Extended-spectrum ␤-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were >2 g/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of >5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by >3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were >32 g/ml, suggesting either the presence of an AmpC-type ␤-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a ␤-lactamase with a pI of >8.3 suggestive of an AmpC-type ␤-lactamase; 6 of the 7 isolates were shown by PCR to contain both ampC-type and bla OXA genes. The IEF profiles of the remaining 16 isolates showed a variety of ␤-lactamase bands, all of which had pIs of <7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type, bla OXA , and bla CTX-M genes. In summary, 83.5% of the K. pneumoniae isolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained ␤-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained ␤-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.

Disk diffusion and broth microdilution (BMD) were used to perform clindamycin (CLI) induction testing on؉ phenotype by disk diffusion were also detected by BMD using a variety of CLI and ERY concentrations; however, isolates with the D phenotype were more difficult to detect by BMD and will likely require optimization of ERY and CLI concentrations in multilaboratory studies to ensure adequate sensitivity. Thus, at present, disk diffusion is the preferred method for testing S. aureus isolates for inducible CLI resistance.Erythromycin (ERY) (a macrolide) and clindamycin (CLI) (a lincosamide) represent two distinct classes of antimicrobial agents that inhibit protein synthesis by binding to the 50S ribosomal subunits of bacterial cells. In staphylococci, resistance to both of these antimicrobial agents can occur through methylation of their ribosomal target site (25). Such resistance is typically mediated by erm genes. Resistance to macrolides also can occur by efflux, typically mediated by the msrA gene (16). Another resistance mechanism, inactivation of lincosamides by chemical modification (such as mediated by the inuA gene), appears to be rare (1,8,15). The target site modification mechanism, also called macrolide-lincosamide-streptogramin B (MLS B ) resistance, results in resistance to ERY, CLI, and streptogramin B. This mechanism can be constitutive, where the rRNA methylase is always produced, or can be inducible, where methylase is produced only in the presence of an inducing agent. ERY is an effective inducer, but CLI is a weak inducer. In vitro, Staphylococcus aureus isolates with constitutive resistance are resistant to ERY and CLI, and isolates with inducible resistance are resistant to ERY but appear susceptible to CLI. In vivo, therapy with CLI may select for constitutive erm mutants (7), which may lead to clinical failure (2,20,24). Isolates with msrA-mediated efflux also appear ERY resistant and CLI susceptible by in vitro tests; however, such isolates do not typically become CLI resistant during therapy.An in vitro induction test can distinguish staphylococci that have inducible erm-mediated resistance from those with msrAmediated resistance. The test is performed by disk diffusion, placing a 15-g ERY disk in proximity to a 2-g CLI disk on an agar plate that has been inoculated with a staphylococcal isolate; the plate is then incubated overnight (5,9,22,23). A flattening of the zone of inhibition around the CLI disk proximal to the ERY disk (producing a zone of inhibition shaped like the letter D) is considered a positive result and indicates that the ERY has induced CLI resistance (a positive "D-zone test").

Detecting β-lactamase-mediated carbapenem resistance among Klebsiella pneumoniae isolates and other Enterobacteriaceae is an emerging problem. In this study, 15 bla KPC -positive Klebsiella pneumoniae that showed discrepant results for imipenem and meropenem from 4 New York City hospitals were characterized by isoelectric focusing; broth microdilution (BMD); disk diffusion (DD); and MicroScan, Phoenix, Sensititre, VITEK, and VITEK 2 automated systems. All 15 isolates were either intermediate or resistant to imipenem and meropenem by BMD; 1 was susceptible to imipenem by DD. MicroScan and Phoenix reported 1 (6.7%) and 2 (13.3%) isolates, respectively, as imipenem susceptible. VITEK and VITEK 2 reported 10 (67%) and 5 (33%) isolates, respectively, as imipenem susceptible. By Sensititre, 13 (87%) isolates were susceptible to imipenem, and 12 (80%) were susceptible to meropenem. The VITEK 2 Advanced Expert System changed 2 imipenem MIC results from >16 µg/mL to <2 µg/mL but kept the interpretation as resistant. The recognition of carbapenem-resistant K. pneumoniae continues to challenge automated susceptibility systems.

Using a set of 55 Staphylococcus aureus challenge organisms, we evaluated six routine methods (broth microdilution, disk diffusion, oxacillin agar screen, MicroScan conventional panels, MicroScan rapid panels, and Vitek cards) currently used in many clinical laboratories and two new rapid methods, Velogene and the MRSAScreen, that require less than a day to determine the susceptibility of S. aureus to oxacillin. The methods were evaluated by using the presence of the mecA gene, as detected by PCR, as the "gold standard." The strains included 19 mecA-positive heterogeneously resistant strains of expression class 1 or 2 (demonstrating oxacillin MICs of 4 to >16 g/ml) and 36 mecA-negative strains. The oxacillin MICs of the latter strains were 0.25 to 4 g/ml when tested by broth microdilution with 2% NaCl-supplemented cation-adjusted Mueller-Hinton broth as specified by the NCCLS. However, when tested by agar dilution with 4% salt (the conditions used in the oxacillin agar screen method), the oxacillin MICs of 16 of the mecA-negative strains increased to 4 to 8 g/ml. On initial testing, the percentages of correct results (% sensitivity/% specificity) were as follows: broth microdilution, 100/100; Velogene, 100/100; Vitek, 95/97; oxacillin agar screen, 90/92; disk diffusion, 100/89; MicroScan rapid panels, 90/86; MRSA-Screen, 90/100; and MicroScan conventional, 74/97. The MRSA-Screen sensitivity improved to 100% if agglutination reactions were read at 15 min. Repeat testing improved the performance of some but not all of the systems.The oxacillin agar screen test has been used for many years to aid in the identification of oxacillin-resistant staphylococci. Recently, this test was shown to be ineffective for coagulasenegative staphylococci (20), and the NCCLS no longer recommends its use for these organisms. In a previous study, the inoculation methods for the oxacillin agar screen test were more clearly defined (19a), and recommendations were made to use a 1-l loop or an expressed swab inoculated into an area of 10 to 15 mm in diameter. In that study, a challenge group of Staphylococcus aureus isolates was tested, including several borderline oxacillin-resistant mecA-positive strains (n ϭ 19) that would be difficult to detect and mecA-negative strains (n ϭ 36) that might be falsely detected as resistant. Since several lots of oxacillin screen agar lacked specificity, the issue of the reliability of all methods for detection of oxacillin resistance was raised. Thus, using that same group of challenge organisms, we chose to examine the accuracy of the other routine methods for detecting oxacillin resistance. These methods were broth microdilution, disk diffusion, oxacillin agar screen, MicroScan conventional panels, MicroScan rapid panels, and Vitek. We also evaluated two new rapid methods, Velogene, a cycling probe assay, and MRSA-Screen, a latex agglutination method that detects the presence of PBP-2a (also known as PBP-2Ј), the penicillin-binding protein (PBP) responsible for the most common form of oxacillin resis...

A challenge panel of enterococci (n ‫؍‬ 50) and staphylococci (n ‫؍‬ 50), including 17 and 15 isolates that were nonsusceptible to linezolid, respectively, were tested with the Clinical and Laboratory Standards Institute broth microdilution and disk diffusion reference methods. In addition, all 100 isolates were tested in parallel by Etest (AB Biodisk, Solna, Sweden), MicroScan WalkAway (Dade, West Sacramento, CA), BD Phoenix (BD Diagnostic Systems, Sparks, MD), VITEK (bioMérieux, Durham, NC), and VITEK 2 (bioMérieux) by using the manufacturers' protocols. Compared to the results of the broth microdilution method for detecting linezolidnonsusceptible staphylococci and enterococci, MicroScan results showed the highest category agreement (96.0%). The overall categorical agreement levels for VITEK 2, Etest, Phoenix, disk diffusion, and VITEK were 93.0%, 90.0%, 89.6%, 88.0%, and 85.9%, respectively. The essential agreement levels (results within ؎1 doubling dilution of the MIC determined by the reference method) for MicroScan, Phoenix, VITEK 2, Etest, and VITEK were 99.0%, 95.8%, 92.0%, 92.0%, and 85.9%, respectively. The very major error rates for staphylococci were the highest for VITEK (35.7%), Etest (40.0%), and disk diffusion (53.3%), although the total number of resistant isolates tested was small. The very major error rate for enterococci with VITEK was 20.0%. Three systems (MicroScan, VITEK, and VITEK 2) provided no interpretations of nonsusceptible results for staphylococci. These data, from a challenge panel of isolates, illustrate that the recent emergence of linezolidnonsusceptible staphylococci and enterococci is providing a challenge for many susceptibility testing systems.

We assessed the in vitro activities of daptomycin, linezolid, and quinupristin-dalfopristin (QD) against a contemporary challenge panel of 88 staphylococcal and 90 enterococcal isolates. The staphylococci selected included vancomycin-intermediate Staphylococcus aureus (VISA), methicillin-resistant S. aureus, and coagulasenegative staphylococci. Enterococcal isolates included vancomycin-resistant Enterococcus faecium (VREF) containing either vanA, vanB1, or vanD. The MICs of daptomycin, linezolid, and QD were determined using commercial broth microdilution panels. All three VISA isolates were susceptible to daptomycin, linezolid, and QD. QD was the most active agent against staphylococcal isolates (MIC50 < or = 0.5 microg/ml and MIC90 = 1 microg/ml), including those with decreased susceptibility to vancomycin. QD was also the most active agent against VREF (MIC90 < or = 0.5 microg/ml). No differences were seen for susceptibility of vanA, vanB1, and vanD VREF strains for daptomycin, linezolid, or QD. Daptomycin was the most effective against E. faecalis. On the basis of manufacturer-suggested interpretive criteria, 92% of isolates were susceptible (MIC90 = 4 microg/ml). All isolates tested were susceptible to at least one antimicrobial agent for which interpretive criteria have been defined. Population analysis of three S. aureus isolates for which the daptomycin MICs were 8 microg/ml showed a pattern of homogeneous resistance.

To determine whether confirmatory tests for extended-spectrum ␤-lactamase (ESBL) production in Escherichia coli are necessary, we selected 131 E. coli isolates that met the National Committee for Clinical Laboratory Standards (NCCLS) screening criteria for potential ESBL production from the Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) strain collection. For all 131 isolates, the broth microdilution (BMD) MIC of at least one extended-spectrum cephalosporin was >2 g/ml. For 21 of 131 (16%) isolates, the ESBL confirmatory test was positive; i.e., the BMD MICs of ceftazidime or cefotaxime decreased by >3 doubling dilutions in the presence of clavulanic acid (CA) or the disk diffusion zone diameters increased by >5 mm around ceftazidime or cefotaxime disks in the presence of CA. All 21 isolates were shown by PCR to contain at least one of the genes bla TEM , bla SHV , and bla OXA , and in isoelectric focusing (IEF) tests, all isolates demonstrated at least one ␤-lactamase band consistent with a TEM, SHV, or OXA enzyme. Of the 21 isolates, 3 showed a CA effect for cefotaxime by BMD but not by disk diffusion testing. A total of 59 (45%) of the 131 isolates demonstrated decreased susceptibility to cefpodoxime alone (MIC ‫؍‬ 2 to 4 g/ml), and none had a positive ESBL confirmatory test result. These were classified as false positives according to ESBL screen test results. For the remaining 51 (39%) isolates, the cefpodoxime MICs ranged from 16 to >128 g/ml and the MICs for the other extended-spectrum cephalosporins were highly variable. All 51 isolates gave negative ESBL confirmatory test results. Most showed IEF profiles consistent with production of both a TEM and an AmpC ␤-lactamase, and representative isolates of several phenotypic groups showed changes in porin profiles; these 51 isolates were considered true negatives. In all, only 16% of 131 E. coli isolates identified as potential ESBL producers by the current NCCLS screening criteria were confirmed as ESBL producers. Thus, changing the interpretation of extended-spectrum cephalosporins and aztreonam results from the susceptible to the resistant category without confirming the presence of an ESBL phenotype would lead to a large percentage of false resistance results and is not recommended. However, by increasing the cefpodoxime MIC screening breakpoint to >8 g/ml, 45% of the false-positive results could be eliminated. NCCLS has incorporated this change in the cefpodoxime screening breakpoint in its recent documents.

C.). The results of each test method were compared to the results of BMD testing using in-house-prepared panels. Seven imipenem-resistant and five meropenem-resistant isolates ofEnterobacteriaceae and 43 imipenem-resistant and 21 meropenem-resistant isolates of P. aeruginosa were identified by BMD. For Enterobacteriaceae, the imipenem and meropenem test methods produced low numbers of very major and major errors. All test systems in the study produced low numbers of very major and major errors when P. aeruginosa was tested against imipenem and meropenem, except for Vitek testing (major error rate for imipenem, 20%). Further testing conducted in 11 of the participating ICARE hospital laboratories failed to pinpoint the factors responsible for the initial overdetection of imipenem resistance. However, this study demonstrated that carbapenem testing difficulties do exist and that laboratories should consider using a second, independent antimicrobial susceptibility testing method to validate carbapenem-intermediate and -resistant results.