Comparison of detection methods for heteroresistant vancomycin-intermediate Staphylococcus aureus, with the population analysis profile method as the reference method

Staphylococcus aureus clinical isolates with vancomycin MICs of 2 μg/ml have been associated with vancomycin therapeutic failure and the heteroresistant vancomycin-intermediate S. aureus (hVISA) phenotype. A population analysis profile (PAP) with an area under the curve (AUC) ratio of ≥ 0.9 for the AUC of the clinical isolate versus the AUC for hVISA strain Mu3 is most often used for determining hVISA, but it is time-consuming and labor-intensive. A collection of 140 MRSA blood isolates with vancomycin MICs of 2 μg/ml by reference broth microdilution and screened for hVISA using PAP-AUC (21/140 [15%] hVISA) were tested by additional methods to detect hVISA. The methods included (i) Etest macromethod using vancomycin and teicoplanin test strips, brain heart infusion (BHI) agar, and a 2.0 McFarland inoculum; (ii) Etest glycopeptide resistance detection (GRD) using vancomycin-teicoplanin double-sided gradient test strips on Mueller-Hinton agar (MHA) with 5% sheep blood and a 0.5 McFarland inoculum; and (iii) BHI screen agar plates containing 4 μg/ml vancomycin and 16 g/liter casein using 0.5 and 2.0 McFarland inocula. Each method was evaluated using PAP-AUC as the reference method. The sensitivity of each method for detecting hVISA was higher when the results were read at 48 h. The Etest macromethod was 57% sensitive and 96% specific, Etest GRD was 57% sensitive and 97% specific, and BHI screen agar was 90% sensitive and 95% specific with a 0.5 McFarland inoculum and 100% sensitive and 68% specific with a 2.0 McFarland inoculum. BHI screen agar with 4 μg/ml vancomycin and casein and a 0.5 McFarland inoculum had the best sensitivity and specificity combination, was easy to perform, and may be useful for clinical detection of hVISA.     

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.     

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.     

A multicenter study evaluating the current strategies for isolating Staphylococcus aureus strains with reduced susceptibility to glycopeptides

Glycopeptide-intermediate Staphylococcus aureus (GISA) and heterogeneous GISA (hGISA) strains are notoriously difficult to detect in the diagnostic laboratory. The clinical importance of GISA, and particularly hGISA, will only be obvious when a definitive detection method is available. A few novel GISA and hGISA detection methods have been proposed; however, their validity has never been tested on a significant scale and in different laboratories. This study compares three screening methods for detecting GISA and hGISA strains in 12 laboratories, using a blind panel of 48 strains with known glycopeptide susceptibilities. The three screening methods used were brain heart infusion agar with 6 mg/liter vancomycin (BHIA6V) (CDC/CLSI), Mueller-Hinton agar with 5 mg/liter teicoplanin (MHA5T) (European Antimicrobial Resistance Surveillance System [EARSS]), and the macrodilution method Etest (MET) (EARSS), with population analysis profile-area under the curve analysis as the gold standard. Sensitivity and specificity were highest for MHA5T and MET, which identified 82.5% and 85.9% of strains, respectively. BHIA6V had poor sensitivity, particularly for hGISA (11.5% of strains were detected), and gave the largest interlaboratory variation in performance. MET exhibited the least interlaboratory variation. It is essential that laboratories use appropriate methods to detect GISA/hGISA strains so that the prevalence and clinical importance of these strains can be assessed properly.     

Prevalence, molecular epidemiology, and clinical significance of heterogeneous glycopeptide-intermediate Staphylococcus aureus in liver transplant recipients

We investigated the prevalence, molecular epidemiology, and clinical significance of heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) isolates in 48 liver transplant recipients infected or colonized with methicillin-resistant S. aureus over a 5-year period. Strains were screened for hGISA on Mueller-Hinton agar containing 5 mg of teicoplanin per liter. Heterogeneous glycopeptide resistance was confirmed by the E-test method with a dense inoculum and a simplified method of population analysis. hGISA strains were found in 13 (27%) of the 48 patients studied. Eleven of the 13 strains shared a common multiresistant phenotype with homogeneous methicillin resistance and gentamicin resistance, and they were closely related according to the results of pulsed-field gel electrophoresis. Only 2 of the 13 patients infected or colonized with hGISA strains had previously received glycopeptide therapy. Most patients were successfully treated with vancomycin, but one patient who failed to respond to vancomycin subsequently died. These results suggest that the high prevalence of hGISA among our patients was due to the clonal spread of a multiresistant strain.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Incidence of glycopeptide hetero-intermediate Staphylococcus aureus strains in Maltese hospitals

The incidence of hetero-intermediate glycopeptide susceptibility among Staphylococcus aureus isolates in Malta, a country with a high incidence of methicillin resistance, was studied by screening 454 non-repetitive S. aureus isolates on teicoplanin-supplemented agar plates, followed by Etests and genotypic studies. All strains were susceptible to vancomycin, but four (0.88%) exhibited teicoplanin MICs of > 12 mg/L. High methicillin-resistant S.aureus endemicity was not an accurate predictor of the emergence of non-susceptibility to glycopeptides.     

Heterogeneity of Brain Heart Infusion agar media (BHI): effects on the determination of the vancomycin and the teicoplanin minimal inhibitory concentrations (MIC) of Staphylococcus aureus strains

The influence of BHI media commercially available on the results of glycopeptides MIC measured by E-test method was studied on 36 S. aureus isolates (21 MRSA and 15 MSSA). The MIC obtained with the vancomycin and the teicoplanin determined by the E-test method, on the ready prepared BHI plates (AES) and the plate prepared at the laboratory among the four dehydrated bases (AES, Biorad, Oxoid, and Becton Dickinson), were compared. The mean of the MIC showed variations from 3.14 (Biorad) to 5.25 mg/L (Oxoid) and from 3.33 (Biorad) to 9.75 mg/L (ready prepared AES) respectively for the vancomycin and for the teicoplanin. A variance analysis (Test de Friedman) showed a significant difference between the five media (p <0.001) with the two antibiotics. The comparison of media 2 by 2 allowed that all combinations excepted one (Biorad vs Becton with the vancomycin) were statistically different (p <0.001). The variation of the MIC observed in relation to the origin of the product of BHI media requires the inclusion of glycopeptide-intermediate S. aureus reference strains to control the prepared culture media.     

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.     

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.     

Heterogeneous glycopeptide intermediate Staphylococcus epidermidis isolated from prosthetic joint infections

Methicillin-resistant Staphylococcus epidermidis (MRSE) poses a major problem in prosthetic joint infections (PJIs). Vancomycin is often considered the drug of choice in the empirical treatment of staphylococcal PJIs. As recent decades have seen reports of heterogeneous glycopeptide intermediate S. aureus (hGISA), our aim was to examine the prevalence of heterogeneous glycopeptide intermediate S. epidermidis (hGISE) in PJIs. S. epidermidis isolates (n?=?122) from 119 patients in three Swedish counties between 1993 and 2012 were included. All were isolated from perioperative tissue samples from revision surgery in clinically verified PJIs. Antimicrobial susceptibility testing against staphylococcal antibiotics was performed. The macromethod Etest (MME) and glycopeptide resistance detection (GRD) Etest were used to detect hGISE. Standard minimal inhibitory concentration (MIC) determination revealed no vancomycin-resistant isolates, while teicoplanin resistance was detected in 14 out of 122 isolates (11.5 %). hGISE was found in 95 out of 122 isolates (77.9 %), 64 out of 67 of isolates with teicoplanin MIC >2 mg/L (95.5 %) and 31 out of 55 of isolates with teicoplanin MIC ≤2 mg/L (56.4 %). Thus, the presence of hGISE cannot be ruled out by teicoplanin MIC ≤2 mg/L alone. Multidrug resistance was detected in 86 out of 95 hGISE isolates (90.5 %) and in 16 out of 27 isolates (59.3 %), where hGISE could not be detected. In conclusion, hGISE detected by MME or GRD was common in this material. However, hGISE is difficult to detect with standard laboratory diagnostic routines. Glycopeptide treatment may not be sufficient in many of these PJIs, even if standard MIC classifies the isolated S. epidermidis as susceptible.     

Outbreak of methicillin-resistant Staphylococcus aureus with reduced susceptibility to glycopeptides in a Parisian hospital

Epidemiological relationships were investigated between 40 methicillin-resistant Staphylococcus aureus (MRSA) strains with decreased glycopeptide susceptibility isolated from November 1998 to March 1999 from 39 patients (17 infected and 22 colonized patients) in nine wards of the Broussais Hospital, Paris, France. Reduced glycopeptide susceptibility was readily detected on brain heart infusion (BHI) agar containing 6 microg of teicoplanin per ml and on gradient plates, but not by the standard disk diffusion method. The MICs of vancomycin and teicoplanin, determined on BHI agar, were 4 and 8 to 32 microg/ml, respectively (standard antibiotic dilution), and 4 to 8 and 8 to 32 microg/ml, respectively (E-test). All strains were resistant to macrolides, aminoglycosides, tetracycline, rifampin, sulfonamides, and pefloxacin, showed reduced susceptibility to fusidic acid and fosfomycin, and were susceptible to trimethoprim and chloramphenicol. Pulsed-field gel electrophoresis and lysotyping revealed that a multidrug-resistant MRSA clone with decreased susceptibility to glycopeptides has been discretely endemic since at least 1996 in our institution, where it was responsible for an outbreak in November and December 1998.     

Molecular Epidemiology of Oxacillin-resistant Staphylococcus aureus and in vitro Activity of Glycopeptides against Staphylococcus species

Oxacillin resistant Staphylococcus aureus and coagulase negative Staphylococcus species (MRSA and MRSCoN)have become major pathogens in nosocomial infections.The first MRSA isolate in the world was identified in En gland in 1961 [1]. Since that tim…