Detection of Staphylococcus aureus isolates with heterogeneous intermediate-level resistance to vancomycin in the United States

The prevalence of heterogeneous intermediate-level resistance to vancomycin (hVISA) in Staphylococcus aureus was assessed by screening a large collection of recent isolates. Susceptibility testing by the Clinical and Laboratory Standards Institute broth microdilution method and the Etest GRD (glycopeptide resistance detection) method (bioMérieux) was performed on 4,210 clinically significant S. aureus isolates obtained in 2009 from 43 U.S. centers. Isolates with Etest GRD-positive results for hVISA were evaluated further by repeat GRD testing and population analysis profiling-area under the curve (PAP-AUC) analysis. No VISA (vancomycin MIC, 4 to 8 μg/ml) or vancomycin-resistant (MIC ≥ 16 μg/ml) strains were detected. The Etest GRD screen for hVISA was initially positive for 68 isolates (1.6%; all by teicoplanin MIC ≥ 8 μg/ml at 24 or 48 h). Among those 68 isolates, 45 were reproducibly GRD positive. PAP-AUC testing confirmed only 11 isolates as hVISA (all had reproducible GRD-positive results). The 11 hVISA isolates were from nine medical centers and appeared genetically diverse (ten different PFGE types). The rates of resistance (including intermediate) for hVISA were as follows: oxacillin, 82%; erythromycin, 82%; clindamycin, 73%; levofloxacin, 73%; trimethoprim-sulfamethoxazole, 9%; and daptomycin, 9%. All hVISA isolates were susceptible to linezolid, tigecycline, and ceftaroline. Our data suggest that the overall prevalence of hVISA in the United States is low (0.3%). The hVISA isolates represented 10.5% of isolates with vancomycin MICs of 2 μg/ml and 0.1% of isolates with vancomycin MICs of 1 μg/ml. The positive predictive value of GRD Etest for hVISA was 16.2% for initial screen positive and 24.4% for reproducibly positive results.     

Vancomycin susceptibility trends and prevalence of heterogeneous vancomycin-intermediate Staphylococcus aureus in clinical methicillin-resistant S. aureus isolates

Due to the rise in methicillin-resistant Staphylococcus aureus (MRSA) infections and widespread use of vancomycin, MRSA isolates with reduced susceptibility to vancomycin are emerging (i.e., MIC creep). However, the prevalence of heterogeneous vancomycin-intermediate S. aureus (hVISA) is unknown due to the difficulty in detecting this phenotype. Recently, Etest glycopeptide resistance detection (GRD) strips have been developed to detect hVISA. This study assessed vancomycin susceptibility in MRSA isolates and determined the prevalence of hVISA by Etest GRD and population analysis profile-area under the curve ratio (PAP-AUC). The genetic backgrounds of 167 MRSA isolates collected from 2000 to 2008 were identified by pulsed-field gel electrophoresis. Vancomycin MICs were determined using Etest and two broth microdilution assays, MicroScan and Sensititre. Etest GRD was performed on all isolates, and those exhibiting a hVISA phenotype were further tested by PAP-AUC. The vancomycin MIC modes remained consistent at 1 μg/ml, as assessed by Sensititre and MicroScan. Etest reported a significant increase (mode MIC = 1.5 μg/ml) in the MIC between 2000 and 2008 (P < 0.01); however, this increase did not reflect a ≥ 2-fold change. In addition, the slight MIC increase did not increase linearly from 2000 to 2008, suggesting biological fluctuation, and is inconsistent with the concept of MIC creep. Etest GRD identified six hVISA isolates, two of which were confirmed to be hVISA by PAP-AUC. In conclusion, reduced vancomycin susceptibility was not detected in our hospital over a 9-year period using three different MIC methodologies, and the hVISA incidence was 1.2%, as determined by Etest GRD and PAP-AUC.     

Performance of various testing methodologies for detection of heteroresistant vancomycin-intermediate Staphylococcus aureus in bloodstream isolates

The best screening method for detecting heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) remains unclear. Using population analysis profiling utilizing the area under the concentration-time curve (PAP-AUC) as the gold standard, we screened 458 consecutive methicillin-resistant S. aureus (MRSA) bloodstream isolates to determine the most accurate and cost-effective testing strategy to detect the presence of heteroresistance. All isolates were also tested using the macromethod Etest (MET) and glycopeptide resistance detection (GRD) Etest. The MIC was determined by several methods, including standard vancomycin Etest, vancomycin broth microdilution (BMD), and Vitek2 testing. Fifty-five (12%) hVISA and 4 (1%) VISA isolates were detected by PAP-AUC. Compared to PAP-AUC, the sensitivities and specificities of MET, GRD Etest, BMD (using a MIC cutoff of ≥ 2 mg/liter), and standard vancomycin Etest (using a MIC cutoff of ≥ 2 mg/liter) were 89 and 55%, 71 and 94%, 82 and 97%, and 71 and 94%, respectively. Combination testing increased the overall testing accuracy by reducing the number of false-positive results. Cost was determined predominately by the number of PAP-AUC runs required following a screening assay. The most cost-effective strategy was BMD (using a MIC cutoff of ≥ 2 μg/ml) as a standalone assay or in combination with PAP-AUC, provided that BMD testing was batched. GRD Etest remained an alternative, with 71% of hVISA isolates detected. Prevalence influenced both cost and test accuracy, with results remaining unchanged for hVISA prevalences of up to 25%. Implementation of any testing strategy would therefore be dependent on balancing cost with accuracy in a given population and clinical context.     

Endemic heteroresistant glycopeptide intermediate Staphylcoccus aureus (hGISA) comprising unrelated clonal types and not associated with vancomycin therapy

The detection of methicillin-resistant S. aureus (SA) (MRSA) refractory to glycopeptides is a serious clinical issue. The prevalence of hetero-resistant GISA (hGISA) strains at H. Maréchal Joffre, France is reported.858 non-repeat SA were isolated during 1999. 367 (43%) of these, from 257 patients, were MRSA (mean incidence 11.9/1000 admissions). All MSRA detected during 1999 were screened for vancomycin (VAN) resistance (BHI+4 mg/l VAN). Isolates recovered were retested using Etest strips (2 McFarland inoculum on BHI) and population analysis profile/area under the curve (PAP-AUC) analysis with hGISA SA Mu3 as a comparator. 58 selected strains were screened for teicoplanin resistance(TEI) using SFM recommended screen (2 McFarland inoculum on MH+5 mg/L TEI) and MIC (0.5 MF inoculum swabbed on MH agar) methods. 188 (51.3%) grew on VAN screen agar (6.1/1000 admissions). 58 strains (7.6%) possessed Etest VAN MIC > 8 mg/l all others being VAN < 8 mg/l. Of these 58 isolates, 10 were stably heterogeneously resistant to both VAN and teicoplanin (MIC > 8 mg/l). PAP-AUC showed 12 strains to have PAP-AUC ratios > 0.95 but < 1.5 (ie. hGISA, not GISA). All 7 isolates defined as hGISA by both Etest and PAP-AUC comprised 1 PFGE clone (< 3 bands difference).Additionally 2 distinct PFGE types were detected among the other 5 hGISA identified PAP-AUC. The 12 hGISAs, were derived from 12 patients with severe underlying disease. None were on glycopeptide therapy prior to hGISA isolation.This is the first report of endemic hGISA, comprising 3 clonal types. The isolation of hVISA seems not to be associated with patient-specific glycopeptide therapies.     

Investigation of reduced susceptibility to glycopeptides among methicillin-resistant Staphylococcus aureus isolates from patients in Ireland and evaluation of agar screening methods for detection of heterogeneously glycopeptide-intermediate S. aureus

Methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 3,189) from 2,990 patients were investigated by agar screening and by the Etest macromethod for reduced susceptibility to glycopeptide. No vancomycin-resistant S. aureus or glycopeptide-intermediate S. aureus (GISA) isolates were detected, but 178 isolates were confirmed as hetero-GISA (hGISA) by vancomycin population analysis profile (vPAP)-area under the curve (AUC) ratio determination and/or teicoplanin PAP (tPAP) methods. Of 139 isolates detected using the recommended Etest macromethod cutoff values of > or =8 mg/liter for both vancomycin and teicoplanin or > or =12 mg/liter for teicoplanin alone, 73 were confirmed as hGISA by vPAP-AUC, 95 were confirmed as hGISA by tPAP, and 108 were confirmed as hGISA by both methods. An Etest macromethod cutoff value of 8 mg/liter for teicoplanin alone detected a further 70 hGISA (17 were confirmed by vPAP-AUC and 70 were confirmed by tPAP). Agar screening utilizing brain heart infusion (BHI) agar containing 6 mg of vancomycin/liter (BHIV6) and Mueller-Hinton (MH) agar containing 8 mg of teicoplanin/liter (MHT8) failed to detect hGISA. MH agar containing 5 mg of teicoplanin/liter (MHT5) and BHI containing 5 mg of teicoplanin/liter (BHIT5) were evaluated using 10-microl volumes of three inoculum concentrations (with densities equivalent to 0.5 and 2.0 McFarland turbidity standards and stationary-phase BHI broth subcultures [MHT5(0.5), MHT5(2.0), MHT5(S), BHIT5(0.5), BHIT5(2.0), and BHIT5(S)]). The sensitivity of all methods except MHT5(0.5) and MHT5(2.0) was 100%. The specificity ranged from 4 to 82%. BHIT5(0.5) yielded the best performance, with a specificity of 84% for detecting isolates with teicoplanin Etest macromethod values of > or =8 mg/liter. Screening on BHIT5(0.5) is useful where screen-positive isolates are investigated with the Etest macromethod and confirmed by vPAP-AUC and tPAP. The prevalence of hGISA among patients with blood culture isolates recovered in Irish hospitals between 1999 and 2003 was 2.6%, whereas the prevalence among patients with isolates from all specimen sites collected during a 2-week survey in 1999 was 12%. The prevalence in one hospital decreased from 5.3% in 2003 to 1.5% in 2004.     

Evaluation of a new Etest vancomycin-teicoplanin strip for detection of glycopeptide-intermediate Staphylococcus aureus (GISA), in particular, heterogeneous GISA

Glycopeptide-intermediate Staphylococcus aureus (GISA) and, in particular, heterogeneous GISA (hGISA) are difficult to detect by standard MIC methods, and thus, an accurate detection method for clinical practice and surveillances is needed. Two prototype Etest strips designed for hGISA/GISA resistance detection (GRD) were evaluated using a worldwide collection of hGISA/GISA strains covering the five major clonal lineages. A total of 150 strains comprising 15 GISA and 60 hGISA strains (defined by population analysis profiles-area under the curve [PAP-AUC]), 70 glycopeptide-susceptible S. aureus (GSSA) strains, and 5 S. aureus ATCC reference strains were tested. For standardized Etest vancomycin (VA) MIC testing, the modified Etest macromethod with VA and teicoplanin (TP) strips tested with a heavier inoculum using brain heart infusion agar (BHI) and two glycopeptide screening agar plates (6 μg/ml VA/BHI and 5 μg/ml Mueller-Hinton agar [MHA]) were tested in parallel with the two new Etest GRD strips: a VA 32 (0.5-μg/ml)-TP 32 (0.5-μg/ml) double-sided gradient (E-VA/TP) with one prototype overlaid with a nutrient (E-VA/TP+S) to enhance the growth of hGISA. The Etest GRD strips were tested with a standard 0.5-McFarland standard inoculum using MHA and MHA plus 5% blood (MHB) and were read at 18 to 24 and 48 h. The interpretive MIC cutoffs used for the new Etest GRD strips at 24 and 48 h were as follows: for GISA, TP or VA, ≥8, and a standard VA MIC of ≥6; for hGISA, TP or VA, ≥8, and a standard VA MIC of ≤4. The results on MHB at 48 h showed that E-VA/TP+S had high specificity (94%) and sensitivity (95%) in comparison to PAP-AUC and was able to detect all GISA (n = 15) and 98% of hGISA (n = 60) strains. In contrast, the glycopeptide screening plates performed poorly for hGISA. The new Etest GRD strip (E-VA/TP+S), utilizing standard media and inocula, is a simple and acceptable tool for detection of hGISA/GISA for clinical and epidemiologic purposes.     

Detection of intermediately vancomycin-susceptible and heterogeneous Staphylococcus aureus isolates: comparison of Etest and Agar screening methods

Detection of Staphylococcus aureus isolates with intermediate vancomycin susceptibility (VISA) and heteroresistance (hVISA) remains problematic. The population analysis profile/area under the curve (PAP/AUC) is the gold standard but is cumbersome. We compared the performance of two Etest screening methods (macromethod [MAC] and glycopeptide resistance detection [GRD]) plus brain heart infusion (BHI) agars supplemented with 3 (BHI-V3) or 4 (BHI-V4) mg/liter vancomycin in detecting hVISA and/or VISA phenotypes. Etest hVISA screenings were done in parallel for 485 saved methicillin-resistant S. aureus (MRSA) blood isolates according to the manufacturer's instructions. The PAP/AUC was measured for all isolates according to the modified method. PAP/AUC test isolate/Mu3 ratios of <0.9, 0.9 to 1.3, and >1.3 were considered positive for susceptible MRSA (S-MRSA), hVISA, and VISA, respectively. PAP/AUC revealed seven VISA and 33 hVISA phenotypes. MAC screening was positive for 30 (75.0%) hVISA/VISA and 49 (11.0%) S-MRSA isolates. GRD screening was positive for 28 (70.0%) hVISA/VISA and 63 (14.2%) S-MRSA isolates. Growth on BHI-V3 was noted in all hVISA/VISA and 24 (5.4%) S-MRSA isolates. Growth on BHI-V4 was noted in all VISA and four (12.1%) hVISA isolates. None of the S-MRSA isolates grew on BHI-V4 agar. The sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were 75.0%, 89.0%, 38.0%, and 97.5% for MAC; 70.0%, 85.8%, 30.8%, and 97.0% for GRD; 100%, 94.6%, 62.5%, and 100% for BHI-V3; and 100, 99.2%, 63.6%, and 100% for BHI-V4 (for detecting VISA). These findings suggest that both Etest screening methods have excellent NPV, but positive results require confirmation. BHI-V3 and BHI-V4 agars provide more precise identification of hVISA and VISA, respectively; they may be reasonable alternatives to PAP/AUC.     

Evaluation of Current Methods for Detection of Staphylococci with Reduced Susceptibility to Glycopeptides

The sensitivity and specificity of seven methods (agar dilution, broth microdilution, Etest at 0.5 and 2.0 McFarland (McF) inocula, two agar screening methods, and population studies [PS]) were evaluated in a double-blind study involving 284 methicillin-resistant Staphylococcus aureus (MRSA) strains and 45 Staphylococcus strains with reduced susceptibilities to vancomycin (SRSV). The results were compared to the population analysis profile-area under the curve ratio method (PAP-AUC ratio compared to that of Mu3) as described by Wootton et al. The agar screening method using brain heart infusion agar (6 microg of vancomycin per ml) gave a sensitivity of 22% and a specificity of 97%. A similar method using Mueller-Hinton agar (5 microg of vancomycin per ml) gave a sensitivity of 20% and a specificity of 99%. The PS method detected 34 false positives (12%) and gave a sensitivity of 71% and a specificity of 88%. Etest using 0.5 and 2.0 McF inocula gave sensitivities and specificities of 82 and 93% and of 96 and 97%, respectively. The best Etest interpretative criteria for the 2.0 McF inoculum was > or =8 mg of vancomycin per liter and > or =8 microg teicoplanin per ml or > or =12 microg of teicoplanin per ml. The direct colony suspension inoculum for this method was found to be equally accurate in detecting (hetero-)glycopeptide-intermediate S. aureus compared to the overnight broth inoculum preparation method. Agar dilution and broth microdilution using the NCCLS breakpoint criteria for vancomycin gave sensitivities and specificities of 20 and 100% and of 11 and 100%, respectively. Using the Etest with a 2.0 McF inoculum, six different media were assessed against a selection of SRSV (n = 48) and MRSA (n = 12). Brain heart infusion agar yielded the highest sensitivity and specificity values: 88 and 88%, respectively.     

Prevalence and characteristics of heteroresistant vancomycin-intermediate Staphylococcus aureus bacteremia in a tertiary care center

Infections with S. aureus with heterogeneous intermediate resistance to vancomycin (hVISA) are occurring more frequently. The detection of these infections, their prevalence, clinical characteristics, and significance are controversial. During 2003 and 2004, all blood culture isolates of methicillin-resistant Staphylococcus aureus (264 patients) at the Sheba Medical Center, Tel Hashomer, Israel, were assessed for hVISA by using the Etest macromethod. A total of 16 patients (6%) were positive for hVISA. Resistance to teicoplanin alone and to vancomycin alone using the Etest macromethod was found in 14 and 10 patients, respectively. Standard MICs to vancomycin were between 1 to 4 mg/ml. Most of these isolates (12 of 16 [75%]) would have been missed without specific testing. The median number of bacteremic days was 4. Seven patients had positive blood cultures for more than 5 days. Twelve patients died, and for eight of these the deaths were directly related to hVISA sepsis. We found that hVISA bacteremia was prevalent in our institution, and we suggest seeking hVISA in patients with persistent S. aureus bacteremia.     

High percentage of methicillin-resistant Staphylococcus aureus isolates with reduced susceptibility to glycopeptides in The Netherlands

While testing the in vitro activities of 14 antimicrobial agents against 107 methicillin-susceptible Staphylococcus aureus (MSSA) and 250 methicillin-resistant S. aureus (MRSA) isolates collected in The Netherlands, we found to our surprise that 19 (7.6%) MRSA isolates were suspected of having reduced susceptibilities to the glycopeptides when the Etest system (AB Biodisk, Solna, Sweden) was used with a large inoculum (no. 2 McFarland standard) and an extended incubation time (48 h) on brain heart infusion agar for MIC testing. Eventually, 15 of these isolates were classified as heterogeneously resistant to glycopeptides (heterogeneously glycopeptide-intermediate S. aureus [hGISA] isolates) according to the population analysis profile-area under the curve analysis. The MICs at which 50 and 90% of isolates are inhibited obtained with the Etest system with the large inoculum were as follows: for MSSA isolates, 3.0 and 4.0 micro g/ml, respectively, for both teicoplanin and vancomycin; for MRSA isolates, 3.0 and 8.0 micro g/ml, respectively, for teicoplanin, and 3.0 and 4.0 micro g/ml, respectively, for vancomycin. This is the first report of hGISA isolates in The Netherlands.     

Low prevalence of methicillin-resistant Staphylococcus aureus with reduced susceptibility to glycopeptides in Belgian hospitals

Staphylococcus aureus strains with decreased susceptibility to glycopeptides (GISA) have been associated with increased risk of glycopeptide treatment failure. To assess the prevalence of these strains in hospitalised patients in Belgium, 455 methicillin-resistant S. aureus (MRSA) isolates collected in 2001 were screened by two assays: (i) growth on vancomycin agar screen (VAS; brain heart infusion agar (BHI) + vancomycin 6 mg/L); and (ii) a synergy/antagonism test with aztreonam/cefazolin on Mu3 agar (BHI + vancomycin 3 mg/mL). Isolates growing on VAS or Mu3 agar were characterised further by analysis of population susceptibility profiles. MICs of glycopeptides were determined by agar dilution, broth microdilution and Etest (low and high inocula) methods. The isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and determination of staphylococcal cassette chromosome mec (SCCmec) type. No GISA isolates were found. Three (0.7%) hetero-vancomycin intermediate S. aureus (hVISA) and ten (2.2%) hetero-teicoplanin intermediate S. aureus (hTISA) isolates were identified by population analysis. All but one hetero-GISA isolate belonged to either epidemic PFGE group A/SCCmec type I (69%) or PFGE group D/SCCmec type I (23%), both of which were resistant to gentamicin. The sensitivity and specificity for the detection of hetero-GISA by the two assays were 15.4% and 99.8%, respectively, for VAS, and 84.6% and 95.9%, respectively, for Mu3. The data indicated that hetero-GISA strains were uncommon among Belgian MRSA isolates from hospitalised patients. Use of Mu3 agar was more sensitive, but less specific, than VAS as a screening method.     

Synergy of β-Lactams with Vancomycin against Methicillin-Resistant Staphylococcus aureus: Correlation of Disk Diffusion and Checkerboard Methods

Modified disk diffusion (MDD) and checkerboard tests were employed to assess the synergy of combinations of vancomycin and β-lactam antibiotics for 59 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Mu50 (ATCC 700699). Bacterial inocula equivalent to 0.5 and 2.0 McFarland standard were inoculated on agar plates containing 0, 0.5, 1, and 2 μg/ml of vancomycin. Oxacillin-, cefazolin-, and cefoxitin-impregnated disks were applied to the surface, and the zones of inhibition were measured at 24 h. The CLSI-recommended checkerboard method was used as a reference to detect synergy. The MICs for vancomycin were determined using the Etest method, broth microdilution, and the Vitek 2 automated system. Synergy was observed with the checkerboard method in 51% to 60% of the isolates when vancomycin was combined with any β-lactam. The fractional inhibitory concentration indices were significantly lower in MRSA isolates with higher vancomycin MIC combinations (P < 0.05). The overall agreement between the MDD and checkerboard methods to detect synergy in MRSA isolates with bacterial inocula equivalent to McFarland standard 0.5 were 33.0% and 62.5% for oxacillin, 45.1% and 52.4% for cefazolin, and 43.1% and 52.4% for cefoxitin when combined with 0.5 and 2 μg/ml of vancomycin, respectively. Based on our study, the simple MDD method is not recommended as a replacement for the checkerboard method to detect synergy. However, it may serve as an initial screening method for the detection of potential synergy when it is not feasible to perform other labor-intensive synergy tests.     

Utility of the Etest GRD for detecting Staphylococcus aureus with reduced susceptibility to glycopeptides in cystic fibrosis patients

Glycopeptide-intermediate S. aureus (GISA), particularly heterogeneous GISA (hGISA), remain difficult to detect in the routine practice of medical microbiology. Novel tools have been evaluated comparatively to the population analysis profile-area under the curve (PAP-AUC) reference method for detecting GISA/hGISA. Among them, the Etest GRD showed relatively high specificity (85.8–97%) and negative predictive value (97%) but lower sensibility (57–95%) and positive predictive value (30.8%). We investigated the utility of the Etest GRD for detecting GISA/hGISA among 180 strains isolated from 106 cystic fibrosis (CF) patients. Etest GRD was performed on all isolates, and those exhibiting a GISA/hGISA phenotype were further tested by PAP-AUC and other agar routine assays for GISA/hGISA detection. The Etest GRD allowed the detection of 15 GISA/hGISA strains, of which eight were confirmed by the reference method. Despite the 3.9% level of false positive results, the Etest GRD constitutes a useful routine tool for detecting GISA/hGISA overlooked by other routine assays, two strains being detected by the Etest GRD only. GISA/hGISA represented 7.7% of MRSA and 2.1% of MSSA, and were found in 4.7% of CF patients colonized/infected by S. aureus, which is the highest rate reported to date in this population.     

Trends in the susceptibility of methicillin-resistant Staphylococcus aureus to nine antimicrobial agents,including ceftobiprole,nemonoxacin, and tyrothricin: results from the Tigecycline In Vitro Surveillance in Taiwan (TIST) study, 2006–2010

This study investigated the in vitro susceptibilities of methicillin-resistant Staphylococcus aureus (MRSA) to nine antimicrobial agents in Taiwan. A total of 1,725 isolates were obtained from 20 hospitals throughout Taiwan from 2006 to 2010. The minimum inhibitory concentrations (MICs) of the nine agents were determined by the agar dilution method. The MICs of mupirocin and tyrothricin were determined for 223 MRSA isolates collected from 2009 to 2010. For vancomycin, 99.7 % were susceptible; however, 30.0 % (n?=?517) exhibited MICs of 2 μg/ml and 0.3 % (n?=?6) demonstrated intermediate susceptibility (MICs of 4 μg/ml). Nearly all isolates (≥99.9 %) were susceptible to teicoplanin, linezolid, and daptomycin. The MIC₉₀ values were 2 μg/ml for ceftobiprole and 1 μg/ml for nemonoxacin. The MIC₉₀ values of mupirocin and tyrothricin were 0.12 and 4 μg/ml, respectively. MIC creep was noted for daptomycin during this period, but not for vancomycin, teicoplanin, linezolid, or tigecycline. For isolates with vancomycin MICs of 2 μg/ml, the MIC₉₀ values were 2 μg/ml for teicoplanin, 0.5 μg/ml for daptomycin, and 0.5 μg/ml for tigecycline. Those values were four- to eight-fold higher than those among isolates with vancomycin MICs of 0.5 μg/ml (2, 0.06, and 0.12 μg/ml, respectively). Of the nine MRSA isolates exhibiting non-susceptibility to vancomycin (n?=?6), teicoplanin (n?=?1), daptomycin (n?=?2), or tigecycline (n?=?1), all had different pulsotypes, indicating the absence of intra-hospital or inter-hospital spread. The presence of a high proportion of MRSA isolates with elevated MICs (2 μg/ml) and MIC creep of daptomycin might alert clinicians on the therapy for serious MRSA infections in Taiwan.     

Glycopeptide resistance in coagulase-negative staphylococci isolated in blood cultures from patients with hematological malignancies during three decades

The aim of this study was to determine if there was a long-term increase in glycopeptide minimum inhibitory concentration (MIC) values, MIC creep, among bloodstream isolates of Staphylococcus epidermidis and S. haemolyticus isolated from patients with hematological malignancies. We conducted a retrospective single-center study where all positive blood cultures of S. epidermidis (n = 387) and S. haemolyticus (n = 19) isolated from patients with hematological malignancies during three decades, 1980 to 2009, were re-evaluated for the presence of reduced susceptibility to vancomycin and teicoplanin. Three different methods for the detection of reduced susceptibility to glycopeptides were used; standard Etest, macromethod Etest, and glycopeptide resistance detection (GRD) Etest. The median MIC value for vancomycin was 2 mg/L. MIC values for vancomycin and teicoplanin did not show any statistically significant increase during the study period. The presence of heterogeneously glycopeptide-intermediate staphylococci (hGIS) was analyzed among 405 coagulase-negative staphylococci (CoNS) isolates. hGIS were found in 31–45% of the CoNS isolates by the macromethod Etest and in 53–67% by the GRD Etest during the three decades. In conclusion, we did not observe any long-term glycopeptide MIC creep determined by the standard Etest, although a high and increasing proportion of heterogeneous vancomycin resistance was observed.     

Correlation of methicillin-resistant Staphylococcus aureus vancomycin minimal inhibitory concentration results by Etest and broth microdilution methods with population analysis profile: lack of Etest overestimation of the MIC

Methicillin-resistant Staphylococcus aureus (MRSA) vancomycin minimum inhibitory concentrations (V-MICs) are sometimes reported to be higher according to Etest versus broth microdilution (BMD). These observations are often interpreted as an Etest overestimation of the actual MIC. We measured V-MIC of 484 MRSA blood isolates using Etest, BMD, and a modified BMD (M-BMD) with incremental dilutions parallel to the Etest scale, correlated the results with population analysis profile–area under the curve (PAP-AUC). All MIC tests were done in parallel. The mean V-MIC was comparable (1.83?±?0.44 [Etest], 1.88?±?0.67 [BMD] and 1.75?±?0.57 mg/L [M-BMD]; p?=?0.9 [ANOVA]). The V-MICs/PAP-AUC correlation coefficient was 0.555 (Etest), 0.513 (BMD), and 0.586 (M-BMD). Etest MICs were equal (44.2 %), one dilution higher (21.9 %), two dilutions higher (2.5 %), one dilution lower (29.8 %), and two dilutions lower (1.6 %) than BMD MICs and were equal (61.5 %), one dilution higher (28.3 %), two dilutions higher (0.4 %), one dilution lower (9.5 %), and two dilutions lower (0.2 %) than M-BMD MICs. The mean PAP-AUC for Etest vs M-BMD among isolates with similar Etest/M-BMD MIC values was 0.25?±?0.15 vs 0.35?±?0.13 (p?=?0.8), 0.46?±?0.16 vs 0.50?±?0.17 (p?=?0.8), 0.64?±?0.19 vs 0.67?±?0.21 (p?=?0.9), and 0.90?±?0.31 vs 0.88?±?0.25 (p?=?1.0) for isolates with V-MIC of ≤1, 1.5, 2, and ≥3 mg/L respectively. These results suggest that Etest might not overestimate V-MIC in comparison to M-BMD or BMD; Etest and M-BMD tests depict comparable PAP-AUC and have a higher correlation with PAP-AUC than the conventional BMD, probably because of the more detailed results. Etest may be more suitable than conventional BMD for MIC outcome assessment because of the more detailed MICs.     

Divergent rifamycin susceptibilities of Clostridium difficile strains in Canada and Italy and predictive accuracy of rifampin Etest for rifamycin resistance

We tested the activities of rifampin (RIF) and rifaximin (RFX) against 180 Clostridium difficile clinical isolates selected from Canadian and Italian culture collections. MICs were determined by CLSI agar dilution for both drugs and by Etest for RIF. Sixteen of 85 Italian isolates (18.8%) showed high-level resistance to both rifamycins (MICs, >16 μg/ml), compared to 2 of 95 (2.1%) Canadian isolates. Two new rpoB mutations were identified in rifamycin-resistant isolates. RIF susceptibility by Etest correlated completely with susceptibility to both rifamycins determined by agar dilution.     

血培养中异质性万古霉素中介金黄色葡萄球菌的分布及特征分析

目的研究血培养中异质性万古霉素中介金黄色葡萄球菌(heterogeneous vancomycin-intermediate Staphylococcus aureus,hVISA)的流行性及分子生物学特点。方法采用MHA5T(含有5μg/ml替考拉宁的MH琼脂)和菌群曲线分析法(populats profiles/area under the curve,PAP/AUC)检测hVISA,PCR方法对hVISA菌株进行SCCmec(staphylococcal cassette chromosome mec)﹑多位点序列分型(multilocus-sequence typing,MLST)﹑金黄色葡萄球菌蛋白A(Staphylococcus aureus protein A,spa)和附属基因调节子(accessory gene regulator,agr)分型检测,TritonX-100诱导自溶性检测hVISA菌株和万古霉素敏感金黄色葡萄球菌(vancomycin-sensitive Staphylococcus aureus,VSSA)菌株的自溶性差异,real-time PCR方法检测hVISA和VSSA菌株中vraR、mgrA、icaA、icaR、pbp4和agr基因的表达差异。结果血培养中甲氧西林耐药金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)的检出率为39.5%,136株MRSA中共检出hVISA 31株,hVISA的阳性率为22.8%,hVISA菌株的万古霉素最小抑菌浓度(MIC)主要集中在1.5μg/ml(占54.8%)和2μg/ml(占25.8%),而VSSA菌株万古霉素MIC主要分布在0.5μg/ml(占46.7%)和0.75μg/ml(占39.0%)。hVISA的主要流行克隆为ST239-SCCmecⅢ-t030-agrⅠ型,有22株,占71.0%,与VSSA相比,hVISA菌株自溶性有所下降(χ^2=13.583,P=0.032)。RT-PCR结果显示与VSSA菌株相比,hVISA菌株中vraR、mgrA和icaA表达水平分别升高了1.58倍、1.53倍和1.06倍(P<0.01),而icaR、agr和pbp4基因的表达水平分别下降了0.85倍、0.61倍和1.03倍(P<0.05)。结论hVISA的流行率高达22.8%,主要流行克隆为ST239-SCCmecⅢ-t030-agrⅠ型,应引起临床高度重视,注意抗生素的合理使用,加强院感防控,避免hVISA菌株流行克隆的传播以及VISA和万古霉素耐药金黄色葡萄球菌(VRSA)的产生。     

Antimicrobial susceptibility testing of Bacillus anthracis: comparison of results obtained by using the National Committee for Clinical Laboratory Standards broth microdilution reference and Etest agar gradient diffusion methods

We determined the patterns of antimicrobial susceptibility of 65 isolates of Bacillus anthracis (50 historical and 15 recent U.S. clinical isolates) to nine antimicrobial agents using the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution reference method. The results for the 50 historical B. anthracis isolates obtained by the broth microdilution method were compared to those generated by the Etest agar gradient diffusion method. One isolate of B. anthracis was beta-lactamase positive and resistant to penicillin (MIC, 128 microg/ml); a second isolate, which was beta-lactamase negative, was borderline penicillin resistant, with the penicillin MICs for the isolate varying from 0.12 to 0.25 microg/ml; and the remainder of the isolates were beta-lactamase negative and penicillin susceptible (MICs, or=16 microg/ml). All B. anthracis isolates were susceptible to chloramphenicol (MICs, 相似文献   

Nationwide Survey Shows that Methicillin-Resistant Staphylococcus aureus Strains Heterogeneously and Intermediately Resistant to Vancomycin Are Not Disseminated throughout Japanese Hospitals

A total of 6,625 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates obtained from 278 hospitals throughout Japan were obtained between November and December 1997 and were examined for their sensitivities to vancomycin using Mueller Hinton (MH), brain heart infusion (BHI), agar plates, or the broth microdilution method. A concentrated inoculum of an MRSA strain or the use of highly enriched medium, such as BHI medium, allows an individual cell to grow on agar plates containing a vancomycin concentration greater than the MIC for the parent strain. However, cells of the colonies which grew on BHI agar plates containing the higher vancomycin concentrations did not acquire a level of vancomycin resistance greater than that of the parent strain and were not subpopulations of heterogeneously vancomycin-resistant MRSA. There was no significance in the fact that these colonies grew on the higher concentration of vancomycin: none showed stable resistance to vancomycin at a concentration above the MIC for the parent strain, and no cell from these colonies showed a relationship between the MIC and the ability of these colonies to grow on higher concentrations of vancomycin. The vancomycin MIC was not above 2 microg/ml for any of the cells originating from these colonies. No Mu3-type heterogeneously resistant MRSA strains, which constitutively produce subpopulations from MRSA clinical isolates with intermediate vancomycin resistance at a high frequency, were detected. There was a unipolar distribution of the MICs ranging from 0.25 to 2 microg of vancomycin/ml among the 6,625 MRSA clinical isolates, indicating that there was no Mu50-type intermediately vancomycin-resistant MRSA (MIC, 8 microg/ml by National Committee for Clinical Laboratory Standards criteria) among the clinical isolates, and there was no evidence of dissemination of Mu3-type MRSA heteroresistant to vancomycin.