Ability of laboratories to detect emerging antimicrobial resistance in nosocomial pathogens: a survey of project ICARE laboratories.

A proficiency testing project was conducted among 48 microbiology laboratories participating in Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology). All laboratories correctly identified the Staphylococcus aureus challenge strain as oxacillin- resistant and an Enterococcus faecium strain as vancomycin-resistant. Thirty-one (97%) of 32 laboratories correctly reported the Streptococcus pneumoniae strain as erythromycin-resistant. All laboratories testing the Pseudomonas aeruginosa strain against ciprofloxacin or ofloxacin correctly reported the organism as resistant. Of 40 laboratories, 30 (75%) correctly reported resistant MICs or zone sizes for the imipenem- and meropenem-resistant Serratia marcescens. For the extended-spectrum beta-lactamase (ESBL)-producing strain of Klebsiella pneumoniae, 18 (42%) of 43 laboratories testing ceftazidime correctly reported ceftazidime MICs in the resistant range. These results suggest that current testing generally produces accurate results, although some laboratories have difficulty detecting resistance to carbapenems and extended-spectrum cephalosporins. This highlights the need for monitoring how well susceptibility test systems in clinical laboratories detect emerging resistance.

Evaluation of a computerized antimicrobial susceptibility system with bacteria isolated from animals.

The BIOMIC is a computerized system used to calculate the minimal inhibitory concentration (MIC) of an antimicrobial agent from a zone of inhibition generated by a disk diffusion test. This system was developed using bacterial pathogens of human origin. This study investigated the use of the BIOMIC system for determining MICs for bacterial pathogens from animals. The MICs generated by the BIOMIC system were compared with the MICs generated using a broth microdilution testing method. A total of 663 drug-organism combinations was tested. These combinations included 3 species of gram-positive bacteria, 5 species of gram-negative bacteria, and the antimicrobial agents ampicillin, gentamicin, cephalothin, ciprofloxacin, enrofloxacin, trimethoprim/sulfamethoxazole, tetracycline, and erythromycin. Overall, the MICs generated by the BIOMIC system correlated with the broth microdilution MICs for 72% of the total drug-organism combinations tested. The Pseudomonas aeruginosa strains tested showed the highest agreement between the 2 systems, with 100% for all antibacterial agents tested, whereas Pasteurella haemolytica, Pasteurella multocida, and enterococci showed the least agreement (76%, 57%, and 47%, respectively). Among these organisms, trimethoprim-sulfa showed the least agreement (31%) and ciprofloxacin showed the greatest (91%). These results indicate that the BIOMIC system could be a useful tool in veterinary medicine for producing quantitative antimicrobial susceptibility results. However, it is currently unreliable for some drug-bacteria combinations. This discrepancy possibly could be corrected by modification of the software using data points generated by a large-scale study.

Antimicrobial resistance and genetic relatedness among Salmonella from retail foods of animal origin: NARMS retail meat surveillance.

Salmonella isolates were recovered from a monthly sampling of chicken breasts, ground turkey, ground beef, and pork chops purchased from selected grocery stores in six participating FoodNet sites (Connecticut, Georgia, Maryland, Minnesota, Oregon, and Tennessee) in 2002 and an additional two sites in 2003 (California and New York). In 2002 and 2003, a total of 6,046 retail meats were examined, including 1,513 chicken breasts, 1,499 ground turkey samples, 1,522 ground beef samples, and 1,502 pork chops. Retail meat samples tested increased to 3,533 in 2003 as compared to 2,513 in 2002. Overall, six percent of 6,046 retail meat samples (n = 365) were contaminated with Salmonella, the bulk recovered from either ground turkey (52%) or chicken breast (39%). Salmonella isolates were serotyped and susceptibility tested using a panel of 16 antimicrobial agents. S. Heidelberg was the predominant serotype identified (23%), followed by S. Saintpaul (12%), S. Typhimurium (11%), and S. Kentucky (10%). Overall, resistance was most often observed to tetracycline (40%), streptomycin (37%), ampicillin (26%), and sulfamethoxazole (25%). Twelve percent of isolates were resistant to cefoxitin and ceftiofur, though only one isolate was resistant to ceftriaxone. All isolates were susceptible to amikacin and ciprofloxacin; however, 3% of isolates were resistant to nalidixic acid and were almost exclusive to ground turkey samples (n = 11/12). All Salmonella isolates were analyzed for genetic relatedness using pulsed-field gel electrophoresis (PFGE) patterns generated by digestion with Xba1 or Xba1 plus Bln1. PFGE fingerprinting profiles showed that Salmonella, in general, were genetically diverse with a total of 175 Xba1 PFGE profiles generated from the 365 isolates. PFGE profiles showed good correlation with serotypes and in some instances, antimicrobial resistance profiles. Results demonstrated a varied spectrum of antimicrobial resistance and PFGE patterns, including several multidrug resistant clonal groups among Salmonella isolates, and signify the importance of sustained surveillance of foodborne pathogens in retail meats.

Characterization of Salmonella Typhimurium of animal origin obtained from the National Antimicrobial Resistance Monitoring System.

Salmonella Typhimurium remains one of the most common causes of salmonellosis in animals and humans in the United States. The emergence of multi-drug resistant Salmonella reduces the therapeutic options in cases of invasive infections, and has been shown to be associated with an increased burden of illness. In this study, 588 S. Typhimurium (including var. Copenhagen) isolates obtained from either animal diagnostic specimens (n = 199) or food animals after slaughter/processing (n = 389) were examined for antimicrobial susceptibility, presence of class-1 integrons, and characterized using pulsed-field gel electrophoresis and phage typing. Seventy-six percent (448/588) of isolates were resistant to at least one antimicrobial. Salmonella isolates displayed resistance most often to streptomycin (63%), tetracycline (61%), ampicillin (61%), and to a lesser extent, chloramphenicol (36%), ceftiofur (15%), gentamicin (9%), and nalidixic acid (4%), with more resistance observed among diagnostic isolates. Salmonella recovered from turkeys (n = 38) exhibited the highest rates of resistance, with 92% of isolates resistant to least one antimicrobial, and 58% resistant to > or =10 antimicrobials. Class 1 integrons were present in 51% of all isolates. Five integron associated resistance genes (aadA, aadB, pse-1, oxa-2 and dhfr) were identified. A total of 311 PFGE patterns were generated using XbaI, indicating a genetically diverse population. The largest PFGE cluster contained 146 isolates, including DT104 isolates obtained from all seven animal species. Results demonstrated a varied spectrum of antimicrobial resistance, including several multidrug resistant clonal groups, among S. Typhimurium and S. Typhimurium var. Copenhagen isolates recovered from both diagnostic and slaughter/processing samples.

Glycopeptide-intermediate Staphylococcus aureus: evaluation of a novel screening method and results of a survey of selected U.S. hospitals.

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

Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum beta-lactamase detection methods.

Extended-spectrum beta-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 > or =2 microg/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 > or =5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by > or =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 > or =32 microg/ml, suggesting either the presence of an AmpC-type beta-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a beta-lactamase with a pI of > or =8.3 suggestive of an AmpC-type beta-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 beta-lactamase bands, all of which had pIs of < or =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 beta-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained beta-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.