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").

Cefpodoxime is one of five antimicrobial agents recommended by the National Committee for Clinical Laboratory Standards for screening isolates of Klebsiella spp. and Escherichia coli for extended-spectrum ␤-lactamase (ESBL) production. In a prior study, we noted that among 131 E. coli isolates for which the MIC of at least one extended-spectrum cephalosporin (ESC) or aztreonam was >2 g/ml (suggesting the presence of ESBL production), there were 59 isolates (45.0%) for which the MIC of cefpodoxime was 2 to 4 g/ml (i.e., a positive ESBL screening test), but the MICs of ceftazidime, cefotaxime, and ceftriaxone were <1 g/ml (below the ESBL screening breakpoint). Thus, the results appeared to be false-positive ESBL screening tests. These 59 isolates were divided into five phenotypic groups based on the susceptibility patterns of the organisms to a variety of ␤-lactam agents and further characterized. The first group (32 isolates) all produced a TEM-1 ␤-lactamase, and changes in the major outer membrane proteins were detected in representative strains. The second group (18 isolates) lacked bla TEM but showed a number of porin changes; some also showed a modest elevation in production of the AmpC chromosomal ␤-lactamase. In the third phenotypic group (seven isolates) all expressed an OXA-30 ␤-lactamase. Some also harbored altered porins. The two remaining phenotypes each had a distinct pattern of porin changes with or without ␤-lactamase production. These data indicate that several factors are associated with decreased susceptibility to cefpodoxime in E. coli, but none of the mechanisms are related to ESBL production. Current screening methods produced false-positive ESBL results for these isolates. Such isolates should not be classified as containing ESBLs, nor should interpretations of ESCs or aztreonam susceptibility be changed to resistant on test reports for these isolates.

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.

NCCLS screening and confirmation methods for detecting extended-spectrum ␤-lactamases (ESBLs) apply only to Escherichia coli and Klebsiella spp., yet ESBLs have been found in other members of the family Enterobacteriaceae. We evaluated the effectiveness of NCCLS methods for detecting ESBLs in 690 gramnegative isolates of Enterobacteriaceae that excluded E. coli, Klebsiella pneumoniae, and Klebsiella oxytoca. Isolates were collected between January 1996 and June 1999 from 53 U.S. hospitals participating in Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology). The antimicrobial susceptibility patterns of the isolates were determined by using the NCCLS broth microdilution method (BMD), and those isolates for which the MIC of ceftazidime, cefotaxime, ceftriaxone, or aztreonam was >2 g/ml or the MIC of cefpodoxime was >8 g/ml (positive ESBL screen test) were further tested for a clavulanic acid (CA) effect by BMD and the disk diffusion method (confirmation tests). Although 355 (51.4%) of the isolates were ESBL screen test positive, only 15 (2.2%) showed a CA effect. Since 3 of the 15 isolates were already highly resistant to the five NCCLS indicator drugs, ESBL detection would have an impact on the reporting of only 1.7% of the isolates in the study. Only 6 of the 15 isolates that showed a CA effect contained a bla TEM , bla SHV , bla CTX-M , or bla OXA ␤-lactamase gene as determined by PCR (with a corresponding isoelectric focusing pattern). Extension of the NCCLS guidelines for ESBL detection to Enterobacteriaceae other than E. coli and Klebsiella spp. does not appear to be warranted in the United States at present, since the test has poor specificity for this population and would result in changes in categorical interpretations for only 1.7% of Enterobacteriaceae tested.First described in 1983 (10), extended-spectrum ␤-lactamases (ESBLs) have contributed to the dramatic increase in resistance to ␤-lactam agents among gram-negative bacteria in recent years (2,3,8). These enzymes, encoded by genes that are typically plasmid borne, hydrolyze penicillins, cephalosporins, and aztreonam and are inhibited by clavulanic acid (CA) (2, 3, 5). Although most commonly produced by Escherichia coli and Klebsiella spp., ESBLs may be harbored by many other gram-negative bacilli as well, including but not limited to many other bacteria in the family Enterobacteriaceae (7,8,25).While most of the currently known ESBLs are derived from the older ␤-lactamases TEM-1, TEM-2, and SHV-1 (2, 11, 25), CTX-M enzymes are also inhibited by CA and are in the category of ESBLs. In addition, some of the enzymes of the OXA family, although belonging to functional group 2d (5), show a CA effect and are considered ESBLs (2).While a variety of phenotypic and molecular methods have been successfully employed to confirm the presence of ESBLs in clinical isolates, their use is too labor-intensive to be practical for routine screening in microbiology laboratories. Since routine susceptibility testing does not always detect the presence of ...

Accurate antimicrobial susceptibility testing (AST) and appropriate reporting of AST results for pathogens isolated from blood cultures are critical functions of the microbiology laboratory. We studied AST performance and reporting from positive blood cultures at hospital microbiology laboratories in Iowa. One hundred sixteen episodes of bacteremia from 14 participating hospitals were examined. We detected AST or identification errors for 18 episodes (16%) and judged reporting of AST results to be inappropriate for 38 episodes (33%). Further study is necessary to determine the impact of testing errors and suboptimal reporting of results on the management of bloodstream infection.Bloodstream infections cause substantial morbidity and mortality, with up to one-quarter of affected patients dying as a result of their infection (14,15). Early receipt of appropriate antimicrobial therapy has been demonstrated to improve the outcome of bloodstream infection (1, 7). Timely and accurate detection and reporting of bloodstream infection are therefore some of the most important functions of a clinical microbiology laboratory (2,3,5,8,11). In addition, antimicrobial susceptibility test (AST) results can affect both the clinician's choice of antimicrobial therapy and the patient's outcome (12,13). If the laboratory reports results that are erroneous, patients with bloodstream infection may not receive optimal therapy. In addition, reporting all AST results for an organism, rather than reporting selectively (e.g., "cascading," or only reporting the narrowest-spectrum agents to which the organism is susceptible), may encourage inappropriate antibiotic use. Thus, prompt detection of bloodstream infection, accurate microbial identification and susceptibility testing, and appropriate reporting of results are important patient safety issues (6).We assessed the preanalytical, analytical, and postanalytical errors made by microbiology laboratories related to the detection, identification, AST, and reporting of results from positive blood cultures. The goal of the study was to identify key areas for improvement in order to guide the development and implementation of an educational intervention for laboratory personnel.The microbiology laboratories of 14 Iowa hospitals participated in this study. The hospitals were all participating in the Emerging Infections and Epidemiology of Iowa Organisms study, a statewide antimicrobial resistance surveillance network (4). These hospitals were chosen based upon geographic location, population distribution, and hospital bed size. Hospital size ranged from 89 to 820 acute care beds (median of 175 beds) and included rural, rural-referral, and urban hospitals.We studied organisms from five consecutive positive blood cultures from unique patients from whom gram-positive pathogens were isolated and five consecutive positive blood cultures from unique patients from whom gram-negative pathogens were isolated. All isolates were deemed clinically significant. A research assistant, infection control profess...

A series of 3-descladinosyl-2,3-anhydro-6-O-methylerythromycin A 11, 12-carbamate analogues have been synthesized and evaluated for antibacterial activity. These compounds were found to be potent antibacterial agents against Gram-positive organisms in vitro, many having MIC values below 1 microg/mL for the macrolide-susceptible Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae, as well as improved activity compared to erythromycin A against the inducibly MLS (macrolide, lincosamide, and streptogramin B)-resistant organisms. Structure-activity studies revealed that arylalkyl carbamates with two and four carbon atoms between the aromatic moiety and carbamate nitrogen have the best in vitro activity. All of the C-10 epi analogues evaluated were found to have substantially less activity than the corresponding natural C-10 isomer. Several analogues demonstrated moderate antibacterial activity against the constitutively resistant S.aureus A-5278, S. pneumoniae5979, and S.pyogenes 930. However, despite potent in vitro activity, these analogues showed only moderate in vivo activity in mouse protection studies.