Improved detection of vanB2-containing enterococcus faecium with vancomycin susceptibility by Etest using oxgall supplementation

We have isolated a number of vanB-containing Enterococcus faecium isolates on bile esculin screening agar containing 6 mg/liter vancomycin, which on subsequent susceptibility testing using Etest have repeatedly demonstrated vancomycin MICs of 相似文献   

Colistin MIC Variability by Method for Contemporary Clinical Isolates of Multidrug-Resistant Gram-Negative Bacilli

In vitro evaluation of colistin susceptibility is fraught with complications, due in part to the inherent cationic properties of colistin. In addition, no reference method has been defined against which to compare the results of colistin susceptibility testing. This study systematically evaluated the available methods for colistin MIC testing in two phases. In phase I, colistin MICs were determined in 107 fresh clinical isolates of multidrug-resistant (MDR) Gram-negative bacilli (GNB) by broth microdilution with polysorbate 80 (BMD-T), broth macrodilution (TDS), and the Etest. In phase II, 50 of these isolates, 10 of which were colistin resistant, were tested in parallel using BMD-T, TDS, agar dilution, broth microdilution without polysorbate 80 (BMD), and the TREK Gram-negative extra MIC format (GNXF) Sensititre. The Etest was also performed on these 50 isolates using Mueller-Hinton agar (MHA) from three different manufacturers. Colistin MIC results obtained from the five methods were compared to the MIC results obtained using BMD-T, the method that enables the highest nominal concentration of colistin in the test medium. Essential agreement ranged from 34% (BMD) to 83% (TDS), whereas categorical agreement was >90% for all methods except for BMD, which was 88%. Very major errors (VMEs) (i.e., false susceptibility) for the Etest were found in 47 to 53% of the resistant isolates, depending on the manufacturer of the MHA that was used. In contrast, VMEs were found for 10% (n = 1) of the resistant isolates by BMD and 0% of the isolates by the TDS, agar dilution, and Sensititre methods. Based on these data, we urge clinical laboratories to be aware of the variable results that can occur when using different methods for colistin MIC testing and, in particular, to use caution with the Etest.     

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.     

Multicenter evaluation of the Etest and disk diffusion methods for differentiating daptomycin-susceptible from non-daptomycin-susceptible Staphylococcus aureus isolates

Daptomycin is a novel cyclic lipopeptide that is approved by the U.S. Food and Drug Administration for the treatment of complicated skin and skin structure infections associated with Staphylococcus aureus and other gram-positive pathogens and also staphylococcal bacteremia, including right-sided endocarditis. The Clinical and Laboratory Standards Institute (CLSI) established "susceptible-only" interpretive criteria for broth microdilution (BMD) and disk diffusion (DD) testing of daptomycin in 2005. However, a series of S. aureus isolates have been recovered with daptomycin MICs in the nonsusceptible range (i.e., MICs of >1 microg/ml). The objective of this study was to determine the ability of the Etest and DD methods to differentiate daptomycin-susceptible from nonsusceptible isolates of S. aureus compared to the results of the CLSI BMD reference method. There was a good correlation between Etest MIC results and the results of BMD among laboratories (r = 0.86 to 0.88), with 95.3% of the Etest MICs within a +/-1 log(2) dilution of the BMD MIC result. A total of 92 of 102 (90.2%) non-daptomycin-susceptible isolates of S. aureus identified by BMD in two participating laboratories were also classified as nonsusceptible by Etest. However, the very major and major error rates reported by one of the participating laboratories were 13.5 and 4.0%, respectively, primarily due to the absence of an intermediate category. The DD method, however, did not reliably differentiate daptomycin-susceptible from non-daptomycin-susceptible isolates. In 2005, daptomycin disks were voluntarily removed from the market by Cubist Pharmaceuticals. The disk diffusion breakpoints were subsequently removed from the CLSI M100 standard in 2006.     

Glycopeptide-Intermediate Staphylococcus aureus: Evaluation of a Novel Screening Method and Results of a Survey of Selected U.S. Hospitals

Isolates of Staphylococcus aureus with decreased susceptibilities to glycopeptide antimicrobial agents, such as vancomycin and teicoplanin, have emerged in the United States and elsewhere. Commercially prepared brain heart infusion agar (BHIA) supplemented with 6 microg of vancomycin per ml was shown in a previous study to detect glycopeptide-intermediate S. aureus (GISA) with high sensitivity and specificity; however, this medium, when prepared in-house, occasionally showed growth of vancomycin-susceptible control organisms. This limitation could significantly impact laboratories that prepare media in-house, particularly if they wished to conduct large surveillance studies for GISA. Therefore, a pilot study to detect GISA was performed with vancomycin-containing Mueller-Hinton agar (MHA) prepared in-house in place of commercially prepared BHIA. MHA was selected for this study because this medium is widely available and well standardized. The results of the pilot study showed that supplementation of MHA with 5 microg of vancomycin per ml was both a sensitive and a specific method for screening for GISA isolates. This method was used to screen for GISA among 630 clinical isolates of methicillin-resistant S. aureus collected during 1997 from 33 U.S. hospitals. Although 14 S. aureus isolates grew on the screening agar, all were vancomycin susceptible (MICs were 相似文献   

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.     

Is Vancomycin MIC “Creep” Method Dependent? Analysis of Methicillin-Resistant Staphylococcus aureus Susceptibility Trends in Blood Isolates from North East Scotland from 2006 to 2010

This study investigated "creep" in vancomycin and daptomycin MICs among methicillin-resistant Staphylococcus aureus (MRSA) isolates from blood cultures over a 5-year period in a hospital in the United Kingdom, using different susceptibility testing methods. Trends in vancomycin and daptomycin susceptibility were evaluated by using Etest performed prospectively on isolates in routine clinical practice from December 2007 to December 2010 (n = 102). Comparison was made to results from prospective testing of subcultures at the Scottish MRSA Reference Laboratory, using an automated system (Vitek 2) and retrospective testing (Etest and CLSI reference broth microdilution [BMD] method) of stored isolates from 2006 to 2010 (n = 208). Spearman's rank correlations revealed a significant increase in vancomycin MIC (P = 0.012) and a significant decrease in daptomycin MIC (P = 0.03) by year of study for Etest results from the time of isolation. However, neither trend was replicated in MICs from automated or retrospective testing. The Friedman test revealed a significant difference between vancomycin MICs obtained from the same samples by different testing methods (χ(2) [3 degrees of freedom] = 97; P < 0.001). MICs from automated testing and Etest analysis of stored isolates were significantly lower than those from Etest analysis at the time of isolation for both antibiotics (P < 0.001). Effects of storage on the MIC appeared within the first 6 months of storage. Inconsistent evidence on vancomycin MIC creep and the relevance of the MIC to clinical outcome may arise from differences in susceptibility testing methods, including storage of isolates. There is a need to standardize and streamline susceptibility testing of vancomycin against MRSA.     

Evaluation of the Etest Method for Determining Fluconazole Susceptibilities of 402 Clinical Yeast Isolates by Using Three Different Agar Media

The performance of the Etest for fluconazole susceptibility testing of 402 yeast isolates was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose (RPG), Casitone agar (CAS), and Mueller-Hinton agar (MHA) and were read after incubation for 48 h at 35°C. The yeast isolates included Candida albicans (n = 161), Candida glabrata (n = 41), Candida tropicalis (n = 35), Candida parapsilosis (n = 29), Candida krusei (n = 32), Candida lusitaniae (n = 31), Candida species (n = 19), Cryptococcus neoformans (n = 40), and miscellaneous yeast species (n = 14). The Etest results correlated well with reference MICs. Overall agreement was 94% with RPG, 97% with CAS, and 53% with MHA. When RPG was used, agreement ranged from 89% for Candida spp. to 100% for C. krusei. When CAS was utilized, agreement ranged from 93% for Cryptococcus neoformans to 100% for C. tropicalis, C. parapsilosis, C. lusitaniae, Candida spp., and miscellaneous yeast species. With MHA, agreement ranged from 17% for C. parapsilosis to 90% for C. krusei. Both RPG and CAS supported growth of all yeast species, whereas growth on MHA was comparatively weaker. Etest results were somewhat easier to read on CAS. The Etest method using either RPG or CAS, but not MHA, appears to be a viable alternative to the NCCLS reference method for determining fluconazole susceptibilities of yeasts.     

Assessment of two commercial susceptibility test methods for determination of daptomycin MICs

Daptomycin is a lipopeptide antibiotic with activity against several important gram-positive bacterial pathogens, including drug-resistant staphylococci and enterococci. Because the mechanism of action of daptomycin is calcium-dependent depolarization of the cell membrane, susceptibility testing requires medium supplemented with a physiological level of calcium. This study assessed two Food and Drug Administration-cleared commercial test devices for determination of daptomycin MICs, Etest and JustOne. A collection of 220 selected isolates, including Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, E. faecium, E. avium, E. durans, E. casseliflavus, and E. gallinarum, were tested by both methods. Included in the collection were 22 S. aureus and 14 Enterococcus sp. isolates that were recovered from patients and were nonsusceptible on the basis of the daptomycin MICs. As the reference method for comparison, all isolates were tested by the Clinical and Laboratory Standards Institute broth microdilution method incorporating cation-adjusted Mueller-Hinton broth with 50 microg/ml calcium. Daptomycin MICs agreed, within 1 twofold dilution, for 97% of the isolates by Etest and for 100% by JustOne. However, daptomycin MICs determined by Etest were 1 dilution lower than the reference MICs for 65% of the Enterococcus sp. isolates tested. This resulted in 28.5% very major (VM) errors (4/14) with enterococci (all E. faecium) but none (0/22) with staphylococci. Use of JustOne yielded MICs that were 1 dilution lower than the reference MICs for 69% of the staphylococci and 25% of the enterococci. This resulted in 13.6% VM errors (3/22) with staphylococci and 14.3% VM errors (2/14) with enterococci. The manufacturer-recommended JustOne inoculum preparation resulted in mean colony counts of only 5 x 10(4) to 1 x 10(5) CFU/ml in the wells of the strip. Increasing the inoculum to 3 x 10(5) to 4 x 10(5) CFU/ml eliminated two of five VM errors upon retesting. No major interpretive errors occurred with either device. In summary, daptomycin MICs generated by the Etest or JustOne method generally agreed within 1 dilution of the reference daptomycin MICs. However, both devices produced slightly lower MICs that resulted in some VM errors.     

Systematic Evaluation of Commercial Susceptibility Testing Methods for Determining the In Vitro Activity of Daptomycin versus Staphylococcus aureus and Enterococci

We systematically evaluated 5 methods for testing daptomycin versus 48 Enterococcus faecalis, 51 Enterococcus faecium, and 50 Staphylococcus aureus isolates using (i and ii) broth microdilution (BMD) with 50-mg/liter calcium medium supplementation (reference method) and 30-mg/liter calcium medium supplementation (BMD30 method), (iii) Etest, and (iv and v) MicroScan panel 33 using 2 methods to prepare the bacterial inoculum (MicroScan turbidity and MicroScan Prompt). Isolates were categorized as susceptible (S) or nonsusceptible (NS) based on measured MICs. Essential (±1 dilution) agreement (EA) and categorical (S/NS) agreement (CA) for each method were compared to the reference method. For E. faecium, categorical agreement was poor between the reference method and BMD30 as well as with the three commercial methods, with frequent false-NS results (30 for BMD30, 18 for Etest, 22 for MicroScan Prompt, and 25 for MicroScan turbidity). All E. faecalis isolates were judged to be S by the reference method; two of these isolates were categorized as NS using the BMD30 method, and one was categorized as NS by all three commercial methods. All S. aureus isolates were judged to be S using all five methods. MIC values determined by the comparator methods tended to be higher than those for the reference method, especially for E. faecium isolates. EAs between the reference BMD and BMD30, Etest, MicroScan Prompt, and MicroScan turbidity were 63%, 63%, 63%, and 56%, respectively, for E. faecium, 87%, 83%, 98%, and 80%, respectively, for E. faecalis, and all 100% for S. aureus.     

Surveillance and detection of erythromycin resistance in Bordetella pertussis isolates recovered from a pediatric population in the Intermountain West region of the United States.

Forty-seven Bordetella pertussis isolates recovered from January 1985 to June 1997 at Primary Children's Medical Center were tested for erythromycin resistance. Agar dilution MICs were determined on Regan-Lowe agar. Forty-six isolates were found to be erythromycin susceptible (all MICs were less than or equal to 0.12 microg/ml). One isolate was found to be erythromycin resistant (MIC, 32 microg/ml). In addition, we compared Etest MIC results and disk diffusion zone diameter measurements, performed on commercially prepared Regan-Lowe agar, to the agar dilution MIC result. Etest MIC and/or disk diffusion testing on commercial Regan-Lowe agar appears to be an adequate method for erythromycin resistance screening of B. pertussis isolates.     

Evaluation of Etest method for determining fluconazole and voriconazole MICs for 279 clinical isolates of Candida species infrequently isolated from blood

The performance of Etest in fluconazole and voriconazole testing of 279 isolates of uncommon Candida spp. was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS)-approved standard broth microdilution (BMD) method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 h at 35 degrees C. The isolates include Candida krusei, C. lusitaniae, C. guilliermondii, C. kefyr, C. rugosa, C. lipolytica, C. pelliculosa, C. dubliniensis, C. famata, C. zeylanoides, C. inconspicua, and C. norvegensis. Overall agreement between Etest and BMD MICs was 96% for fluconazole and 95% for voriconazole. Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values with both fluconazole and voriconazole. The Etest method using RPMI agar appears to be a useful method for determining fluconazole and voriconazole susceptibilities of uncommon species of Candida.     

Cation concentration variability of four distinct Mueller-Hinton agar brands influences polymyxin B susceptibility results

Polymyxins have been the only alternative therapeutic option for the treatment of serious infections caused by multidrug-resistant Acinetobacter baumannii or Pseudomonas aeruginosa isolates. For this reason, it is of crucial importance that susceptibility tests provide accurate results when testing these drug-pathogen combinations. In this study, the effect of cation concentration variability found on different commercial brands of Mueller-Hinton agar (MHA) for testing polymyxin B susceptibility was evaluated. The polymyxin B susceptibilities determined using Etest and disk diffusion were compared to those determined by the CLSI reference broth microdilution method. In general, the polymyxin B MIC values were higher when determined by Etest than when determined by broth microdilution against both A. baumannii and P. aeruginosa isolates. A high very major error rate (10%) was observed, as well as a trend toward lower MICs, compared to those determined by broth microdilution when the Merck MHA was tested by Etest. Poor essential agreement rates (10 to 70%) were observed for P. aeruginosa when all MHA brands were tested by Etest. Although an excellent categorical agreement rate (100%) was seen between the disk diffusion and broth microdilution methods for P. aeruginosa, larger zones of inhibition were shown obtained using the Merck MHA. The high cation concentration variability found for the MHA brands tested correlated to the low accuracy, and discrepancies in the polymyxin B MICs were determined by Etest method, particularly for P. aeruginosa isolates.     

Determination of vancomycin and daptomycin MICs by different testing methods for methicillin-resistant Staphylococcus aureus

Vancomycin and daptomycin MICs from 161 isolates of methicillin-resistant Staphylococcus aureus (MRSA) were compared using commercial and in-house broth microdilution, Etest, and common automated methods. Vancomycin Etest MICs were higher than those of other methods, whereas the MICs for daptomycin testing were comparable. Vancomycin MICs vary depending on the testing methodology.     

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.     

Evaluation of the Etest and disk diffusion methods for determining susceptibilities of 235 bloodstream isolates of Candida glabrata to fluconazole and voriconazole

The performances of the Etest and the disk diffusion methods for testing of the susceptibilities of 235 Candida glabrata isolates to fluconazole and voriconazole were compared with that of the National Committee for Clinical Laboratory Standards (NCCLS) approved standard broth microdilution (BMD) method. The NCCLS method used RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI 1640 agar containing 2% glucose (RPG agar) and with Mueller-Hinton agar containing 2% glucose and 0.5 microg of methylene blue per ml (MBE agar) and were read after incubation for 48 h at 35 degrees C. Disk diffusion testing was performed with MBE agar, 25-microg fluconazole disks, and 1- microg voriconazole disks and by incubation at 35 degrees C for 24 h. Overall agreements between the Etest and the BMD MICs obtained with RPG and MBE agars were 91 and 96%, respectively, for fluconazole and 93 and 95%, respectively, for voriconazole. Categorical agreements between the agar-based methods and BMD were 52.3 to 64.7% with fluconazole and 94.8 to 97.4% with voriconazole. The vast majority of the discrepancies by the disk diffusion and Etest methods with fluconazole were minor errors. The agar-based methods performed well in identifying isolates with resistance to fluconazole and decreased susceptibility to voriconazole.     

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.     

Influence of variations in test methods on susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin

The National Committee for Clinical Laboratory Standards standard broth microdilution method for testing the susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin was evaluated by altering one variable at a time. Variables that were tested included age of colony for inoculum preparation, inoculum density, test medium, incubation atmosphere, and incubation time. For the macrolide, azalide, and ketolide agents, incubation in 5 to 7% CO(2) most significantly affected the MICs, producing nearly twofold increases for clarithromycin and telithromycin and a greater than threefold increase for azithromycin. For ampicillin, a 10-fold increase in inoculum density increased the geometric mean MICs for beta-lactamase-negative strains from 1. 50 to 2.45 microg/ml. In addition, 206 H. influenzae strains were tested for their susceptibilities to the same drugs by the broth microdilution tests in two media, as well as by agar dilution tests, disk diffusion tests, and Etests, on six different agar media. The three standard methods with Haemophilus test medium (HTM) compared favorably with each other except for a high minor discrepancy rate (27%) by the disk diffusion test with ampicillin and clarithromycin. Agar dilution test MICs on the five comparative media were generally higher than those on HTM agar but were only rarely more than one twofold concentration higher. Etest MICs of azithromycin and telithromycin were more than twofold higher than agar dilution and broth microdilution MICs on HTM; ampicillin Etest MICs were nearly twofold lower. The use of media other than HTM agar appears to have a minimal effect on susceptibility test results for the ketolide, azalide, or macrolide drugs that we tested against H. influenzae.     

In vitro activity of beta-lactam antibiotics to community-associated methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (CA-MRSA)

Community-associated (CA) MRSA often display low MIC values against oxacillin. The in vitro activity of various beta-lactam antibiotics against heterogeneous CA-MRSA (n = 98) isolated in a low endemic area was determined by Etest, and Mueller–Hinton agar (MUHAP) was compared with Mueller–Hinton agar supplemented with 2% NaCl (MUHSP). In general, the CA-MRSA isolates showed higher MIC values for the various beta-lactam antibiotics on MUHSP compared with MUHAP. MIC values for oxacillin ranged from 1 to >256 mg/L on MUHSP. Cephalothin, representing the first generation of cephalosporins, showed MICs from 0.75 to 96 mg/L and the MIC₅₀ and MIC₉₀ for cefuroxime, cefotaxime and cefepime, representing the second, third and fourth generations, respectively, were rather high. However, the MIC₅₀ and MIC₉₀ for ceftobiprole (fifth generation) were 1.5 and 2 mg/L, respectively, on MUHSP. The MIC₅₀ and MIC₉₀ for imipenem were 0.75 and 2 mg/L, respectively, on MUHSP. Only 3/98 (3%) CA-MRSA isolates showed a MIC >4 mg/L. Consequently, low MIC values for imipenem, lower than those of the newly developed fifth generation cephalosporins, were found among CA-MRSA. These findings may be considered for further studies including clinical trials in order to evaluate carbapenems as a potential treatment option for infections caused by CA-MRSA.     

Area of technical uncertainty for susceptibility testing of amoxicillin/clavulanate against Escherichia coli: analysis of automated system,Etest and disk diffusion methods compared to the broth microdilution reference

ObjectivesThe European Committee on Antimicrobial Susceptibility Testing (EUCAST) recently warned about an area of technical uncertainty (ATU) of amoxicillin/clavulanate (AMX/C) disk susceptibility testing against members of the Enterobacterales. Thus, we aimed to compare the reliability of three routine methods and to evaluate the impact of the ATU.Methods286 Escherichia coli strains (including 159 AMX-resistant strains) were categorized for the two EUCAST AMX/C breakpoints by disk diffusion (Bio-Rad), the Phoenix automated system (Becton Dickinson) and the Etest (AES) compared to the broth microdilution reference method.ResultsBy microdilution, 84.2% of strains were AMX/C-susceptible using the urinary breakpoint (MIC ≤32 mg/L) and 62.2% using the systemic breakpoint (MIC ≤8 mg/L), with 63.6% of MICs between 4 and 16 mg/L. For the systemic breakpoint, category agreement (CA) and very major error (VME) were unacceptable for the Etest (71.7% and 27.3%), disk (73.1% and 23.4% at 19-mm cut-off) and to a lesser extent for the Phoenix system (83.6% and 10.5%). For disks, an unacceptable VME rate was observed for diameters up to 22 mm, probably due to overcharged disks. For the Etest, VMEs were high at 6 mg/L (46/63) and 8 mg/L (22/29). For the urinary breakpoint, CA was more acceptable for disk (88.9%) and Etest (84.3%) but was unevaluable for Phoenix.ConclusionAMX/C susceptibility testing of E. coli for systemic breakpoint was unreliable with the three routine methods, explained mainly by the high prevalence (~60%) of strains with microdilution MICs around the breakpoint (8 mg/L). Our data confirmed the EUCAST 19–20-mm ATU for disk and suggest introducing ATU for Etest MIC values of 6 and 8 mg/L.