prevalent.ConclusionThe prevalence of hVISA/VISA has been increasing in recent years, but has been grossly underestimated. Its incidence is higher in Asia than in Europe/America. hVISA is isolated from blood culture samples more often than from other samples. These strains are highly prevalent in epidemic MRSA strains. This study clarifies the epidemiology of hVISA/VISA and indicates that the detection of these strains and the control of nosocomial infections must be strengthened.

Prevalence of vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA among methicillin-resistant S. aureus with high vancomycin minimal inhibitory concentrations in Taiwan: A multicenter surveillance study, 2012–2013

Intermediate-resistance and heteroresistance to vancomycin in methicillin-resistant Staphylococcus aureus (MRSA) is reported worldwide. A surveillance study in 2003 showed that the prevalence rates of vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA) in Taiwan were 0.2% and 0.7%, respectively. This study aimed to investigate the updated prevalence of VISA and hVISA in Taiwan. MRSA isolates from sterile sites with minimal inhibitory concentrations (MICs) of 1μg/mL or more to vancomycin were collected from 15 participating hospitals in Taiwan. Enrolled MRSA isolates were submitted to antimicrobial susceptibility testing, staphylococcal cassette chromosome mec (SCCmec) element typing, and multilocus sequence typing. Isolates with vancomycin MIC of 1μg/mL or 2μg/mL were screened for vancomycin heterogeneous resistance by Etest glycopeptide-resistance detection (GRD). Those with positive GRD screening results were then analyzed by modified population analysis profiling-area under the curve method for confirmation of vancomycin heteroresistance. Between 2012 and 2013, a total of 622 MRSA isolates from sterile sites with vancomycin MIC of 1μg/mL or more were studied. The prevalence rates of hVISA and VISA among these isolates were 10.0% and 2.7%, respectively. The hVISA prevalence increased significantly compared to that in 2003. Compared with vancomycin-susceptible S. aureus, hVISA and VISA isolates were less susceptible to ciprofloxacin, clindamycin, daptomycin, gentamicin, rifampin, and trimethoprim/sulfamethoxazole, and are thus, more likely to have SCCmec II or III element. A twofold increase in either vancomycin or teicoplanin MIC doubled the probability of being hVISA. Growing hVISA prevalence was highly suspected. Longitudinal surveillance of this phenomenon and monitoring of its clinical impact are necessary.

The Prevalence of Vancomycin-Intermediate Staphylococcus aureus and Heterogeneous VISA Among Methicillin-Resistant Strains Isolated from Pediatric Population in a Turkish University Hospital.

There are limited data regarding the prevalence of vancomycin-intermediate Staphylococcus aureus (VISA)/heterogeneous VISA (hVISA) among pediatric population. Our objective was to determine the distribution of vancomycin and daptomycin minimum inhibitory concentrations (MICs) and explore the phenomenon of vancomycin MIC creep and the VISA/hVISA prevalence among the methicillin-resistant Staphylococcus aureus (MRSA) strains belonging to pediatric population by population analysis profile-area under the curve (PAP-AUC) and Etest macromethod. Vancomycin and daptomycin susceptibilities of 94 pediatric isolates of MRSA were tested by broth microdilution (BMD) and Etest methods. To determine the prevalence of VISA/hVISA, Etest macromethod and PAP-AUC was performed on all isolates. All isolates were susceptible to vancomycin and daptomycin by both BMD and Etest methods. Twenty-eight (29.8%) isolates had vancomycin MICs of 2 μg/ml by BMD. No increase in vancomycin MICs was observed over time. There were no VISA among 94 MRSA tested but 20 (21.3%) hVISA isolates were identified by PAP-AUC. Results of Etest macromethod were compared to PAP-AUC. Etest macromethod was 60.0% sensitive and 90.5% specific. The hVISA isolates represented 53.6% of isolates with vancomycin MICs of 2 μg/ml. Also, 75% of hVISA isolates had vancomycin MICs of 2 μg/ml. To our knowledge, this is the first study investigating the prevalence of VISA/hVISA among MRSA isolated from pediatric patients by PAP-AUC method. Based on our findings, MRSA isolates, which have vancomycin MIC of 2 μg/ml can be investigated for the presence of hVISA. In this study, daptomycin showed potent activity against all isolates and may represent a therapeutic option for MRSA infections.

Clinical, microbiological, and genetic characteristics of heteroresistant vancomycin-intermediate Staphylococcus aureus bacteremia in a teaching hospital.

The emergence of vancomycin intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) is of major concern worldwide. Our objective was to investigate the prevalence, phenotypic and molecular features of hVISA strains isolated from bacteremic patients and to determine the clinical significance of the hVISA phenotype in patients with bacteremia. A total of 104 S. aureus blood isolates were collected from a teaching hospital of Argentina between August 2009 and November 2010. No VISA isolate was recovered, and 3 out of 92 patients (3.3%) were infected with hVISA, 2 of them methicillin-resistant S. aureus (MRSA) (4.5% of MRSA). Macro Etest and prediffusion method detected 3/3 and 2/3 hVISA respectively. Considering the type of bacteremia, the three cases were distributed as follows: two patients had suffered multiple episodes of bacteremia (both hVISA strains recovered in the second episode), while only one patient had suffered a single episode of bacteremia with hVISA infection. MRSA bloodstream isolates exhibiting the hVISA phenotype were related to HA-MRSA Cordobes clone (ST5-SCCmec I-spa t149) and MRSA Argentinean pediatric clone (ST100-SCCmec IVNV-spa t002), but not to CA-MRSA-ST30-SCCmec IV-spa t019 clone that was one of the most frequent in our country. Although still relatively infrequent in our hospital, hVISA strains were significantly associated with multiple episodes of bacteremia (p=0.037) and genetically unrelated.

Reduced susceptibility to vancomycin in isogenic Staphylococcus aureus strains of sequence type 59: tracking evolution and identifying mutations by whole-genome sequencing

Vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) phenotypes are increasingly reported in methicillin-resistant S. aureus (MRSA) strains of distinct genetic backgrounds. This study tracked genetic evolution during the development of vancomycin non-susceptibility in a prevalent Asian community-associated MRSA clone of sequence type (ST) 59. ST59 strains were consecutively isolated from a patient who failed chemotherapy for a septic knee over 15 months. The genetic mutations associated with the VISA phenotype were identified by whole-genome sequencing of two strains, which had the vancomycin-susceptible S. aureus (VSSA) and VISA phenotypes. The mutations were subsequently screened in other strains. By correlating the accumulated mutations with vancomycin susceptibility, genetic evolution was tracked at the whole-genome scale. Nine non-synonymous mutations and two steps of genetic evolution were identified during the development of the VISA phenotype. The first step involved a nonsense mutation in agrC and point mutations at five other loci, which were associated with the VSSA-to-hVISA conversion. Mutations of rpoB and fusA following the use of rifampicin and fusidic acid were identified in the second step of evolution, which corresponded to the development of dual resistance to rifampicin and fusidic acid and the conversion of hVISA to VISA. In vivo genetic evolution of S. aureus occurred in stepwise order during the development of incremental vancomycin non-susceptibility and was related to the use of antimicrobial agents.

Novel Combinations of Vancomycin plus Ceftaroline or Oxacillin against Methicillin-Resistant Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA

We demonstrated a significant inverse correlation between vancomycin and beta-lactam susceptibilities in vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) isolates. Using time-kill assays, vancomycin plus oxacillin or ceftaroline was synergistic against 3 of 5 VISA and 1 of 5 hVISA isolates or 5 of 5 VISA and 4 of 5 hVISA isolates, respectively. Beta-lactam exposure reduced overall vancomycin-Bodipy (dipyrromethene boron difluoride [4,4-difluoro-4-bora-3a,4a-diaza-s-indacene] fluorescent dye) binding but may have improved vancomycin-cell wall interactions to improve vancomycin activity. Further research is warranted to elucidate the mechanism behind vancomycin and beta-lactam synergy.

Contribution of Selected Gene Mutations to Resistance in Clinical Isolates of Vancomycin-Intermediate Staphylococcus aureus

Infections with vancomycin-intermediate Staphylococcus aureus (VISA) have been associated with vancomycin treatment failures and poor clinical outcomes. Routine identification of clinical isolates with increased vancomycin MICs remains challenging, and no molecular marker exists to aid in diagnosis of VISA strains. We tested vancomycin susceptibilities by using microscan, Etest, and population analyses in a collection of putative VISA, methicillin-resistant S. aureus, and methicillin-sensitive S. aureus (VSSA) infectious isolates from community- or hospital-associated S. aureus infections (n = 77) and identified 22 VISA and 9 heterogeneous VISA (hVISA) isolates. Sequencing of VISA candidate loci vraS, vraR, yvqF, graR, graS, walR, walK, and rpoB revealed a high diversity of nonsynonymous single-nucleotide polymorphisms (SNPs). For vraS, vraR, yvqF, walK, and rpoB, SNPs were more frequently present in VISA and hVISA than in VSSA isolates, whereas mutations in graR, graS, and walR were exclusively detected in VISA isolates. For each of the individual loci, SNPs were only detected in about half of the VISA isolates. All but one VISA isolate had at least one SNP in any of the genes sequenced, and isolates with an MIC of 6 or 8 μg/ml harbored at least 2 SNPs. Overall, increasing vancomycin MICs were paralleled by a higher proportion of isolates with SNPs. Depending on the clonal background, SNPs appeared to preferentially accumulate in vraS and vraR for sequence type 8 (ST8) and in walK and walR for ST5 isolates. Taken together, by comparing VISA, hVISA, and VSSA controls, we observed preferential clustering of SNPs in VISA candidate genes, with an unexpectedly high diversity across these loci. Our results support a polygenetic etiology of VISA.

Vancomycin-intermediate Staphylococcus aureus selected during vancomycin therapy of experimental endocarditis are not detected by culture-based diagnostic procedures and persist after treatment arrest

Laboratory detection of vancomycin-intermediate Staphylococcus aureus (VISA) and their heterogeneous VISA (hVISA) precursors is difficult. Thus, it is possible that vancomycin failures against supposedly vancomycin-susceptible S. aureus are due to undiagnosed VISA or hVISA. We tested this hypothesis in experimental endocarditis. Rats with aortic valve infection due to the vancomycin-susceptible (MIC 2 mg/L), methicillin-resistant S. aureus M1V2 were treated for 2 days with doses of vancomycin that mimicked the pharmacokinetics seen in humans following intravenous administration of 1 g of the drug every 12 h. Half of the treated animals were killed 8 h after treatment arrest and half 3 days thereafter. Population analyses were done directly on vegetation homogenates or after one subculture in drug-free medium to mimic standard diagnostic procedures. Vancomycin cured 14 of 26 animals (54%; P<0.05 versus controls) after 2 days of treatment. When vegetation homogenates were plated directly on vancomycin-containing plates, 6 of 13 rats killed 8 h after treatment arrest had positive cultures, 1 of which harboured hVISA. Likewise, 6 of 13 rats killed 3 days thereafter had positive valve cultures, 5 of which harboured hVISA. However, one subculture of vegetations in drug-free broth was enough to revert all the hVISA phenotypes to the susceptible pattern of the parent. Thus, vancomycin selected for hVISA during therapy of experimental endocarditis due to vancomycin-susceptible S. aureus. These hVISA were associated with vancomycin failure. The hVISA phenotype persisted in vivo, even after vancomycin arrest, but was missed in vitro after a single passage of the vegetation homogenate on drug-free medium. hVISA might escape detection in clinical samples if they are subcultured before susceptibility tests.

Heterogeneously vancomycin-intermediate Staphylococcus aureus (hVISA) emerged before the clinical introduction of vancomycin in Japan: a retrospective study

Vancomycin-intermediate Staphylococcus aureus (VISA) and its precursor, heterogeneous VISA (hVISA), are increasingly the cause of vancomycin treatment failure. Prolonged glycopeptide treatment causes the emergence of these pathogens. However, we recently reported that hVISA can be generated by methicillin-resistant S. aureus (MRSA) exposure to imipenem (Katayama et al., Antimicrob Agents Chemother. 53:3190-6). We report here a retrospective prevalence study of VISA and hVISA on 750 MRSA clinical strains isolated from 31 Japanese national university hospitals in 1990, the year before the introduction of injectable vancomycin into clinical use in Japan in 1991. No VISA strain was identified, but population analysis identified 38 hVISA strains (5.1%) from 19 hospitals. We also determined the nucleotide sequences of vraSR, walRK, clpP, and rpoB genes whose mutations are known to be associated with vancomycin resistance. When compared with vancomycin-susceptible MRSA strain N315, six of the 38 hVISA strains possessed nonsynonymous mutations in vraSR, seven in walRK, and two in rpoB genes, Thirteen of 38 (34.2%) hVISA strains possessed at least one of these mutations. Results were consistent with our hypothesis that hVISA was present in Japanese hospitals before the clinical introduction of vancomycin.

Incidence and Characteristics of Vancomycin Nonsusceptible Strains of Methicillin-Resistant Staphylococcus aureus at Hershey Medical Center

All 982 methicillin-resistant Staphylococcus aureus strains collected from August 2006 to December 2007 were tested for vancomycin susceptibility by using 3-microg/ml vancomycin brain heart infusion screening plates, a vancomycin Etest, and a vancomycin/teicoplanin macro Etest. Three vancomycin-intermediate Staphylococcus aureus (VISA) (0.3%) and two heterogeneous VISA (0.2%) isolates were identified. The screening method yielded 895 cases of < or =1 colony and 87 positive results (with growth of >1 colony after 48 h); further Etests showed 82/87 isolates with growth on screening plates to be false positive. Repeat testing showed a false-positivity rate of only 15 of the original 87 isolates by plate screening.

Comparative Proteomics Analyses Reveal a Potential Biomarker for the Detection of Vancomycin-Intermediate Staphylococcus aureus Strains

Vancomycin-intermediate Staphylococcus aureus (VISA) strains tend to develop during glycopeptide treatment of infections caused by methicillin-resistant S. aureus (MRSA). Rapid and effective detection methods for VISA strains are lacking, and mechanisms of resistance are unclear. Here, global comparative proteomic approaches have been used to identify potential biomarkers of intermediate vancomycin resistance. With the use of high-resolution two-dimensional gels and iTRAQ mass tagging, numerous proteins were found to be differentially expressed between clinical MRSA and VISA isolates of the same multilocus sequence type. One of these, the predicted lytic transglycosylase SAV2095 (SceD-like protein), was selected for further study based on both its high level of induction in Mu50 and its predicted role in modeling the cell wall, which is the target of vancomycin. Relative SAV2095 mRNA expression levels were compared between 25 MRSA and VISA/heterogeneous VISA clinical isolates by real-time RT-PCR. The SAV2095 mRNA was significantly induced in all VISA isolates relative to all MRSA strains ( p < 0.001), and significant induction of SAV2095 was also seen for several potential heterogeneous VISA strains that appear vancomycin-sensitive by standard minimum inhibitory concentration-determining methods. Furthermore, strains selected in vitro for increasing levels of resistance from four unrelated clinical MRSA isolates displayed concomitant increases in levels of SAV2095 expression. Together, these results suggest that SAV2095 expression level could serve as a molecular diagnostic marker for the rapid detection of VISA.

Evidence for Reduction in Breakpoints Used To Determine Vancomycin Susceptibility in Staphylococcus aureus

Questions regarding the prevalence and clinical significance of both vancomycin-intermediate Staphylococcus aureus (VISA) and, more importantly, heterogeneous VISA (hVISA) are difficult to address, mainly due to the lack of accurate methods and guidelines for the identification of hVISA. VISA strains have been isolated from many areas of the world but are few in number (<30); however, hVISA strains appear more common. Current susceptibility techniques, along with CSLI (S/I/R-4/8-16/32) and EUCAST (S/R-4/8) recommended breakpoints, are generally inadequate for the identification of hVISA (10, 11). Although glycopeptides remain the recommended therapy for patients with methicillin-resistant Staphylococcus aureus infection, treatment failure is common, with 10 to 20% in cases of endocarditis and 40% in lower respiratory tract infections (3, 6). Treatment failure cause is not fully understood; however, clinical failures have been reported for patients with VISA (8, 1) and, more recently, for hVISA infection (2, 12, 13). Evidence suggests that hVISA infections are clinically relevant, with patients with hVISA bacteremia more likely to have high-bacterial-load infections, vancomycin treatment failure, bacteremia for >7 days, and a significantly higher mortality (63%) than those patients infected with vancomycin-susceptible methicillin-resistant S. aureus (VSSA) bacteremia (12%) (4, 5). More recently, treatment failure in patients with infections caused by strains with MICs within the susceptibility range (≤2 mg/liter) has been reported (9). Also, current CDC guidelines for testing S. aureus with vancomycin recommend that strains exhibiting MICs of ≤2 mg/liter plus growth on vancomycin screening plates are possibly VISA (www.cdc.gov/ncidod/hip/vanco/vanco.htm). These data suggest that current susceptibility breakpoints for glycopeptides require review. We used an international collection of VSSA, VISA, and hVISA strains with CSLI agar dilution techniques to determine vancomycin MICs (7). Each data set was compared with a view to recalculating a vancomycin breakpoint. VISA and hVISA phenotypes were identified by the more accurate modified population analysis method (population analysis profile-area under the curve) (14). Of 106 glycopeptide-susceptible S. aureus strains, 10.4% had vancomycin MICs of 0.5 mg/liter, 85.8% had vancomycin MICs of 1 mg/liter, and 3.8% had vancomycin MICs of 2 mg/liter. The percentage of isolates classified as susceptible by the CSLI and EUCAST breakpoint (4 mg/liter) and a reduced breakpoint (2 mg/liter) was 100%, suggesting that no false positives would emerge from breakpoint reduction. Of 20 VISA strains, 55% exhibited vancomycin MICs of 4 mg/liter and 45% had vancomycin MICs of 8 mg/liter. Using the current breakpoint, 55% are classified as susceptible and 45% as intermediate. However, when using the reduced breakpoint, 100% are classified as intermediate (CSLI) or resistant (EUCAST). Of 157 hVISA strains, 2% exhibited vancomycin MICs of 1 mg/liter, 80.2% had vancomycin MICs of 2 mg/liter, and 17.8% had vancomycin MICs of 4 mg/liter. Using the current breakpoint, 100% are classified as susceptible; however, when using the reduced breakpoint, 82.2% would still be classified as susceptible but 17.8% would be classified as intermediate (CSLI) or resistant (EUCAST). By use of the reduced breakpoint, the correct classifications of hVISA and VISA would increase from 45% and 0% to 100% and 17.2%, respectively. Distribution of vancomycin MICs in the different phenotypes shows that 2 mg/liter is the most predominant in hVISA (Fig. ​(Fig.1).1). The effect of a reduced breakpoint on the classification of coagulase-negative staphylococci would be minimal, with only 1.56% currently exhibiting a vancomycin MIC of 4 mg/liter (www.EUCAST.org). This evidence supports a modification of the contemporary interpretative guidelines for vancomycin susceptibility testing from ≤4 mg/liter for susceptible isolates to ≤2 mg/liter. FIG. 1. Frequency distribution of vancomycin MICs in VSSA, hVISA, and VISA strains.

Evidence for Reduction in Breakpoints Used To Determine Vancomycin Susceptibility in Staphylococcus aureus

Questions regarding the prevalence and clinical significance of both vancomycin-intermediate Staphylococcus aureus (VISA) and, more importantly, heterogeneous VISA (hVISA) are difficult to address, mainly due to the lack of accurate methods and guidelines for the identification of hVISA. VISA strains have been isolated from many areas of the world but are few in number (<30); however, hVISA strains appear more common. Current susceptibility techniques, along with CSLI (S/I/R-4/8-16/32) and EUCAST (S/R-4/8) recommended breakpoints, are generally inadequate for the identification of hVISA (10, 11). Although glycopeptides remain the recommended therapy for patients with methicillin-resistant Staphylococcus aureus infection, treatment failure is common, with 10 to 20% in cases of endocarditis and 40% in lower respiratory tract infections (3, 6). Treatment failure cause is not fully understood; however, clinical failures have been reported for patients with VISA (8, 1) and, more recently, for hVISA infection (2, 12, 13). Evidence suggests that hVISA infections are clinically relevant, with patients with hVISA bacteremia more likely to have high-bacterial-load infections, vancomycin treatment failure, bacteremia for >7 days, and a significantly higher mortality (63%) than those patients infected with vancomycin-susceptible methicillin-resistant S. aureus (VSSA) bacteremia (12%) (4, 5). More recently, treatment failure in patients with infections caused by strains with MICs within the susceptibility range (≤2 mg/liter) has been reported (9). Also, current CDC guidelines for testing S. aureus with vancomycin recommend that strains exhibiting MICs of ≤2 mg/liter plus growth on vancomycin screening plates are possibly VISA (www.cdc.gov/ncidod/hip/vanco/vanco.htm). These data suggest that current susceptibility breakpoints for glycopeptides require review. We used an international collection of VSSA, VISA, and hVISA strains with CSLI agar dilution techniques to determine vancomycin MICs (7). Each data set was compared with a view to recalculating a vancomycin breakpoint. VISA and hVISA phenotypes were identified by the more accurate modified population analysis method (population analysis profile-area under the curve) (14). Of 106 glycopeptide-susceptible S. aureus strains, 10.4% had vancomycin MICs of 0.5 mg/liter, 85.8% had vancomycin MICs of 1 mg/liter, and 3.8% had vancomycin MICs of 2 mg/liter. The percentage of isolates classified as susceptible by the CSLI and EUCAST breakpoint (4 mg/liter) and a reduced breakpoint (2 mg/liter) was 100%, suggesting that no false positives would emerge from breakpoint reduction. Of 20 VISA strains, 55% exhibited vancomycin MICs of 4 mg/liter and 45% had vancomycin MICs of 8 mg/liter. Using the current breakpoint, 55% are classified as susceptible and 45% as intermediate. However, when using the reduced breakpoint, 100% are classified as intermediate (CSLI) or resistant (EUCAST). Of 157 hVISA strains, 2% exhibited vancomycin MICs of 1 mg/liter, 80.2% had vancomycin MICs of 2 mg/liter, and 17.8% had vancomycin MICs of 4 mg/liter. Using the current breakpoint, 100% are classified as susceptible; however, when using the reduced breakpoint, 82.2% would still be classified as susceptible but 17.8% would be classified as intermediate (CSLI) or resistant (EUCAST). By use of the reduced breakpoint, the correct classifications of hVISA and VISA would increase from 45% and 0% to 100% and 17.2%, respectively. Distribution of vancomycin MICs in the different phenotypes shows that 2 mg/liter is the most predominant in hVISA (Fig. ​(Fig.1).1). The effect of a reduced breakpoint on the classification of coagulase-negative staphylococci would be minimal, with only 1.56% currently exhibiting a vancomycin MIC of 4 mg/liter (www.EUCAST.org). This evidence supports a modification of the contemporary interpretative guidelines for vancomycin susceptibility testing from ≤4 mg/liter for susceptible isolates to ≤2 mg/liter. FIG. 1. Frequency distribution of vancomycin MICs in VSSA, hVISA, and VISA strains.