Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus

We compared the results obtained with six commercial MIC test systems (Etest, MicroScan, Phoenix, Sensititre, Vitek Legacy, and Vitek 2 systems) and three reference methods (agar dilution, disk diffusion, and vancomycin [VA] agar screen [VScr]) with the results obtained by the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method for the detection of VA-intermediate Staphylococcus aureus (VISA). A total of 129 S. aureus isolates (VA MICs by previous BMD tests, ≤1 μg/ml [n = 60 strains], 2 μg/ml [n = 24], 4 μg/ml [n = 36], or 8 μg/ml [n = 9]) were selected from the Centers for Disease Control and Prevention strain collection. The results of BMD with Difco Mueller-Hinton broth were used as the standard for data analysis. Essential agreement (percent ±1 dilution) ranged from 98 to 100% for all methods except the method with the Vitek Legacy system, for which it was 90.6%. Of the six commercial MIC systems tested, the Sensititre, Vitek Legacy, and Vitek 2 systems tended to categorize VISA strains as susceptible (i.e., they undercalled resistance); the MicroScan and Phoenix systems and Etest tended to categorize susceptible strains as VISA; and the Vitek Legacy system tended to categorize VISA strains as resistant (i.e., it overcalled resistance). Disk diffusion categorized all VISA strains as susceptible. No susceptible strains (MICs ≤ 2 μg/ml) grew on the VScr, but all strains for which the VA MICs were 8 μg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 μg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 μg/ml was difficult for most systems and methods, including the reference methods.In January 2006, the Clinical and Laboratory Standards Institute (CLSI) published new interpretive criteria for vancomycin and Staphylococcus aureus. The breakpoints were lowered from ≤4 μg/ml to ≤2 μg/ml for susceptible, 8 to 16 μg/ml to 4 to 8 μg/ml for intermediate, and ≥32 μg/ml to ≥16 μg/ml for resistant (2). The vancomycin breakpoints for coagulase-negative staphylococci were not changed. The rationale for lowering the S. aureus intermediate breakpoint to 4 μg/ml was (i) that intermediate S. aureus isolates, although they are rare, likely represented a population of organisms that demonstrate heteroresistance, and (ii) limited outcome data suggested that infections with these isolates are likely to fail vancomycin therapy (9). The results of broth microdilution performed by use of the CLSI reference method were the primary S. aureus susceptibility data evaluated before the CLSI breakpoint change was made. We undertook the study described here to determine the accuracy of commercial systems and reference methods for the detection of decreased vancomycin susceptibility among isolates of S. aureus.(This work was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 17 to 20 September 2007.)     

In Vitro Antimicrobial Susceptibility of Aerococcus urinae

Aerococcus urinae may cause urinary tract infections, bacteremia, and endocarditis. No standardized susceptibility test methods or interpretive criteria have been proposed for this organism. This study reports the MIC results for 128 A. urinae isolates tested by broth microdilution. The isolates had low MICs to amoxicillin, cefotaxime, ceftriaxone, doxycycline, linezolid, meropenem, penicillin, rifampin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin. However, 55% of the isolates had MICs to clindamycin of >0.25 μg/ml, 44% had MICs to erythromycin of >0.25 μg/ml, and 16% had MICs to levofloxacin of >2 μg/ml.     

Characterization of Staphylococci with Reduced Susceptibilities to Vancomycin and Other Glycopeptides

During the last several years a series of staphylococcal isolates that demonstrated reduced susceptibility to vancomycin or other glycopeptides have been reported. We selected 12 isolates of staphylococci for which the vancomycin MICs were ≥4 μg/ml or for which the teicoplanin MICs were ≥8 μg/ml and 24 control strains for which the vancomycin MICs were ≤2 μg/ml or for which the teicoplanin MICs were ≤4 μg/ml to determine the ability of commercial susceptibility testing procedures and vancomycin agar screening methods to detect isolates with reduced glycopeptide susceptibility. By PCR analysis, none of the isolates with decreased glycopeptide susceptibility contained known vancomycin resistance genes. Broth microdilution tests held a full 24 h were best at detecting strains with reduced glycopeptide susceptibility. Disk diffusion did not differentiate the strains inhibited by 8 μg of vancomycin per ml from more susceptible isolates. Most of the isolates with reduced glycopeptide susceptibility were recognized by MicroScan conventional panels and Etest vancomycin strips. Sensititre panels read visually were more variable, although with some of the panels MICs of 8 μg/ml were noted for these isolates. Vitek results were 4 μg/ml for all strains for which the vancomycin MICs were ≥4 μg/ml. Vancomycin MICs on Rapid MicroScan panels were not predictive, giving MICs of either ≤2 or ≥16 μg/ml for these isolates. Commercial brain heart infusion vancomycin agar screening plates containing 6 μg of vancomycin per ml consistently differentiated those strains inhibited by 8 μg/ml from more susceptible strains. Vancomycin-containing media prepared in-house showed occasional growth of susceptible strains, Staphylococcus aureus ATCC 29213, and on occasion, Enterococcus faecalis ATCC 29212. Thus, strains of staphylococci with reduced susceptibility to glycopeptides, such as vancomycin, are best detected in the laboratory by nonautomated quantitative tests incubated for a full 24 h. Furthermore, it appears that commercial vancomycin agar screening plates can be used to detect these isolates.     

Carbapenem Heteroresistance in VIM-1-Producing Klebsiella pneumoniae Isolates Belonging to the Same Clone: Consequences for Routine Susceptibility Testing

Susceptibility results with low reproducibility by the same or different methods have been observed for metallo-beta-lactamase (MBL)-producing Enterobacteriaceae. Eighteen VIM-1-producing Klebsiella pneumoniae isolates (one per patient) belonging to a single epidemic clone in our hospital (2005 to 2008) but with different susceptibilities to carbapenems were studied. Imipenem MICs ranged from 8 to >128 mg/liter by standard CLSI microdilution, from ≤1 to >8 mg/liter by the semiautomatic Wider system, and from 0.75 to >32 mg/liter by Etest. Meropenem MICs ranged from 0.5 to 128, ≤1 to >8, and 0.38 to >32 mg/liter, respectively. Ertapenem MICs by CLSI microdilution and Etest ranged from 1 to 64 and 0.75 to >32 mg/liter, respectively. The rates of essential agreement (±1 log₂ dilution) for imipenem and meropenem MICs between the Wider system and the reference microdilution method were 45% and 49%, respectively. Those between Etest and the reference microdilution method for imipenem, meropenem, and ertapenem MICs were 33%, 67%, and 84%. The rates of very major errors for the Wider system and Etest were 33% and 28% for imipenem and 25% and 75% for meropenem, respectively. Low MIC reproducibility was observed even when the same inoculum was used (differences up to 4-fold dilutions). Heteroresistance was suspected due to the presence of colonies in the Etest inhibition zone. It was confirmed by population analysis profiles of 4 isolates displaying different imipenem MICs, with the exception of an OmpK36-porin-deficient isolate that homogeneously expressed carbapenem resistance (MIC, >128 mg/liter). Low carbapenem MIC reproducibility could be due to the presence of resistant subpopulations and variable expression of the resistance mechanisms. Since carbapenem MICs are not good markers of MBL production, reliable and reproducible phenotypic methods are needed to detect the presence of this mechanism.Resistance to carbapenems due to metallo-beta-lactamases (MBLs) in Enterobacteriaceae is increasingly recognized, and different levels of resistance to carbapenems have been observed in these isolates (18). The detection of MBL-carrying isolates in clinical laboratories presents practical difficulties when routine susceptibility testing is performed (10, 28). Automated systems do not accurately detect enzyme-mediated carbapenem resistance when it is expressed at low levels, and MBL-carrying organisms can even appear to be fully susceptible to these compounds (9, 28). Additionally, reproducible results within the same method or among different methods, such as disk diffusion or Etest, are not always obtained, and discrepancies in the susceptibility to imipenem and meropenem of carbapenemase (KPC or VIM types)-producing Klebsiella pneumoniae isolates have already been reported (10, 28).In addition, some studies describing heteroresistance to carbapenems in Acinetobacter baumannii isolates demonstrate its presence by the growth of colonies in the inhibition zone of the Etest strip (12, 19). This phenomenon has also been reported for Klebsiella pneumoniae (28), Pseudomonas aeruginosa (20), and, more recently, Enterobacter aerogenes isolates (11).The objective of this work was to analyze the carbapenem susceptibility profiles of VIM-1-producing K. pneumoniae isolates by different susceptibility testing methods. These isolates belong to a single clone that was involved in an epidemic in our hospital (2005 to 2008) affecting 18 patients in different wards (25).     

Effects of Aggregate and Individual Antibiotic Exposure on Vancomycin MICs for Staphylococcus aureus Isolates Recovered from Pediatric Patients

We evaluated the evolution of vancomycin MICs for Staphylococcus aureus and their relationship with vancomycin use among hospitalized children. S. aureus isolates recovered from sterile sites were prospectively tested for vancomycin susceptibility using the Etest between 1 April 2000 and 31 March 2008. Vancomycin MICs were grouped into three categories: ≤1, 1.5, and 2 μg/ml. The association between vancomycin MICs and aggregate vancomycin use and individual patient vancomycin exposure 6 months prior to the documented infection was assessed. The geometric mean values for vancomycin MICs for S. aureus fluctuated over time without a significant trend (P = 0.146). Of the 436 patients included in the study, 363 (83%) had methicillin-susceptible S. aureus (MSSA) and 73 (17%) had methicillin-resistant S. aureus (MRSA) infections. The rate of isolates with a vancomycin MIC of 2 μg/ml increased from 4% (2 of 46) in 2000 to 2001 to 24% (11 of 46) in 2007 to 2008, despite a decrease in vancomycin use (r = −0.11; P = 0.825). The percentage of isolates with a vancomycin MIC of 2 μg/ml was higher for MRSA (15%; 11 of 73) than for MSSA strains (5.2%; 19 of 363) (χ² = 9.2; P = 0.01). Individual patient vancomycin exposure was not associated with a higher vancomycin MIC. In the unadjusted model, in which we compared patients with S. aureus infections with MICs of ≤1 μg/ml, the odds ratios of exposure rates for patients with isolates with MICs of 1.5 μg/ml and 2 μg/ml were 1.02 (P = 0.929) and 1.13 (P = 0.767), respectively. In our experience, the geometric means of vancomycin MICs from S. aureus isolates recovered from hospitalized children oscillated over time and were not associated with previous individual patient vancomycin exposure or aggregate vancomycin use.     

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.     

Comparison of Meropenem MICs and Susceptibilities for Carbapenemase-Producing Klebsiella pneumoniae Isolates by Various Testing Methods

We describe the levels of agreement between broth microdilution, Etest, Vitek 2, Sensititre, and MicroScan methods to accurately define the meropenem MIC and categorical interpretation of susceptibility against carbapenemase-producing Klebsiella pneumoniae (KPC). A total of 46 clinical K. pneumoniae isolates with KPC genotypes, all modified Hodge test and bla_KPC positive, collected from two hospitals in NY were included. Results obtained by each method were compared with those from broth microdilution (the reference method), and agreement was assessed based on MICs and Clinical Laboratory Standards Institute (CLSI) interpretative criteria using 2010 susceptibility breakpoints. Based on broth microdilution, 0%, 2.2%, and 97.8% of the KPC isolates were classified as susceptible, intermediate, and resistant to meropenem, respectively. Results from MicroScan demonstrated the most agreement with those from broth microdilution, with 95.6% agreement based on the MIC and 2.2% classified as minor errors, and no major or very major errors. Etest demonstrated 82.6% agreement with broth microdilution MICs, a very major error rate of 2.2%, and a minor error rate of 2.2%. Vitek 2 MIC agreement was 30.4%, with a 23.9% very major error rate and a 39.1% minor error rate. Sensititre demonstrated MIC agreement for 26.1% of isolates, with a 3% very major error rate and a 26.1% minor error rate. Application of FDA breakpoints had little effect on minor error rates but increased very major error rates to 58.7% for Vitek 2 and Sensititre. Meropenem MIC results and categorical interpretations for carbapenemase-producing K. pneumoniae differ by methodology. Confirmation of testing results is encouraged when an accurate MIC is required for antibiotic dosing optimization.Carbapenems are considered first-line therapy for infection with multidrug-resistant Enterobacteriaceae (14). However, the increasing emergence of serine-based carbapenemase-producing Klebsiella pneumoniae (KPC) worldwide is of growing concern. This problem is particularly worrisome due to the fact that this K. pneumoniae is one of the leading causes of hospital-acquired infections in severely ill patients, and few antibiotics retain microbiological activity against isolates that produce bla_KPC (15). Additionally, studies have demonstrated increased mortality rates in patients infected with carbapenem-resistant Enterobacteriaceae compared with those infected with susceptible strains (1, 12, 13).Detection of KPC based strictly on susceptibility testing is challenging due mostly to the heterogeneous expression of β-lactam resistance (15). Many automated systems report KPC as susceptible to meropenem, and while some isolates truly are, the MICs for most KPC are above the Food and Drug Administration (FDA) susceptibility breakpoint (4 μg/ml) (11). To address testing and reporting issues, the Clinical Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing changed the susceptibility breakpoint for meropenem, imipenem, and doripenem to ≤1 μg/ml against Enterobacteriaceae in January 2010 (8). At the time of writing, the FDA breakpoint remained at ≤4 μg/ml for meropenem.Given the lack of options for antibiotics that retain susceptibility against pathogens that produce KPC, selection of a dosing regimen that could potentially treat infections caused by these organisms depends on the ability to accurately determine the antibiotic MIC. With respect to KPC, the accurate determination of the meropenem MIC may permit the application of pharmacodynamic principles to dosing regimen optimization by administering higher doses and using prolonged or continuous infusions, as has been accomplished against other resilient bacteria (3, 10, 14).Herein, we describe the levels of agreement between commonly used testing methods (broth microdilution [BMD], Etest, Vitek 2, Sensititre, and MicroScan) in their abilities to accurately determine the meropenem MIC and further classify categorical susceptibilities of carbapenemase-producing K. pneumoniae isolates based on the 2010 CLSI breakpoints compared with FDA breakpoints.     

Multicenter Study To Determine Disk Diffusion and Broth Microdilution Criteria for Prediction of High- and Low-Level Mupirocin Resistance in Staphylococcus aureus

Mupirocin susceptibility testing of Staphylococcus aureus has become more important as mupirocin is used more widely to suppress or eliminate S. aureus colonization and prevent subsequent health care- and community-associated infections. The present multicenter study evaluated two susceptibility testing screening methods to detect mupirocin high-level resistance (HLR), broth microdilution (BMD) MICs of ≥512 μg/ml, and a 6-mm zone diameter for a disk diffusion (DD) test with a 200-μg disk. Initial testing indicated that with Clinical and Laboratory Standards Institute methods for BMD and DD testing, the optimal conditions for the detection of mupirocin HLR were 24 h of incubation and reading of the DD zone diameters with transmitted light. Using the presence or absence of mupA as the “gold standard” for HLR, the sensitivity and specificity of a single-well 256 μg/ml BMD test were 97 and 99%, respectively, and those for the 200-μg disk test were 98 and 99%, respectively. Testing with two disks, 200 μg and 5 μg, was evaluated for its ability to distinguish HLR isolates (MICs ≥ 512 μg/ml), low-level-resistant (LLR) isolates (MICs = 8 to 256 μg/ml), and susceptible isolates (MICs ≤ 4 μg/ml). Using no zone with both disks as an indication of HLR and no zone with the 5-μg disk plus any zone with the 200-μg disk as LLR, only 3 of the 340 isolates were misclassified, with 3 susceptible isolates being classified as LLR. Use of standardized MIC or disk tests could enable the detection of emerging high- and low-level mupirocin resistance in S. aureus.Mupirocin is a topical antibacterial agent that is used both for the treatment of skin infections and for the suppression or elimination of nasal carriage of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) (8). The recommendations of the Healthcare Infection Control Practices Advisory Committee suggest the use of a tiered approach to the prevention and control of infections with multidrug-resistant organisms, including MRSA, in acute-care settings (20). In their recommendations, decolonization is presented as one intervention that may be considered when intensified MRSA control measures are needed; if decolonization is used, susceptibility testing and monitoring for the emergence of resistance to the decolonization agent are recommended in one study (21).There are two levels of resistance to mupirocin: low-level resistance (LLR), for which the MICs are 8 to 256 μg/ml, and high-level resistance (HLR), for which the MICs are ≥512 μg/ml (11). The mupirocin MICs of strains susceptible to mupirocin are MICs ≤4 μg/ml. HLR is associated with the presence of the plasmid-mediated mupA gene, which encodes a mupirocin-resistant isoleucyl-tRNA synthetase, although S. aureus strains with HLR that lack mupA have occurred (this study) and can also be created in the laboratory (23). LLR results from mutation of the native, chromosomal isoleucyl-tRNA synthetase ileS gene (1). Studies suggest that S. aureus strains with HLR to mupirocin cannot be successfully eliminated with mupirocin and that the occurrence of HLR is increasing (22). It has been suggested that S. aureus strains demonstrating LLR could be eliminated by topical application of mupirocin because of the high concentrations achieved locally, but this has not been demonstrated definitively (11, 21).Until recently, methods for testing topical agents have not been included in susceptibility testing documents published by the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS), although guidelines for testing by various methods have been suggested by others (9, 10, 12, 13, 16, 17). The British Society for Antimicrobial Chemotherapy has formal recommendations for the testing of mupirocin (www.bsac.org.uk) that include testing of a 5-μg and a 20-μg mupirocin disk. Their recommendations require MIC testing to determine the level of resistance if a 5-μg disk is used alone. An initial investigation at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, showed that a 200-μg mupirocin disk was able to differentiate isolates with LLR from those with HLR (15). We undertook the study described here to determine the MIC and disk diffusion criteria for the detection of S. aureus strains with high- or low-level mupirocin resistance and to validate quality control tests. Using data from this study, a screen test for prediction of high-level mupirocin resistance is now included in CLSI susceptibility testing documents (3, 6, 7).     

Vancomycin MIC for Methicillin-Resistant Coagulase-Negative Staphylococcus Isolates: Evaluation of the Broth Microdilution and Etest Methods

Vancomycin MIC results were determined by the broth microdilution (BMD) method and by Etest using 130 methicillin-resistant coagulase-negative staphylococcus bloodstream isolates obtained from a tertiary hospital. The majority (98.5%) of MIC results determined by BMD were ≤1 μg/ml, in contrast to MIC results determined by Etest (72.3% were ≥1.5 μg/ml). The MICs obtained by Etest were, in general, 1- to 2-fold higher than the MICs obtained by BMD.Coagulase-negative staphylococci (CoNS) have emerged as important nosocomial pathogens during the last decade, particularly in nosocomial bloodstream infections (4). Resistance to methicillin in CoNS is very common among isolates recovered from hospitalized individuals (4, 19). For this reason, vancomycin is usually the drug of choice for treatment of infections by methicillin-resistant CoNS (MRCoNS) (17).A reduction in the efficacy of vancomycin has been described in studies of methicillin-resistant Staphylococcus aureus (MRSA) infections treated with this antibiotic, and it has been suggested that slight increases in vancomycin MICs of between 1 and 2 μg/ml, which are within the susceptible range, may be related to suboptimal clinical outcomes (12, 16). Therefore, the determination of the MIC of vancomycin has been recommended for these pathogens (3). Some of these studies have used the broth microdilution (BMD) method for determining vancomycin MICs, while others have used the commercial Etest technique (AB Biodisk, Solna, Sweden). It has been reported that the Etest provides higher MICs than those obtained with BMD in S. aureus, mainly MRSA (14, 15). Nevertheless, there is no study comparing both methodologies for determination of the MIC of vancomycin in CoNS.Considering the increasing incidence of MRCoNS, the need for MIC determination of vancomycin, and the absence of studies assessing the performance of the Etest with these organisms, we aimed to compare the Etest and BMD methods for determination of the MICs of vancomycin in MRCoNS isolates.A total of 130 clinical isolates of CoNS recovered from blood samples obtained from patients who were hospitalized from May 2004 to August 2005 at the Hospital de Clínicas de Porto Alegre were analyzed. Only one isolate per patient was included. Blood cultures were performed using BacT/Alert (bioMérieux, Marcy l''Etoile, France). The colony morphology, Gram stain reaction, catalase testing, and absence of the coagulase enzyme were used to identify CoNS. Isolates were identified as Staphylococcus epidermidis by PCR using primers for tuf (10). The isolates that were not identified as S. epidermidis by PCR were identified using the API ID 32 Staph (bioMérieux, Marcy l''Etoile, France) semiautomated system, according to the instructions of the manufacturer. Results yielding a quality of identification of 85% or higher were accepted. The presence of the mec A gene was assessed by PCR using specific primers (13). The MICs of vancomycin were determined in duplicate by reference BMD, as recommended by CLSI, using in-house-prepared panels. The following dilutions of vancomycin were tested: 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/ml. The standard Etest procedure was performed using Mueller-Hinton agar (Becton Dickinson, Sparks, MD), with an inoculum density equivalent to a 0.5 McFarland standard. Vancomycin Etest strips were placed onto the agar with sterile forceps. The cultures were incubated for 24 h at 35°C. S. aureus ATCC 29213 was used for quality control (3).The Wilcoxon test was used to compare the MICs obtained by Etest and BMD. Two sets of comparisons were done, one with the exact MIC values determined by Etest and the other with the MICs determined by Etest rounded up to the next dilution.All 130 isolates of CoNS proved to be mec A positive and were identified as follows: 87 (66.9%) were S. epidermidis isolates, 13 (10.0%) were S. haemolyticus isolates, 12 (9.2%) were S. hominis isolates, and 11 (8.5%) were S. capitis isolates. Seven isolates (5.4%) were not identified to the species level. The MICs of vancomycin ranged from 0.25 to 2 μg/ml by BMD and from 0.38 to 3 μg/ml by Etest. No discrepancies were observed in duplicates performed by BMD. The MIC₅₀s and MIC₉₀s for vancomycin were both 1 μg/ml by BMD and 1.5 and 2 μg/ml, respectively, by Etest (P < 0.001 for both comparisons). Most MICs determined by Etest were ≥1.5 μg/ml (94 isolates, 72.3%), while most MICs determined by BMD were ≤1 μg/ml (128 isolates, 98.5%). A total of 113 (86.9%) and 5 (3.8%) isolates presented MICs determined by Etest that were 1- and 2-fold dilutions higher than those determined by BMD, respectively. Only 10 isolates (7.7%) presented the same MIC using the two methods (Fig. ​(Fig.1).1). When the Etest MICs of 0.38, 0.75, 1.5, and 3 μg/ml were converted to 0.5, 1, 2, and 4 μg/ml, respectively, there was an even higher discrepancy between the two methods. In this case, almost all MRCoNS had MICs of ≥1 μg/ml (124 isolates, 95.4%) (Fig. ​(Fig.2).2). MICs determined by Etest were higher than those determined by BMD for all MRCoNS species, when analyzed separately (Table ​(Table1).1). No isolate had a MIC higher than 4 μg/ml, regardless of the method used.Open in a separate windowFIG. 1.Scattergram of correlation between vancomycin MICs obtained by the broth microdilution and Etest methods.Open in a separate windowFIG. 2.Scattergram of correlation between vancomycin MICs obtained by the broth microdilution method and the Etest method after rounding up.

TABLE 1.

Distribution of vancomycin MICs according to CoNS species

Major Variation in MICs of Tigecycline in Gram-Negative Bacilli as a Function of Testing Method

Tigecycline is one of the few remaining therapeutic options for extensively drug-resistant (XDR) Gram-negative bacilli (GNB). MICs of tigecycline to Acinetobacter baumannii have been reported to be elevated when determined by the Etest compared to determinations by the broth microdilution (BMD) method. The study aim was to compare the susceptibility of GNB to tigecycline by four different testing methods. GNB were collected from six health care systems (25 hospitals) in southeast Michigan from January 2010 to September 2011. Tigecycline MICs among A. baumannii, carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, and susceptible Enterobacteriaceae isolates were determined by Etest, BMD, Vitek-2, and MicroScan. Nonsusceptibility was categorized as a tigecycline MIC of ≥4 μg/ml for both A. baumannii and Enterobacteriaceae. The study included 4,427 isolates: 2,065 ESBL-producing Enterobacteriaceae, 1,105 A. baumannii, 888 susceptible Enterobacteriaceae, and 369 CRE isolates. Tigecycline nonsusceptibility among A. baumannii isolates was significantly more common as determined by Etest compared to that determined by BMD (odds ratio [OR], 10.3; P < 0.001), MicroScan (OR, 12.4; P < 0.001), or Vitek-2 (OR, 9.4; P < 0.001). These differences were not evident with the other pathogens. Tigecycline MICs varied greatly according to the in vitro testing methods among A. baumannii isolates. Etest should probably not be used by laboratories for tigecycline MIC testing of A. baumannii isolates, since MICs are significantly elevated with Etest compared to those determined by the three other methods.     

Performance of Fusidic Acid (CEM-102) Susceptibility Testing Reagents: Broth Microdilution,Disk Diffusion,and Etest Methods as Applied to Staphylococcus aureus

Fusidic acid (CEM-102) is an established antistaphylococcal agent that has been used in clinical practice for more than 4 decades. The activity of fusidic acid against 778 isolates of Staphylococcus aureus collected from U.S. (53.8% were methicillin-resistant S. aureus [MRSA]) and Canadian (46.5% were MRSA) medical centers was assessed to determine the intermethod accuracy of the Clinical and Laboratory Standards Institute (CLSI) and Etest methods. Broth microdilution MIC results were compared by scattergram analysis to zone diameters around commercially available 5- and 10-μg disks. Acceptable correlation (r = 0.74 to 0.76) was observed for the two disk concentrations, and applying breakpoints of ≤1 μg/ml (≥22 mm) for susceptibility (S) and ≥4 μg/ml (≤19 mm) for resistance (R) provided 99.9% absolute intermethod categorical agreement. Reference CLSI MIC versus Etest MIC results (r = 0.77; 728 strains) showed 55.4% identical results and agreement of 99.7% ± one log₂ dilution. The diagnostic susceptibility testing reagents (including Etest) for fusidic acid (CEM-102) performed at an excellent level of intermethod agreement for the proposed breakpoint criteria.Staphylococcus aureus is a leading cause of skin and skin structure infections (SSSI), hospital- and community-acquired bacterial pneumonia, and nosocomial bloodstream infections (BSI) (3, 9, 14). Resistance to methicillin (oxacillin) among S. aureus (MRSA) isolates ranges from 30% to more than 60% and is present worldwide (9, 10, 17). Furthermore, hospital-associated strains of MRSA (HA-MRSA) are often multidrug-resistant (MDR), exhibiting resistance to all β-lactam agents, penems, carbapenems, aminoglycosides, macrolides, tetracyclines, trimethoprim, and fluoroquinolones (3). The emergence of MRSA strains with reduced vancomycin susceptibility further reduces treatment options (4). Although community-associated MRSA (CA-MRSA) presently remains susceptible to clindamycin, most tetracyclines, and trimethoprim-sulfamethoxazole, its emergence as a cause of infection in health care facilities is a growing source of concern (19). These resistance issues associated with such a virulent and prevalent pathogen have spurred the development of new antistaphylococcal agents, as well as reconsideration of the role of older agents with demonstrated antistaphylococcal activity (4, 10). Regarding MDR-MRSA, it has been suggested that the use of an agent such as fusidic acid may prove useful in treating these difficult infections and could help to delay the inevitable development of resistance to newer agents, such as linezolid and daptomycin (15). A promising feature of fusidic acid is the lack of cross-resistance with other antimicrobial classes as a result of the unique mode of action that inhibits bacterial protein synthesis at the translational stage (2, 12, 15).Although fusidic acid has been used throughout much of the world for more than 40 years (1, 15), U.S. Food and Drug Administration (FDA) licensure has never been obtained, and the drug is not currently available in the United States. As a result, resistance to fusidic acid is extremely uncommon among U.S. strains of S. aureus, including methicillin-susceptible, MRSA, and vancomycin-intermediate and -resistant strains, as well as those strains with decreased susceptibility to linezolid and daptomycin. Despite the fact that in vitro susceptibility testing of fusidic acid has been performed for many years, fusidic acid is not presently included in the tables of the Clinical and Laboratory Standards Institute (CLSI), and interpretive breakpoints for MIC and disk diffusion testing of fusidic acid against S. aureus are not available (11, 20).Previous authors have demonstrated that susceptibility of staphylococci to fusidic acid may be indicated at MICs of ≤0.25, ≤0.5, or ≤1 μg/ml and resistance at MICs of ≥2 μg/ml (11, 15, 20). Recently, Skov et al. (20) utilized CLSI reference broth microdilution and disk diffusion methods to propose staphylococcal susceptibility interpretive criteria of ≤0.5 μg/ml (≥21 mm) and resistance criteria of ≥2 μg/ml (≤18 mm). The EUCAST (13) organization has selected ≤1 μg/ml as susceptibility breakpoint for MIC testing. In the present study, we provide additional fusidic acid MIC and disk diffusion data to support the findings of Skov et al. (20) or EUCAST (13) and, in addition, evaluate the utility of the Etest (AB Biodisk, Solna, Sweden) methodology for testing this agent against a large North American collection of S. aureus strains (13, 20).A total of 778 nonduplicate clinical isolates of S. aureus (52% MRSA) from patients with SSSI or BSI were obtained from more than 30 medical centers in the United States and Canada between 1997 and 2006. A subset of CA-MRSA isolates (50 strains from the United States) were tested as a resistance subset only. All isolates (778 overall) were forwarded to the monitoring laboratory (JMI Laboratories, North Liberty, IA) for subsequent identification confirmation and reference antimicrobial susceptibility testing. Identification was performed using an automated system (Vitek; bioMerieux, Hazelwood, MO) or conventional manual methods, as required.All strains were tested by the CLSI broth microdilution method using prepared and validated frozen-form panels in cation-adjusted Mueller-Hinton broth (6). Fusidic acid (also known as CEM-102; Cempra) reference powder was obtained from Cempra Pharmaceuticals (Chapel Hill, North Carolina). Disk diffusion testing (all strains) was performed according to the CLSI method (5) using Mueller-Hinton agar and two disk concentrations (5 [728 strains] and 10 μg [778 strains]). The zone diameters were measured to the nearest mm using a caliper (Fig. ​(Fig.1a).1a). Etest was performed as recommended by the manufacturer (AB Biodisk) using Mueller-Hinton agar, with inoculums of 1 × 10⁸ to 2 × 10⁸ CFU/ml (5) and incubation at 37°C in air for 18 to 24 h. The MIC was read at 80% inhibition relative to the growth of the control. The organism collection (excluding the CA-MRSA subset) (728 strains) was used to directly compare the two disk tests and the reference versus Etest MIC results (Fig. ​(Fig.1,1, ​,2,2, and ​and3).3). All fusidic acid-nonsusceptible strains (14 total) were found with each test method.Open in a separate windowFIG. 1.(a) Scattergram comparing fusidic acid (CEM-102) broth microdilution MIC results with zone diameters obtained with a 10-μg fusidic acid disk for 778 isolates of S. aureus. The solid lines indicate the interpretive breakpoints proposed by Skov et al. (20). The broken lines indicate alternative MIC (≤1 μg/ml) and disk diffusion (≥22 mm) interpretive criteria consistent with EUCAST guidelines (13). Values show the number of isolates with each result. (b) Scattergram comparing fusidic acid (CEM-102) broth microdilution MIC results with zone diameters obtained with a 5-μg fusidic acid disk for 728 isolates of S. aureus. The solid lines indicate the interpretive breakpoints proposed by Skov et al. (20), and broken lines indicate a proposed higher MIC breakpoint of ≤1 μg/ml. Values show the number of isolates with each result.Open in a separate windowFIG. 2.Scattergram showing the excellent correlation obtained with a 5-μg and 10-μg fusidic acid disk diffusion tests (728 strains). Values show the number of isolates with each result.Open in a separate windowFIG. 3.Comparison of fusidic acid broth microdilution and Etest MIC results for 728 isolates of S. aureus (r = 0.77). Values show the number of isolates with each result. Solid lines represent the ± 1 log₂ dilution values for equivalent or identical MICs.Quality control (QC) was performed concurrently with all testing determinations, using S. aureus ATCC 29213 (MIC) or ATCC 25923 (disks) and S. pneumoniae ATCC 49619. The proposed QC ranges for MIC and disk diffusion (10 μg) tests for S. aureus ATCC 29213 and ATCC 25923 were 0.06 to 0.25 μg/ml and 24 to 32 mm, respectively. The ranges for S. pneumoniae ATCC 49619 were 4 to 32 μg/ml and 8 to 16 mm, respectively (16). Among 61 replicates, all QC values were within control ranges (8, 16).Broth microdilution test results were compared to zone diameters of inhibition around 5- and 10-μg fusidic acid disks by scattergram analysis and regression line equations. Interpretive zone size criteria were established using the error rate-bounded method of Metzler and DeHaan (18) as described by CLSI document M23-A3 (7). Correlation between the MIC methods (broth microdilution and Etest) was performed by scattergram and regression analysis. The essential agreement between the two methods was calculated, as well as the percentage of results within plus-or-minus one log₂ dilution step, optimized to 95% (7).Among strains of S. aureus tested in this study, 14 were resistant to fusidic acid as defined by a breakpoint of ≥2 μg/ml (Fig. ​(Fig.1a).1a). Excellent correlation (r = 0.74) was noted between broth microdilution MICs and zone diameters obtained with the 10-μg disk test (Fig. ​(Fig.1a).1a). Using a susceptible MIC breakpoint of ≤0.5 or ≤1 μg/ml, correlate zone diameter breakpoints could be selected to accurately distinguish susceptible wild-type strains from less-susceptible isolates. Examples of breakpoints for the 10-μg fusidic acid disk and the CLSI method of ≥21 mm for susceptibility and ≤18 mm for resistance (see solid vertical and horizontal lines in Fig. ​Fig.1a)1a) were published by Skov et al. (20). Applying these to the results in Fig. ​Fig.1a,1a, the absolute intermethod categorical agreement was 99.9%, with only one minor error. A slight adjustment to ≥22 mm (S) and ≤19 mm (R) produced complete (100.0%) intermethod accord. Using a higher susceptibility MIC of ≤1 μg/ml (13) and the same correlate zone diameters also yielded a very high level of intermethod agreement (99.7%), but the modification of the zone diameter criteria to ≥22 mm (S) and ≤19 mm (R) returned the agreement to 99.9%. These results are in close agreement with those criteria suggested by Toma and Barriault (21), also using the CLSI method, a 10-μg disk, and Mueller-Hinton medium.Figures ​Figures1b1b and ​and22 demonstrate the excellent agreement for the 5-μg fusidic acid (CEM-102) disk results and reference broth microdilution tests (Fig. ​(Fig.1b)1b) and the outstanding correlation between the 5- and 10-μg disk zone diameters (r = 0.97) (Fig. ​(Fig.2).2). Applying the breakpoint criteria suggested by Skov et al. (20) resulted in perfect (100.0%) intermethod agreement between the CLSI broth microdilution and the 5-μg disk results. Although the 5-μg disk for fusidic acid could certainly be standardized for use, the 10-μg disk is more widely available or preferred, with at least three manufacturers internationally.Figure ​Figure33 shows the correlation of the fusidic acid (CEM-102) reference broth microdilution results with the MICs produced by Etest. The essential agreement was 99.7% ± one log₂ dilution step with 55.4% identical MIC results. A slight trend toward a lower MIC (31.2% of results were one log₂ dilution lower) was noted for the Etest. The Etest proved to be an acceptable alternative method to determine fusidic acid MIC results for S. aureus, with an intermethod agreement comparable to that for the CLSI disk diffusion method (e.g., >99%).In summary, the in vitro diagnostic tests for fusidic acid (CEM-102) and S. aureus performed at an acceptable level of intermethod agreement. The CLSI M07-A8 (6) broth microdilution method performed well, as did the reference agar disk diffusion method of CLSI M02-A10 (5), each showing excellent intermethod categorical accuracy for either 5- or 10-μg disks. For the 10-μg disk, we propose zone diameter breakpoints of ≥22 mm (≤1 μg/ml), 20 to 21 mm (2 μg/ml), and ≤19 mm (≥4 μg/ml) for the susceptible, intermediate, and resistant category, respectively, which would provide harmonization with current EUCAST criteria (13) (Table ​(Table1).1). Alternatively, the interpretive criteria of Skov et al. (20) would provide a comparable level of accurate intermethod performance. The Etest could be applied as an alternative MIC method with near-complete concordance by quantitative measure (MIC) and by category analyses. The potency of fusidic acid can be assessed with confidence by the standardized CLSI MIC and disk diffusion test methods and by the Etest during clinical trials in the United States and elsewhere. Such testing will be important in monitoring emerging resistant subpopulations, such as those that have appeared in several nations over the last few decades (15).

TABLE 1.

Proposed interpretive breakpoints for fusidic acid against S. aureus

Vancomycin MIC plus Heteroresistance and Outcome of Methicillin-Resistant Staphylococcus aureus Bacteremia: Trends over 11 Years

Vancomycin MICs (V-MIC) and the frequency of heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) isolates are increasing among methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates, but their relevance remains uncertain. We compared the V-MIC (Etest) and the frequency of hVISA (Etest macromethod) for all MRSA blood isolates saved over an 11-year span and correlated the results with the clinical outcome. We tested 489 isolates: 61, 55, 187, and 186 isolates recovered in 1996-1997, 2000, 2002-2003, and 2005-2006, respectively. The V-MICs were ≤1, 1.5, 2, and 3 μg/ml for 74 (15.1%), 355 (72.6%), 50 (10.2%), and 10 (2.1%) isolates, respectively. We detected hVISA in 0/74, 48/355 (13.5%), 15/50 (30.0%), and 8/10 (80.0%) isolates with V-MICs of ≤1, 1.5, 2, and 3 μg/ml, respectively (P < 0.001). The V-MIC distribution and the hVISA frequency were stable over the 11-year period. Most patients (89.0%) received vancomycin. The mortality rate (evaluated with 285 patients for whose isolates the trough V-MIC was ≥10 μg/ml) was comparable for patients whose isolates had V-MICs of ≤1 and 1.5 μg/ml (19.4% and 27.0%, respectively; P = 0.2) but higher for patients whose isolates had V-MICs of ≥2 μg/ml (47.6%; P = 0.03). However, the impact of V-MIC and hVISA status on mortality or persistent (≥7 days) bacteremia was not substantiated by multivariate analysis. Staphylococcal chromosome cassette mec (SCCmec) typing of 261 isolates (including all hVISA isolates) revealed that 93.0% of the hVISA isolates were SCCmec type II. These findings demonstrate that the V-MIC distribution and hVISA frequencies were stable over an 11-year span. A V-MIC of ≥2 μg/ml was associated with a higher rate of mortality by univariate analysis, but the relevance of the V-MIC and the presence of hVISA remain uncertain. A multicenter prospective randomized study by the use of standardized methods is needed to evaluate the relevance of hVISA and determine the optimal treatment of patients whose isolates have V-MICs of ≥2.0 μg/ml.The treatment of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) bacteremia with vancomycin is often associated with a poor clinical outcome (6, 15, 28, 40). Treatment failure was reported among patients infected with isolates whose vancomycin MICs were ≥4 μg/ml (6, 9, 12, 25, 28, 42). This prompted the Clinical and Laboratory Standards Institute to lower the cutoffs for S. aureus susceptibility to ≤2 μg/ml for susceptible, 4 to 8 μg/ml for intermediate (vancomycin-intermediate S. aureus [VISA]), and 16 μg/ml for resistance (39). Within the susceptibility range, the MIC is reported to increase over time (14, 25, 35-40). This is often referred to as MIC creep (38). Additionally, isolates with heteroresistance (heteroresistant vancomycin-intermediate S. aureus [hVISA]) are emerging, and this has uncertain implications for laboratory detection and clinical management (2, 5, 15, 24, 40-42). The first isolate of hVISA to be identified was reported from Japan in 1997 (11). Since then, it has been reported worldwide at frequencies of 0 to 50% (2, 4, 6, 9, 12, 19, 20, 21, 24, 26, 27, 31, 40, 42, 44). This disparity in frequency is probably a result of its variable incidence and the different testing methodologies used. Likewise, the frequency of isolates with MICs of 1.5 to <4 μg/ml varies according to the testing method used (3, 32). The relevance of an MIC on the higher side of the susceptibility range and the presence of hVISA isolates remains uncertain (8, 19, 21). Therapeutic failure was reported in patients infected with isolates with vancomycin MICs of 2 μg/ml (6, 12, 28) and 1.5 or 1 μg/ml (25, 34, 37). Most clinical microbiology laboratories use automated testing methods that are known to underestimate the vancomycin MIC (13, 24). Additionally, most previous studies addressing the relevance of such isolates were observational and usually involved only a few patients and poorly selected controls (1, 4, 7, 9, 12, 14, 25, 35, 38, 42). At our institution, we found the frequency of hVISA isolates among isolates from patients with persistent MRSA bacteremia to be 14%; however, heteroresistance did not correlate with the mortality rate (19). In the current study, we tested all blood MRSA isolates collected over 11 years to determine whether the vancomycin MIC and the prevalence of hVISA have changed over time and to evaluate the effects of increasing vancomycin MICs and the hVISA frequency on patient outcomes.     

BD Phoenix and Vitek 2 Detection of mecA-Mediated Resistance in Staphylococcus aureus with Cefoxitin

The BD Phoenix (BD Diagnostics, Sparks, MD) and Vitek 2 (bioMérieux, Durham, NC) automated susceptibility testing systems have implemented the use of cefoxitin to enhance the detection of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). To assess the impact of this change, 620 clinically significant S. aureus isolates were tested in parallel on Phoenix PMIC/ID-102 panels and Vitek 2 AST-GP66 cards. The results for oxacillin and cefoxitin generated by the automated systems were compared to those generated by two reference methods: mecA gene detection and MICs of oxacillin previously determined by broth microdilution according to CLSI guidelines. Testing of isolates with discordant results was repeated to attain a majority or consensus final result. There was 100% final agreement between the results of the two reference methods. For the 448 MRSA and 172 methicillin-susceptible S. aureus isolates tested, the rates of categorical agreement of the results obtained with the automated systems with those obtained by the reference methods were 99.8% for the Phoenix panels and 99.7% for the Vitek 2 cards. A single very major error occurred on each instrument (0.2%) with different MRSA isolates. The only major error was attributed to the Vitek 2 system overcalling oxacillin resistance. In 16 instances (9 on the Phoenix system, 7 on the Vitek 2 system), an oxacillin MIC in the susceptible range was correctly changed to resistant by the expert system on the basis of the cefoxitin result. The inclusion of cefoxitin in the Phoenix and Vitek 2 panels has optimized the detection of MRSA by both systems.The accurate detection of mecA-mediated ß-lactam resistance in Staphylococcus aureus is essential for the treatment of overt infections and the implementation of infection control practices. Although FDA-cleared PCR assays for the rapid detection of methicillin (meticillin)-resistant S. aureus (MRSA) are available for use for surveillance and testing of clinical specimens, isolates causing infections continue to require susceptibility testing to guide therapy.The phenotypic detection of mecA-mediated resistance has presented ongoing challenges due to variable gene expression that is modulated by many factors (1). Variables such as temperature, incubation time, growth medium, and sodium chloride concentrations have been considered in the development of Clinical Laboratory Standards Institute (CLSI) reference susceptibility test methods (3). Among the penicillinase-resistant penicillins, oxacillin is the most stable and sensitive for the detection of mecA-mediated resistance. However, heterogeneously resistant populations may have oxacillin test results indicating susceptibility (1).Recognition that cefoxitin is a stronger inducer of mecA expression than oxacillin led to studies that assessed this agent as a surrogate marker for methicillin resistance (2, 6, 9, 15, 16). For disk diffusion testing of staphylococci, cefoxitin (30 μg) provides more accurate results than oxacillin and zones that are easier to read (2, 3, 6). While cefoxitin has replaced oxacillin in the CLSI disk diffusion test, laboratories may use oxacillin or cefoxitin to predict mecA-mediated resistance by use of the CLSI broth microdilution (BMD) method (4). A resistant oxacillin or cefoxitin MIC test result indicates resistance to penicillins, cephems, carbapenems, and ß-lactams and ß-lactamase inhibitors (4).Manufacturers of automated susceptibility testing instruments have also adapted their products to optimize the detection of mecA-mediated resistance. The BD Phoenix (BD Diagnostics, Sparks, MD) and the Vitek 2 (bioMérieux, Durham, NC) systems now offer panels that include oxacillin and cefoxitin. The instruments'' expert systems interpret any S. aureus isolate that tests positive by the cefoxitin screen (MIC > 4 μg/ml on the Phoenix system, MIC > 6 μg/ml on the Vitek 2 system) as oxacillin resistant.This purpose of this study was to examine the accuracies of the Phoenix and the Vitek 2 instruments for the detection of mecA-mediated resistance in S. aureus. The oxacillin, cefoxitin, and expert system results generated by the Phoenix and Vitek 2 instruments were compared to the results generated by two reference methods: the oxacillin MICs determined by the CLSI BMD method and mecA gene detection by PCR.(This study was presented in part at the 108th General Meeting of the American Society for Microbiology, 2 June 2008, Boston, MA [abstr. C-009].)     

In vitro activity of tigecycline against gram-positive and gram-negative pathogens as evaluated by broth microdilution and Etest

The current surveillance establishes the activity profile of tigecycline against recent clinical U.S. isolates of target pathogens. Findings from a distributed surveillance that utilized Etest yielded a tigecycline activity profile that varied from that observed in a separate centralized broth microdilution (BMD) surveillance (D. C. Draghi et al., Poster D-0701, 46th Intersci. Conf. Antimicrob. Agents Chemother., San Francisco, CA). Differences were noted among Acinetobacter spp. and Serratia marcescens and, to a lesser extent, with Streptococcus pyogenes. To address whether these differences were due to discordance in testing methodology or to variations among the analyzed populations, isolates from the current surveillance were concurrently tested by BMD and Etest. In all, 1,800 Staphylococcus aureus, 259 S. pyogenes, 226 Streptococcus pneumoniae, 93 Enterococcus faecalis, 1,356 Enterobacteriaceae, and 227 Acinetobacter baumannii strains were evaluated. Tigecycline had potent activity by BMD, with >99.6% susceptibility (%S) observed for all pathogens with interpretive criteria, excluding Enterobacter cloacae (98.3% S) and E. faecalis (86.0% S), and MIC₉₀s ranged from 0.03 μg/ml (S. pyogenes/S. pneumoniae) to 1 μg/ml (Enterobacteriaceae/A. baumannii). Similar profiles were observed by Etest, with the exception of A. baumannii, although for most evaluated pathogens Etest MICs trended one doubling-dilution higher than BMD MICs. Major or very major errors were infrequent, and a high degree of essential agreement was observed, excluding A. baumannii, S. marcescens, and S. pneumoniae, for which ≥4-fold differences in MICs were observed for 29, 27.1, and 34% of the isolates, respectively. Further analysis regarding the suitability of the tigecycline Etest for testing S. marcescens, Acinetobacter spp., and S. pneumoniae is warranted.     

Breakthrough Invasive Candidiasis in Patients on Micafungin

For Candida species, a bimodal wild-type MIC distribution for echinocandins exists, but resistance to echinocandins is rare. We characterized isolates from patients with invasive candidiasis (IC) breaking through ≥3 doses of micafungin therapy during the first 28 months of its use at our center: MICs were determined and hot-spot regions within FKS genes were sequenced. Eleven of 12 breakthrough IC cases identified were in transplant recipients. The median duration of micafungin exposure prior to breakthrough was 33 days (range, 5 to 165). Seventeen breakthrough isolates were recovered: FKS hot-spot mutations were found in 5 C. glabrata and 2 C. tropicalis isolates; of these, 5 (including all C. glabrata isolates) had micafungin MICs of >2 μg/ml, but all demonstrated caspofungin MICs of >2 μg/ml. Five C. parapsilosis isolates had wild-type FKS sequences and caspofungin MICs of 0.5 to 1 μg/ml, but 4/5 had micafungin MICs of >2 μg/ml. The remaining isolates retained echinocandin MICs of ≤2 μg/ml and wild-type FKS gene sequences. Breakthrough IC on micafungin treatment occurred predominantly in severely immunosuppressed patients with heavy prior micafungin exposure. The majority of cases were due to C. glabrata with an FKS mutation or wild-type C. parapsilosis with elevated micafungin MICs. MIC testing with caspofungin identified all mutant strains. Whether the naturally occurring polymorphism within the C. parapsilosis FKS1 gene responsible for the bimodal wild-type MIC distribution is also responsible for micafungin MICs of >2 μg/ml and clinical breakthrough or an alternative mechanism contributes to the nonsusceptible echinocandin MICs in C. parapsilosis requires further study.Invasive candidiasis (IC) is an important, life-threatening infection in hospitalized patients. The echinocandins (micafungin, caspofungin, and anidulafungin) are the newest class of medications approved for the prophylaxis and treatment of IC. They act via noncompetitive inhibition of β-1,3-glucan synthase, the enzyme responsible for producing β-1,3-d-glucan in the fungal cell wall (41). These drugs have low toxicity and few drug-drug interactions and possess a broad spectrum of antifungal activity against Candida species, including those resistant to fluconazole. In clinical trials, the echinocandins have demonstrated noninferiority for the treatment of IC versus amphotericin B deoxycholate, liposomal amphotericin B, and fluconazole (25, 32, 44). The echinocandins are considered interchangeable for clinical use, and a recent study comparing micafungin to caspofungin for IC supports this notion (38). Based on the accumulated experience, echinocandins are now considered a first-line therapeutic choice for IC (37).The echinocandins exhibit a bimodal MIC distribution among Candida species. MICs of C. parapsilosis, C. guilliermondii, and C. famata MICs (MIC₉₀, 0.25 to 2 μg/ml) are up to 133 times higher than those of C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyr (MIC₉₀, 0.015 to 0.25 μg/ml) (42). However, this difference has not translated into consistent clinical failure (25, 38, 44), and the MIC breakpoint for echinocandin susceptibility was set at ≤2 μg/ml, which is inclusive of 99% of the wild-type distribution of all Candida species (9). Organisms with MICs of >2 μg/ml are considered “nonsusceptible,” but the breakpoint for resistance has yet to be determined owing to the paucity of clinical isolates available from patients failing echinocandin therapy and with MICs of >2 μg/ml.As echinocandin use has escalated, cases of echinocandin breakthrough IC have been described (6, 7, 13, 25, 39, 50), and nonsusceptible isolates (MIC > 2 μg/ml) have been recovered from patients who demonstrated treatment failure (9). Moreover, several of these nonsusceptible isolates possess nonsynonymous point mutations in genes encoding the β-1,3-glucan synthase enzyme complex (Fksp) (4, 13, 39, 47). These specific FKS “hot-spot” mutations reduce the susceptibility of the β-1,3-glucan synthase enzyme complex to echinocandin drugs, supporting a biological mechanism of resistance (14).In February 2006, micafungin became the formulary echinocandin at our hospital, a tertiary care center with multiple intensive care units, two dedicated hematopoietic stem cell transplant (HSCT) units, and an active solid organ transplant (SOT) service. Multiple patients with breakthrough IC while receiving micafungin therapy were noted. These cases were reviewed, and the Candida isolates recovered from these patients were screened for FKS gene mutations; results were correlated with MIC values.(This work was presented in part at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 12 to 14 September 2009 [slide presentation M-1243]).     

In Vitro Activity of Ceftolozane-Tazobactam as Determined by Broth Dilution and Agar Diffusion Assays against Recent U.S. Escherichia coli Isolates from 2010 to 2011 Carrying CTX-M-Type Extended-Spectrum β-Lactamases

Ceftolozane MIC₅₀/MIC₉₀s were 4/8 μg/ml when tested against 26 CTX-M-14-type-producing isolates and 64/>64 μg/ml against 219 CTX-M-15-type-producing isolates. The addition of 4 μg/ml tazobactam lowered the ceftolozane MIC₅₀/MIC₉₀s to ≤0.25/0.5 μg/ml by broth microdilution and Etest. The zone diameters for the ceftolozane-tazobactam disks were 23 to 29 mm for 92.2% of the isolates.     

Rapid Detection of Staphylococcus aureus Strains with Reduced Susceptibility to Vancomycin by Isothermal Microcalorimetry

Methicillin-resistant Staphylococcus aureus (MRSA) usually harbors a vancomycin-susceptible phenotype (VSSA) but can exhibit reduced vancomycin susceptibility phenotypes that can be heterogeneous-intermediate (hVISA), intermediate (VISA), or fully resistant (VRSA). Current detection techniques (e.g., Etest and population analysis profiles [PAPs]) are slow and time-consuming. We investigated the potential of microcalorimetry to detect reduced susceptibilities to vancomycin in MRSA strains. Representative MSSA, VSSA, hVISA, VISA, and VRSA reference strains, as well as clinical isolates, were used. PAPs were performed by standard methods. Microcalorimetry was performed by inoculating 5 × 10⁷ CFU of overnight cultures into 3-ml vials of brain heart infusion broth supplemented with increasing concentrations of vancomycin, and growth-related heat production was measured at 37°C. For the reference strains, no heat production was detected in the VSSA isolates at vancomycin concentrations of >3 μg/ml during the 72 h of incubation. The hVISA and VISA strains showed heat production with concentration-proportional delays of up to 6 μg/ml in 48 h and up to 12 μg/ml in 72 h, respectively. The VRSA strain showed heat production at concentrations up to 16 μg/ml in 12 h. The testing of clinical strains indicated an excellent negative predictive value, allowing us to rule out a decreased vancomycin susceptibility phenotype in <8 h of incubation. Sequential isolates from a patient undergoing vancomycin therapy showed evolving microcalorimetric profiles up to a VISA phenotype. Microcalorimetry was able to detect strains with reduced susceptibilities to vancomycin in <8 h. The measurement of bacterial heat production might represent a simple and rapid method for the detection of reduced susceptibilities to vancomycin in MRSA strains.     

Antifungal Susceptibility of 205 Candida spp. Isolated Primarily during Invasive Candidiasis and Comparison of the Vitek 2 System with the CLSI Broth Microdilution and Etest Methods

Infections due to Candida spp. are frequent, particularly in immunocompromised and intensive care unit patients. Antifungal susceptibility tests are now required to optimize antifungal treatment given the emergence of acquired antifungal resistance in some Candida species. An antifungal susceptibility automated method, the Vitek 2 system (VK2), was evaluated. VK2 was compared to the CLSI broth microdilution reference method and the Etest procedure. For this purpose, 205 clinical isolates of Candida spp., including 11 different species, were tested for fluconazole, voriconazole, and amphotericin B susceptibility. For azoles, essential agreement ranged from 25% to 100%, depending on the method used and the Candida species tested. Categorical agreements for all of the species averaged 92.2% and ranged from 14.3 to 100%, depending on the 24-h or 48-h MIC reading by the Etest and CLSI methods and on the Candida species. Results obtained for Candida albicans showed excellent categorical and essential agreements with the two comparative methods. For Candida glabrata, the essential agreement was high with the CLSI method but low with the Etest method, and several very major errors in interpretation were observed between VK2 and the Etest method for both azoles. Low MICs of fluconazole were obtained for all of the Candida krusei isolates, but the VK2 expert software corrected all of the results obtained to resistant. Amphotericin B results showed MICs of ≤1 mg/liter for 201 (VK2), 190 (CLSI), and 202 (Etest) isolates. The AST-YS01 Vitek 2 card system (bioMérieux) is a reliable and practical standardized automated antifungal susceptibility test. Nevertheless, more assays are needed to better evaluate C. glabrata fluconazole sensitivity.Invasive candidiasis infections are of increasing concern, particularly in immunodeficient or intensive care unit patients. The emergence of antifungal resistance and the development of new echinocandin class and broad-spectrum azole fungal agents have complicated the choice of antifungal treatment for candidiasis.Antifungal choice is first based on Candida species identification, but antifungal susceptibility testing will play an increasingly important role when selecting which antifungal drug to use (3). Standardized methods for antifungal susceptibility testing have been available for many years. The Clinical and Laboratory Standards Institute (CLSI) standardized broth microdilution method remains a reference for antifungal susceptibility testing (4). Indeed, clinically relevant interpretative breakpoints are available and quality control strains are validated (11). Nevertheless, this method is complex and laborious to use as a routine method. Alternative, standardized and reliable methods adapted to hospital laboratories, such as the Etest, are now commonly used (2).The objective of this study was to evaluate and compare a new automated antifungal susceptibility test system (AST-YS01 Vitek 2 cards; bioMérieux) to both the CLSI reference broth microdilution method and Etest procedures. For this purpose, 208 clinical isolates of Candida spp. isolated primarily from patients with invasive candidiasis were tested with fluconazole, voriconazole, and amphotericin B.(Preliminary data were presented at the 19th European Congress of Clinical Microbiology and Infectious Diseases, Helsinki, Finland, 16 to 19 May 2009, abstr. P1306.)     

Comparing Etest and Broth Microdilution for Antifungal Susceptibility Testing of the Most-Relevant Pathogenic Molds

Invasive mold infections are life-threatening diseases for which appropriate antifungal therapy is crucial. Their epidemiology is evolving, with the emergence of triazole-resistant Aspergillus spp. and multidrug-resistant non-Aspergillus molds. Despite the lack of interpretive criteria, antifungal susceptibility testing of molds may be useful in guiding antifungal therapy. The standard broth microdilution method (BMD) is demanding and requires expertise. We assessed the performance of a commercialized gradient diffusion method (Etest method) as an alternative to BMD. The MICs or minimal effective concentrations (MECs) of amphotericin B, voriconazole, posaconazole, caspofungin, and micafungin were assessed for 290 clinical isolates of the most representative pathogenic molds (154 Aspergillus and 136 non-Aspergillus isolates) with the BMD and Etest methods. Essential agreements (EAs) within ±2 dilutions of ≥90% between the two methods were considered acceptable. EAs for amphotericin B and voriconazole were >90% for most potentially susceptible species. For posaconazole, the correlation was acceptable for Mucoromycotina but Etest MIC values were consistently lower for Aspergillus spp. (EAs of <90%). Excellent EAs were found for echinocandins with highly susceptible (MECs of <0.015 μg/ml) or intrinsically resistant (MECs of >16 μg/ml) strains. However, MEC determinations lacked consistency between methods for strains exhibiting mid-range MECs for echinocandins. We concluded that the Etest method is an appropriate alternative to BMD for antifungal susceptibility testing of molds under specific circumstances, including testing with amphotericin B or triazoles for non-Aspergillus molds (Mucoromycotina and Fusarium spp.). Additional study of molecularly characterized triazole-resistant Aspergillus isolates is required to confirm the ability of the Etest method to detect voriconazole and posaconazole resistance among Aspergillus spp.     

Comparative Evaluation of Tigecycline Susceptibility Testing Methods for Expanded-Spectrum Cephalosporin- and Carbapenem-Resistant Gram-Negative Pathogens

We evaluated the Vitek2, Etest, and MIC Test Strip (MTS) methods of tigecycline susceptibility testing with 241 expanded-spectrum cephalosporin-resistant and/or carbapenem-resistant Enterobacteriaceae and Acinetobacter baumannii clinical isolates by using dry-form broth microdilution (BMD) as the reference method. The MIC_50/90s were as follows: BMD, 1/4 μg/ml; Vitek2, 4/≥8 μg/ml; Etest, 2/4 μg/ml; MTS, 0.5/2 μg/ml. Vitek2 produced 9.1/21.2% major errors, Etest produced 0.4/0.8% major errors, and MTS produced no major errors but 0.4/3.3% very major errors (FDA/EUCAST breakpoints). Vitek2 tigecycline results require confirmation by BMD or Etest for multidrug-resistant pathogens.