Comparison of disk diffusion, VITEK 2, and broth microdilution antimicrobial susceptibility test results for unusual species of Enterobacteriaceae.

We compared the antimicrobial susceptibility testing results generated by disk diffusion and the VITEK 2 automated system with the results of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) reference method for 61 isolates of unusual species of Enterobacteriaceae. The isolates represented 15 genera and 26 different species, including Buttiauxella, Cedecea, Kluyvera, Leminorella, and Yokenella. Antimicrobial agents included aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins, and trimethoprim-sulfamethoxazole. CLSI interpretative criteria for Enterobacteriaceae were used. Of the 12 drugs tested by BMD and disk diffusion, 10 showed >95% categorical agreement (CA). CA was lower for ampicillin (80.3%) and cefazolin (77.0%). There were 3 very major errors (all with cefazolin), 1 major error (also with cefazolin), and 26 minor errors. Of the 40 isolates (representing 12 species) that could be identified with the VITEK 2 database, 36 were identified correctly to species level, 1 was identified to genus level only, and 3 were reported as unidentified. VITEK 2 generated MIC results for 42 (68.8%) of 61 isolates, but categorical interpretations (susceptible, intermediate, and resistant) were provided for only 22. For the 17 drugs tested by both BMD and VITEK 2, essential agreement ranged from 80.9 to 100% and CA ranged from 68.2% (ampicillin) to 100%; thirteen drugs exhibited 100% CA. In summary, disk diffusion provides a reliable alternative to BMD for testing of unusual Enterobacteriaceae, some of which cannot be tested, or produce incorrect results, by automated methods.

Evaluation of the Phoenix 100 ID/AST system and NID panel for identification of Enterobacteriaceae, Vibrionaceae, and commonly isolated nonenteric gram-negative bacilli.

The Phoenix 100 ID/AST system (Becton Dickinson Co., Sparks, Md.) is an automated system for the identification and antimicrobial susceptibility testing of bacterial isolates. This system with its negative identification (NID) panel was evaluated for its accuracy in the identification of 507 isolates of the family Enterobacteriaceae, 57 other nonenteric gram-negative isolates that are commonly isolated in clinical microbiology laboratories, and 138 isolates of the family Vibrionaceae. All of the isolates had been characterized by using approximately 48 conventional tube biochemicals. Of the 507 isolates of the Enterobacteriaceae, 456 (89.9%) were correctly identified to the genus and species levels. The five isolates of Proteus penneri required an off-line indole test, as suggested by the system to differentiate them from Proteus vulgaris. The identifications of 20 (3.9%) isolates were correct to the genus level but incorrect at the species level. Two (0.4%) isolates were reported as "no identification." Misidentifications to the genus and species levels occurred for 29 (5.7%) isolates of the Enterobacteriaceae. These incorrect identifications were spread over 14 different genera. The most common error was the misidentification of Salmonella species. The shortest time for a correct identification was 2 h 8 min. The longest time was 12 h 27 min, for the identification of a Serratia marcescens isolate. Of the 57 isolates of nonenteric gram-negative bacilli (Acinetobacter, Aeromonas, Burkholderia, Plesiomonas, Pseudomonas, and Stenotrophomonas spp.), 48 (84.2%) were correctly identified to the genus and species levels and 7 (12.3%) were correctly identified to the genus level but not to the species level. The average time for a correct identification was 5 h 11 min. Of the Vibrionaceae spp., 123 (89.1%) were correctly identified at the end of the initial incubation period, which averaged 4 h. Based on the findings of this study, the Phoenix 100 ID/AST system NID panel falls short of being an acceptable new method for the identification of the Enterobacteriaceae, Vibrionaceae, and gram-negative nonenteric isolates that are commonly encountered in many hospital microbiology laboratories.

Manual and automated instrumentation for identification of Enterobacteriaceae and other aerobic gram-negative bacilli.

Identification of gram-negative bacilli, both enteric and nonenteric, by conventional methods is not realistic for clinical microbiology laboratories performing routine cultures in today's world. The use of commercial kits, either manual or automated, to identify these organisms is a common practice. The advent of rapid or "spot" testing has eliminated the need for some commonly isolated organisms to be identified with the systems approach. Commercially available systems provide more in-depth identification to the species level as well as detect new and unusual strains. The answers obtained from these systems may not always be correct and must be interpreted with caution. The patient demographics, laboratory workload and work flow, and technologist's skill levels should dictate the system of choice. Cost considerations introduce another variable into the equation affecting choice. Each system has its own strengths and weaknesses, and each laboratory must decide on the level of sophistication that fulfills its particular needs.

Photorhabdus asymbiotica, a pathogen emerging on two continents that proves that there is no substitute for a well-trained clinical microbiologist.

A 54-year-old ranch hand presented to the emergency room with an alleged spider bite and multiple abscesses. Both wound and blood cultures grew Photorhabdus asymbiotica, an enteric gram-negative rod that was initially misidentified by the hospital's rapid identification system. Clinical laboratories should be aware of the limitations of their rapid identification systems and always use them as an adjunct to analysis of morphological and phenotypic traits.

Ability of the MicroScan rapid gram-negative ID type 3 panel to identify nonenteric glucose-fermenting and nonfermenting gram-negative bacilli.

The MicroScan Rapid Neg ID3 panel is designed for the identification of Enterobacteriaceae and nonenteric glucose-fermenting and nonfermenting gram-negative bacilli. We evaluated this panel for its ability to identify gram-negative non-Enterobacteriaceae bacteria. A total of 134 strains, representing 26 genera and 42 species, were taken from storage at -70(o)C, passaged three times before testing, and inoculated into the panels according to the manufacturer's directions before being inserted into a Walk/Away 96 instrument loaded with version 22.28 software. At the end of the initial 2.5-h incubation period, 89 isolates (66.4%) were correctly identified at a probability level of > or =85%. After additional testing recommended by the manufacturer was completed, another 11 isolates (8.2%) were correctly identified at probability levels of > or =85%. Twenty-five (18.7%) isolates were correctly identified after additional testing, but the probability levels were less than 85%. Two isolates were unidentified, and seven (5.2%) were incorrectly identified. The seven misidentified strains were not concentrated in any one genus. With an accuracy approaching 75%, this product may be used for the identification of the commonly isolated non-Enterobacteriaceae bacteria but may present problems in identification of other non-glucose-fermenting gram-negative bacilli.

Evaluation of the Vitek 2 ID-GNB assay for identification of members of the family Enterobacteriaceae and other nonenteric gram-negative bacilli and comparison with the Vitek GNI+ card.

We evaluated the Vitek 2 ID-GNB identification card (bioMérieux, Inc., Durham, N.C.) for its ability to identify members of the family Enterobacteriaceae and other gram-negative bacilli that are isolated in clinical microbiology laboratories. Using 482 enteric stock cultures and 103 strains of oxidase-positive, gram-negative glucose-fermenting and nonfermenting bacilli that were maintained at -70 degrees C and passaged three times before use, we inoculated cards according to the manufacturer's directions and processed them in a Vitek 2 instrument using version VT2-R02.03 software. All panel identifications were compared to reference identifications previously confirmed by conventional tube biochemical assays. At the end of the initial 3-h incubation period, the Vitek 2 instrument demonstrated an accuracy of 93.0% for the identification of enteric strains; 414 (85.9%) were correctly identified at probability levels ranging from excellent to good, and an additional 34 (7.1%) strains were correctly identified but at a low level of discrimination. Nineteen (3.9%) strains were unidentified, and 15 (3.1%) were misidentified. The 19 unidentified strains were scattered among 10 genera. Three of the 15 misidentified strains were lactose-positive Salmonella spp. and were identified as Escherichia coli; another was a lactose-positive, malonate-negative Salmonella enterica subsp. arizonae strain that was identified as E. coli. Of the 103 glucose-fermenting and nonfermenting nonenteric strains, 88 (85.4%) were correctly identified at probability levels ranging from excellent to good, and 10 (9.7%) were correctly identified, but at a low level of discrimination, for a total of 95.1% accuracy with this group. Two strains were unidentified and three were misidentified. The errors occurred for strains in three different genera. With the increased hands-off approach of the Vitek 2 instrument and accuracies of 93% for the identification of enteric organisms and 95.1% for the identification of nonenteric organisms with the ID-GNB card, use of this product presents an acceptable method for the identification of most gram-negative organisms commonly isolated in the clinical laboratory. A comparison of these results to those obtained by testing 454 of the same strains with the Vitek GNI+ card revealed no significant difference in the abilities of the two cards to identify these organisms accurately.

Accuracy of six commercially available systems for identification of members of the family vibrionaceae.

Six commercially available bacterial identification products were tested with Vibrio alginolyticus (12 strains), V. cholerae (30 strains), Photobacterium (Vibrio) damselae (10 strains), V. fluvialis (10 strains), V. furnissii (4 strains), V. hollisae (10 strains), V. metschnikovii (9 strains), V. mimicus (10 strains), V. parahaemolyticus (30 strains), and V. vulnificus (10 strains) to determine the accuracy of each system for identification. The products included API 20E, Crystal E/NF, MicroScan Neg ID2 and Rapid Neg ID3, and Vitek GNI+ and ID-GNB. Each product was tested only with those species that were listed in its database. Overall, the systems correctly identified 63.9, 80.9, 63.1, 73.6, 73.5, and 77.7% of the isolates to species level, respectively. Error rates ranged from 0.8% for the API 20E to 10.4% for the Rapid Neg ID3. The API 20E gave "no identification" for 13.1% of the isolates, while the Neg ID2, GNI+, ID-GNB, and Crystal were unable to identify 1.8, 2.9, 5.0, and 6.9%, respectively. For V. cholerae, specifically, accuracy ranged from 50.0 to 96.7%, with the API 20E having the worst performance and Crystal having the best. V. fluvialis presented the biggest challenge for the API 20E and the GNI+, with probabilities averaging 10%, while V. mimicus was a major problem with the Crystal E/NF, which identified none of the strains correctly. With the Neg ID2, correct answers were often obtained only after a modified inoculation of the panel with a bacterial suspension prepared with 0.85% NaCl. Additional tests required for identification often included growth in the absence of NaCl, which is not readily available in most clinical laboratories. The only product to correctly identify at least 90% of V. cholerae strains was the Crystal E/NF, and only three of the six products, the API 20E and both of the Vitek cards, correctly identified more than 90% of the V. parahaemolyticus strains. Thus, extreme care must be taken in the interpretation of answers from these six commercially available systems for the identification of Vibrio species.

Vancomycin-resistant Staphylococcus aureus isolate from a patient in Pennsylvania.

A vancomycin-resistant Staphylococcus aureus (VRSA) isolate was obtained from a patient in Pennsylvania in September 2002. Species identification was confirmed by standard biochemical tests and analysis of 16S ribosomal DNA, gyrA, and gyrB sequences; all of the results were consistent with the S. aureus identification. The MICs of a variety of antimicrobial agents were determined by broth microdilution and macrodilution methods following National Committee for Clinical Laboratory Standards (NCCLS) guidelines. The isolate was resistant to vancomycin (MIC = 32 micro g/ml), aminoglycosides, beta-lactams, fluoroquinolones, macrolides, and tetracycline, but it was susceptible to linezolid, minocycline, quinupristin-dalfopristin, rifampin, teicoplanin, and trimethoprim-sulfamethoxazole. The isolate, which was originally detected by using disk diffusion and a vancomycin agar screen plate, was vancomycin susceptible by automated susceptibility testing methods. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA indicated that the isolate belonged to the USA100 lineage (also known as the New York/Japan clone), the most common staphylococcal PFGE type found in hospitals in the United States. The VRSA isolate contained two plasmids of 120 and 4 kb and was positive for mecA and vanA by PCR amplification. The vanA sequence was identical to the vanA sequence present in Tn1546. A DNA probe for vanA hybridized to the 120-kb plasmid. This is the second VRSA isolate reported in the United States.