Rapid method for identification of gram-negative, nonfermentative bacilli.

A rapid system (OA), based on oxidative attack of substrates, was developed for identification of gram-negative, nonfermentative bacillia (NFB). One hundred and twelve strains of NFB from 25 species (representing the genera Pseudomonas, Alcaligenes, Acinetobacter, Bordetella, Flavobacterium, Moraxella, and Xanthomonas) were assayed by OA, buffered single substrate, and oxidative/fermentative methods. The 38 substrates consisted of salts of organic acids, nitrogen-containing compounds, alcohols, and carbohydrates. Ninety-four percent of the test strains were identified by the OA method in 24 h, and 99% were identifiable in 48 h. Reproducibility was 99%. Correlation with buffered single substrate was 98% (all substrates) and 90% with the oxidative/fermentative method (carbohydrates only). Biochemical profiles of all strains are presented, as well as tables showing the most useful tests for identification.     

Rapid identification of gram-negative bacilli from blood cultures

Blood-culture broths showing macroscopic or radiometric evidence of growth of gram-negative bacilli were examined by a rapid automated bacterial identification system. A differential centrifugation technique was developed to prepare suitable inocula. The identification results obtained were confirmed by the API 20E method, with single colonies of the strains isolated 24 h later. Of 90 organisms tested, seven did not give the same identification by the two systems. With the rapid automated technique a presumptive identification of gram-negative bacilli can be made 24 h earlier than by more conventional methods.     

Collaborative evaluation of the Abbott Avantage system for identification of frequently isolated nonfermentative or oxidase-positive gram-negative bacilli

The capability of the Abbott Avantage system to identify 10 species of commonly isolated glucose nonfermentative or oxidase-positive gram-negative bacilli in a 5-h test period was evaluated in a collaborative study. The Avantage nonenteric data base uses 20 biochemical test reactions performed in an expanded Abbott bacterial identification cartridge plus the results of a manual oxidase test. The species included in the Avantage data base are Acinetobacter anitratus, Acinetobacter Iwoffi, Aeromonas hydrophila, Flavobacterium meningosepticum-IIb group, Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas fluorescens-putida group, Pseudomonas maltophilia, Pasteurella multocida, and Plesiomonas shigelloides. The collaborative study included the testing of 200 coded challenge strains in all three laboratories and the subsequent testing of an additional group of 100 to 200 clinical isolates recovered independently by each laboratory. Reference identifications for all isolates were determined by conventional biochemical test reactions. The overall accuracy of identification of the coded challenge strains for the three laboratories was 97%, whereas 95% of 437 clinical isolates and selected stock cultures of clinical derivation were identified correctly.     

Rapid automated identification of gram-negative bacilli from blood cultures with the AutoMicrobic system.

Automated identification of gram-negative bacilli directly from blood culture bottles by using the AutoMicrobic System (AMS) was evaluated with a modified procedure for the AMS Enterobacteriaceae-plus nonfermenter identification card. A total of 150 strains were tested (44 clinical and 106 seeded) and compared with a conventional identification procedure. These strains included 107 Enterobacteriaceae and 43 oxidase-positive or glucose-nonfermenting, or both, organisms. AMS identifications on one of these strains were not interpretable owing to equal probability AMS identification values. Of the remaining 149 strains, 138 (92.6%) were correctly identified within 8 to 13 h of the first reading. Of 69 identifications analyzed after 6 h of incubation, 91% were correct. This procedure was found to be rapid, convenient, and nonlabor intensive and is recommended for presumptive identification of gram-negative bacilli in blood cultures.     

autoSCAN-4 system for identification of gram-negative bacilli.

A production model of the autoSCAN-4 system (American MicroScan, Inc., Mahwah, N.J.) was tested with not more than 11 strains each of 73 groups or species of gram-negative bacilli from various Centers for Disease Control culture collections. The strains included typical and atypical strains of enteric fermenters, nonenteric fermenters, and nonfermenters. The autoSCAN-4 system identified 95.3% of all 405 cultures accurately: 95.4% of 307 members of the family Enterobacteriaceae, 96.6% of 29 nonenteric fermenters, and 94.2% of 69 nonfermenters. Manual readings of the same trays provided essentially the same results, with a maximum change of only +1.6% identification accuracy of members of the Enterobacteriaceae. These data were obtained by all required additional tests, including serology and computer consultation when indicated. Only 19 of the cultures tested were misidentified. These were distributed randomly throughout the various groups and species except that Edwardsiella tarda was usually missed because of poor H2S reactions in the test medium. Of six Yersinia enterocolitica isolates, two were not identified. Only one nonenteric fermenter, a Pasteurella sp., and four nonfermenters (three Pseudomonas sp. and one Centers for Disease Control group Ve-2) were misidentified.     

Evaluation of the new Vitek 2 GN card for the identification of gram-negative bacilli frequently encountered in clinical laboratories

The new Vitek 2 GN card (bioMérieux, Marcy-l'Etoile, France) was developed for better identification of fermenting and nonfermenting bacilli. This new card allows the identification of 159 taxa. A total of 426 isolates (331 fermenting and 95 nonfermenting gram-negative bacilli) belonging to 70 taxa covered by the database were evaluated. All isolates were identified in parallel with the ID 32 GN, the API 20E, and the API 20NE methods. The system correctly identified 97.4% (n=415) of the strains. Only 2.1% (n=9) needed additional testing. One strain (0.25%) was misidentified (Klebsiella pneumoniae subsp. pneumoniae), and another one (0.25%) was not identified (Morganella morganii subsp. morganii). The new GN card gives more accurate identifications overall for gram-negative bacilli when compared to the systems described in other similar studies. This study was presented in part at the 104th General Meeting of the American Society for Microbiology, New Orleans, Louisiana, 2004, Abstract C-180.     

Rapid identification and antimicrobial susceptibility testing of gram-negative bacilli from blood cultures by the AutoMicrobic system.

A procedure was developed which allows direct identification and antimicrobial susceptibility testing of fermentative and nonfermentative gram-negative bacilli from positive blood cultures. A 10-ml sample was removed from turbid blood culture bottles, and the bacteria were washed and concentrated by centrifugation. The bacterial pellet was used to inoculate an Enterobacteriaceae Plus Identification Card and a Gram-Negative General Susceptibility Card of the AutoMicrobic system. Results with these cards were compared with results obtained with standard technique for 196 blood cultures seeded with recent clinical isolates. Identification of most cultures was available in 8 h, whereas the antimicrobial susceptibility results were available in an average of 4.7 h for all organisms. Direct identification was correct for 95% of the cultures, whereas the antimicrobial susceptibility data had an average agreement of 87% with 3.8% very major and 1.4% major errors. In using this procedure it was possible to provide accurate preliminary identification and results of antimicrobial susceptibility tests for gram-negative bacilli on the same day that a blood culture was determined to be positive.     

Use of casein,tyrosine, and hypoxanthine in the identification of nonfermentative gram-negative bacilli

Four hundred and five (405) strains of nonfermentative gram-negative bacilli (NFB) were identified using conventional tests. The NFB studied include 323 originating from clinical specimens, 37 from environmental samples, and 45 authentic strains from other workers and from our culture collections. All these strains were studied for their ability to degradate casein, tyrosine, and hypoxanthine. It was found that these tests are useful in identifying NFB within 24 to 48 h when used with other tests.     

Evaluation of a new identification system, Crystal Enteric/Non-Fermenter, for gram-negative bacilli.

A total of 505 fermentative and 201 nonfermentative gram-negative bacilli, identified by conventional methods, were tested by the Crystal Enteric/Non-Fermenter ID kit and by the API 20E or API 20NE identification system. The overall correct results for fermenters were 92.9% by the Crystal kit and 89.1% by the API 20E system. The false identifications (genus and species incorrect) accounted for 3.1 and 7.1% for the Crystal and API systems, respectively. For nonfermenters, figures for correct identifications by the two systems were comparable (Crystal, 75.9%; API 20NE, 75.3%) while the API 20NE system gave twice as many incorrect results (13.8%) as Crystal (6.3%); however, Crystal failed to precisely identify several species included in a "miscellaneous" group. The Crystal Enteric/Non-Fermenter system is an easy-to-use kit which compares favorably with other commercial systems.     

Computer aided identification of eugonic gram-negative bacilli: Clustering for bacteriological screening

A system based on pattern recognition in terms of operationally meaningful criteria has been designed for the identification of eugonic gram-negative bacilli. It includes the selection of a battery of tests to be used as a screening. The results of these tests place each isolate in any one of a number of clusters. A second set of tests is used to identify the isolate as one of the species in the cluster. The system can identify most common species economically and can be used in clinical laboratories not equipped with computers.     

Evaluation of the AutoMicrobic system for identification and susceptibility testing of gram-negative bacilli

The AutoMicrobic system (AMS) (Vitek Systems, Inc., Hazelwood, Mo.) was compared with the API-20E system for the identification of gram-negative bacilli by using 380 stock clinical isolates and 377 immediately encountered fresh clinical isolates. For the stock isolates, with Enterobacteriaceae-Plus Biochemical Cards and automated interpretation, 364 (95.8%) were in agreement to the species level. For the fresh clinical isolates, agreement at the genus and species levels was 89.7 and 85.9%, respectively, when Enterobacteriaceae-Plus Cards were interpreted by the AMS. Manual interpretation of Enterobacteriaceae-Plus Biochemical Cards improved species level agreement to 91.0%. Subsequent retesting of all discrepant isolates with the Gram-Negative Identification Card resulted in significant improvement of results, and for the stock and fresh clinical isolates, species level agreement was 98.7 and 97.3%, respectively. AMS susceptibility testing was evaluated by comparing ampicillin and cephalothin MICs determined in parallel by AMS and a reference broth microdilution test for stock isolates, and by comparison of AMS and standardized disk agar diffusion test results for fresh clinical isolates. For the stock isolates, AMS mean integer MICs approximated microdilution mean integer MICs with AMS, providing excellent MIC replicability. For ampicillin and cephalothin, 50 and 46.8%, respectively, of AMS integer MICs were within +/- 1 microgram/ml of the reference values, and 89.3 and 63.1% of AMS integer MICs were within +/- 2 micrograms/ml of the reference values. For the fresh clinical isolates, AMS and reference results were in disagreement for 4.5% of the antimicrobial agents tested, with 2.3% as a combination of "major" and "very major" errors.     

Evaluation of a commercial automated system for the identification of gram-negative enteric bacilli

A total of 908 distinct clinical isolates and 60 reference strains of aerobic gram-negative bacilli were identified by our own in-house biochemical identification system (RHH) and by a commercial automated system (Mastascan Colour). Overall, both systems performed well in the identification of routine isolates of aerobic gram-negative bacilli, with only six discrepancies between the two systems. These six organisms were species infrequently encountered in the clinical microbiology laboratory. Of the 60 reference strains, many of which were biochemically atypical, the RHH system was unable to identify one and mis-identified two others. The commercial system was unable to identify one strain and misidentified five others. Both systems were inexpensive in terms of consumable materials, and the commercial system was compatible with the routine work of the department.     

Collaborative clinical evaluation of the Autobac IDX system for identification of gram-negative bacilli

The Autobac IDX system (General Diagnostics, Warner-Lambert Co., Morris Plains, N.J.) for rapid, semiautomated identification of gram-negative bacilli was compared with the identification methods in routine use in four laboratories. The study included 1,515 organisms representing 30 species of enteric and nonenteric bacteria. Discrepancies between the results of the IDX system and routine methods were resolved by classical biochemical testing at a reference center. Overall, 98% of the organisms were correctly identified by the routine methods, and 93% were correctly identified by the IDX systems. After adjustment for frequency of clinical occurrence of the organisms tested, the IDX system performed with 95% accuracy. Results with the IDX system were available in 3 to 6 h. Results with the comparative methods were available in 4 to 48 h. A wide variety of organisms, including oxidase positive, oxidase negative, fermentative, and nonfermentative, were identified by a single system by using Autobac. Three or more systems were required to identify the 30 species by the comparative methods. Overall, the results indicate the Autobac IDX system is useful for the rapid identification of enteric and nonfermentative gram-negative bacilli.     

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.     

Accuracy of a rapid carbohydrate oxidation microtube method for identification of nonfermentative gram-negative bacilli

A rapid carbohydrate oxidation microtube system (Carr Microbiologicals, Wichita, Kans.), designed for detecting the saccharolytic activity of gram-negative, nonfermenting bacilli, was evaluated and compared with the conventional oxidation-fermentation method. The oxidation of glucose, maltose, lactose, and xylose was tested with 430 strains of Pseudomonas, Acinetobacter, Achromobacter, Alcaligenes, Moraxella, Flavobacterium, and Bordetella species. More than 95% of the isolates tested gave correct oxidation reactions within 4 h in the rapid carbohydrate oxidation microtubes, whereas oxidation-fermentation media required 24 h to achieve the same sensitivity. The microtube system was found to be simple, accurate, rapid, and economical.