Evaluation of the Oxi/Ferm Tube System with Selected Gram-Negative Bacteria

The Oxi/Ferm test system was evaluated for accuracy and reliability for identification of nonfermentative and oxidase-positive fermentative bacteria by using 375 bacterial strains obtained from stock culture and clinical specimens. The Oxi/Ferm system is a compartmentalized tube containing eight media to provide nine biochemical test results. When combined with the oxidase test, the results corresponding to the positive reactions are totaled and the composite number is located in the coding manual to identify the organisms. The 375 isolates studied were evaluated for accuracy of identification, using both the original and revised code manuals. In comparison with the conventional media used, there was 100% correlation in tests for hydrogen sulfide and indole production, over 96% for nitrogen gas, arginine, and urease, over 92% for xylose and dextrose oxidation, and less than 90% for citrate utilization and dextrose fermentation. There was an overall accuracy in identification of 89.3% using the original manual, with accuracy revised slightly upward to 90.7% using the revised manual. There was 100% accuracy in identification with 44.0% of the strains tested (11 species) using the original manual and with 66.1% (16 species) using the revised manual. Thirteen of the 40 original misidentifications and 14 of 35 revised misidentifications resulted from failure to code and were unidentifiable by Oxi/Ferm. The remainder were incorrectly identified or could not be differentiated from closely related strains. Eleven strains of Alcaligenes odorans were correctly identified using the original code, whereas no code was provided in the revised manual. The Oxi/Ferm system is both simple and rapid and is satisfactory for identification of the more common isolates.     

Comparison of the Oxi/Ferm and N/F systems for identification of infrequently encountered nonfermentative and oxidase-positive fermentative bacilli.

The N/F system (Flow Laboratories, Inc. Rockville, Md) and the Oxi/Ferm Tube, (Roche Diagnostics, Div. Hoffman-La Roche, Inc. Nutley, N.J.) were evaluated in parallel for identification of infrequently encountered nonfermentative gram-negative bacilli and oxidase-positive, fermentative gram-negative bacilli selected from fresh clinical isolates and stock cultures. When compared with conventional media, the Flow N/F system correctly identified 97.7% (86 or 88) of all strains tested. No organisms were misidentified, but this system failed to identify two strains. Six supplemental tests were needed for the complete identification of 16 strains (18%); however, correct results were obtained within 48 h for 85% of the isolates. The Oxi/Ferm method correctly identified 87.5% of the isolates; 7% were incorrectly identified, and the method failed to identify five strains. Seventeen supplemental tests were required to identify 64 strains (73%). Complete identification within 48 h was obtained for 60% of the organisms tested.     

Accuracy and reproducibility of the Oxi/Ferm system in identifying a select group of unusual gram-negative bacilli.

The Oxi/Ferm (O/F) identification system was compared in a double-blind study to a conventional test battery for the characterization of 96 reference and clinical strains consisting of 83 nonfermentative and 13 oxidase-producing, fermentative gram-negative bacilli. The O/F tube and supplemental tests correctly identified 84% of the nonfermentative and 77% of the oxidase-producing, fermentative bacilli. However, when the supplemental tests were excluded and the biochemical profiles generated by all nine O/F tube reactions were examined, the profile accuracy reached 95% (79 of 83) for the nonfermentative and 93% (12 of 13) for oxidase-producing, fermentative bacilli. Seven of the nine O/F substrate reactions demonstrated less than or equal to 89% agreement with conventional reactions, whereas the urea and arginine reactions provided 82 and 85% agreement, respectively. Replicate O/F tests with six selected organisms demonstrated 97% identification reproducibility and 84% overall substrate reproducibility. The mean O/F identification time was 2.6 days as compared to 3.3 days for the conventional system. Although this study suggests that the O/F system is a convenient, rapid, and accurate alternative to conventional identification methods, several modifications are recommended.     

Identification of Pasteurella multocida and Pasteurella haemolytica by API 20E, Minitek, and Oxi/Ferm systems.

Fifty serotyped isolates each of Pasteurella multocida and Pasteurella haemolytica were tested on the API 20E strip (Analytab Products, Plainview, N.Y.), the Oxi/Ferm tube (Roche Diagnostics, Nutley, N.J.), and the Minitek system (BBL Microbiology Systems, Cockeysville, Md.). None of the rapid test systems reliable identified these organisms. With the API system, discrepancies between expected and actual results for the oxidase test and nitrate test frequently resulted in misidentification or no identification. The Minitek system misidentified 68% of the P. haemolytica isolates. The Minitek identification of Pasteurella depends on 100% positive xylose reactions, whereas only 56% of the P. haemolytica strains were positive for xylose fermentation. The Oxy/Ferm system, instead of giving a definitive identification, in most instances merely placed Pasteurella in a category of similar organisms.     

Comparison of the API 20E and Oxi/Ferm systems in identification of nonfermentative and oxidase-positive fermentative bacteria.

The API 20E and Oxi/Ferm systems were tested in parallel to identify nonfermentative bacteria and oxidase-positive fermentative bacteria. Test strains consisted of consecutive clinical isolates, with stock cultures used to supplement those species infrequently recovered. The two microsystems, as well as tubes of triple sugar iron, motility, cetrimide, and oxidative glucose media, were inoculated by each worker for each organism. Identification of each isolate was by the protocol of the manufacturers, with supplemental tests and flagella stains performed when necessary. Concurrent identification was undertaken with a conventional system against which the results of the two systems were compared for accuracy. There was a 95.3% accuracy in identification by the Oxi-Ferm system and 88.9% by the API system. Almost one-fourth of all identification attempts with the API required computer assistance, and most of these were for oxidase positive bacteria. Because of this, and because the API system showed greater accuracy in identification of the oxidase-negative bacteria, it seems best suited for identification of these organisms (P. maltophilia, A. anitratus, and A. lwoffi). The Oxi/Ferm system is technically less cumbersome than the API and is well suited for both groups of organisms.     

Evaluation of two test-kits — API and Oxi Ferm tube — for identification of oxidative-fermentative gram-negative rods

Two test-kits — API and Oxi Ferm tube — have been compared for accuracy in individual tests and for identification on the genus or species level with conventional biochemical tests on 154 oxidative-fermentative gram-negative rods. The two test systems were found to be reliable and permit identification of the clinically most significant oxidative-fermentative strains.     

Characteristics and biotypes of Pasteurella multocida isolated from humans.

Fifty-two isolates of Pasteurella (48 strains of Pasteurella multocida and 4 strains of atypical Pasteurella) were identified by conventional and commercial test systems. All strains fermented glucose, sucrose, and fructose in purple broth base (Difco Laboratories) with bromocresol purple as indicator, although the atypical Pasteurella produced fermentation reactions that were barely perceptible. Eleven different biotypes were identified by fermentation reactions in maltose, mannitol, xylose, sorbitol, and trehalose media. There was a correlation of biotypes to cat bites, with 61% of cat bite isolates falling into biotype A and B. A correlation of biotype and dog bite isolates was not seen. The choice of medium used for fermentation tests was critical as evidenced by the inability of the organisms to grow in a second commercially purchased preparation of purple broth base. The reliability of commercial test systems in identifying Pasteurella was 81% for Oxi/Ferm (Roche Diagnostics, Div. Hoffmann-La Roche, Inc., Nutley, N.J.), 68% for API (Analytab Products, Plainview, N.Y.), and 11% for Minitek (BBL Microbiology Systems, Cockeysville, MD.).     

Nonfermentative bacilli: evaluation of three systems for identification.

Three systems for the identification of nonfermentative bacilli were evaluated for their rapidity and accuracy of identification of 217 strains. Two of the systems, API 20E (API) and Oxi/Ferm tube (OxiF), are available as kits; the oxidative attack (OA) system is not commerically available. The overall accuracies of the OA, API, and OxiF systems were 91, 69, and 50%, respectively. Identification within 48 h was achieved for 98% of the strains by OA, for 50% by API, and for 18% by OxiF. Most of the organisms that were either misidentified or not identified by API and OxiF were those nonfermentative bacilli which are relatively more fastidious or rarely encountered or both. All three systems accurately identified nonfermentative bacilli commonly isolated at Olive View Medical Center, namely, Pseudomonas aeruginosa, Acinetobacter anitratus, Pseudomonas maltophilia, Acinetobacter lwoffi, saccharolytic flavobacteria (CDC IIb), moraxellae, Pseudomonas fluorescens, and Pseudomonas putida. The OA system identified 100% of the above organisms correctly, API identified 99.4%, and OxiF identified 99.3%. Since these organisms comprise 92% of the total number of nonfermentative bacilli isolated at Olive View Medical Center, we conclude that both API and OxiF may be useful alternatives to conventional methods, based on accuracy of identification alone. These two systems were considered substantially inferior to the OA system when both accuracy and rapidity of identification were taken into account.     

Identification of Gram-negative non-fermenters and oxidase-positive fermenters by the Oxi/Ferm Tube

Since the recent introduction of the Roche Oxi/Ferm Tube to the UK two identification schemes have been developed by the manufacturer for use with the kit. We evaluated the success of these two schemes in identifying 222 predominantly culture collection strains belonging to 45 taxa of non-fermenters and nine taxa of oxidase-positive fermenters. The strains were chosen to represent all the taxa included in the two identification schemes developed by the manufacturer and we have therefore been able to assess the overall success of identification by the two schemes. Since, however, our choice of strains does not reflect their incidence in clinical material, our identification rates are not necessarily those that might be obtained in a routine clinical laboratory. The most advanced identification scheme so far developed for the Oxi/Ferm Tube (CCIS System 1977-1432) allowed 62% of the 222 strains to be correctly identified although a disturbing feature was that more of the strains that were not correctly identified were incorrectly identified (24%) rather than not identified (14%); these figures represent an improvement over the earlier identification scheme (CCIS System 1976-621-74346) for which the corresponding figures were 56%, 32%, and 12%. CCIS System 1977-1432 seems likely to give a better performance in a routine clinical laboratory than in this study since for those taxa which, we would judge from the material sent to us for identification, are most commonly seen in a routine laboratory (Acinetobacter calcoaceticus, A. lwoffii, Pseudomonas aeruginosa, P. fluorescens, P. maltophilia, P. pseudoalcaligenes, and P. putida) 89% were correctly identified, none remained unidentified, and 11% were incorrectly identified. Thirty strains, each of a different taxon, were tested in triplicate to assess the reproducibility of reactions in the Oxi/Ferm Tube.     

Comparison of the AutoMicrobic system and a conventional tube system for identification of nonfermentative and oxidase-positive gram-negative bacilli

The AutoMicrobic system (AMS) Enterobacteriaceae-plus Biochemical Card was developed to identify a select group of 10 species of glucose-nonfermentative and oxidase-positive fermentative gram-negative bacilli. In this study, 159 nonenteric clinical isolates were identified by the AMS and conventional tube biochemicals based on E. O. King's (Centers for Disease Control) identification schema. The AMS properly identified 96.7% (117 of 121) of isolates whose taxa were included in the AMS data base. Of 38 isolates (94.7%) in which taxa were not included in the data base, 36 were correctly called unidentified organisms. A principal advantage of the AMS is the automated identification of frequently isolated nonenterics in a period of only 8 to 13 h. The AMS, with the use of the Enterobacteriaceae-plus Biochemical Card appears to be a rapid and accurate system for the identification of the most commonly isolated nonfermentative and oxidase-positive fermentative gram-negative bacilli.     

Comparison of the Minitek system with conventional methods for identification of nonfermentative and oxidase-positive fermentative gram-negative bacilli.

The Minitek system was compared with conventional test methods for identifying a wide range of nonfermentative and oxidase-positive fermentative gram-negative bacilli. A total of 230 isolates representing 33 species and biotypes were tested against 12 Minitek substrates and the corresponding conventional tube media. In addition, supplementary tests were included for 141 (61.3%) of the isolates. Overall, 88% of the positive reactions agreed, and negative reactions agreed 95.5%. Anaerobic dextrose, maltose, lactose, and citrate were responsible for 62.4% of the 93 discrepant positive reactions, and 51.8% of the 83 discrepant negative reactions involved the aerobic dextrose, nitrate, and citrate disks. Some discrepancies were related to specific organisms. The system and supplementary tests correctly identified 88.3% of the isolates to species level and 92.6% to correct genus. No particular organism or substrate was responsible for misidentification errors. In a test challenge with 19 selected organisms, 3 were incorrectly identified to species and 4 were misidentified to genus level. Most of the errors responsible for these incorect identifications were due to factors other than the Minitek disks and pointed out the importance of using appropriate supplemental data, and the need for a coding manual and a more enriched broth for certain types of isolates.     

Four methods for identification of gram-negative nonfermenting rods: organisms more commonly encountered in clinical specimens.

Four commercial kits, Oxi/Ferm (OF), API 20E (AP), Minitek (MT; BBL Microbiology Systems), and Flow N/F (NF), were evaluated, without additional tests, for identification of 258 gram-negative nonfermentative rods. OF and MT were read after 48 h of incubation, and AP and NF were read after both 24 and 48 h of incubation, respectively. Overall, OF correctly identified 51% of strains, with 46% as part (but not first) of a spectrum of identifications (SI), and 3% incorrect species identification. MT yielded 85% correct identification, with 15% SI. Of 126 glucose-positive strains, or those with greater than or equal to 3 positive AP reactions after 24 h, 60% were correctly identified, with 40% SI; incubation for an additional 24 h raised the rate of correct identification to 99%, with 1% SI. A total of 132 strains yield less than 3 positive AP reactions after 24 h and were identified after 48 h only; of these, 82% were correctly identified, with 17% SI and 1% incorrect species identification. NF correctly identified 79% of cultures after 24 h, with 21% SI; corresponding figures after an additional 24 h of incubation were 80% and 20%, respectively. All four commercial methods show promise; OF is easiest to inoculate, but requires extra tests for optimal identification. AP reliably identifies the majority of clinically important nonfermenters, with fairly good species identification of saccharolytic strains after 24 h. MT yields reliable identification of most nonfermenters and has the advantage of flexibility. NF is easy to inoculate, yields satisfactory identification rates, and may be read after 24 h of incubation.     

Comparison of four methods for identification of gram-negative non-fermenters: Organisms less commonly encountered in clinical specimens

Four commercial kits - Oxi/Ferm (OF), API 20E (AP), Minitek (MT), Flow N/F (NF) were evaluated, without additional tests, for identification of 105 opportunistic Gram-negative non-fermentative rods. OF correctly identified 42% of strains, with 35% as part (but not first) of a spectrum of identifications (SI) and 23% incorrect identification. MT yielded 75% correct identification, with 12% SI and 13% incorrect. AP correctly identified 64% of strains, with 26% SI, 10% incorrect. NF correctly speciated 70% of strains, with 24% SI, 6% incorrect. All 4 methods show deficiencies in identification of these rare but increasingly clinically encountered organisms. Addition of new tests/modification of existing ones would render these systems more capable of identifying this organisms group.     

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.     

Evaluation of the MicroScan rapid neg ID3 panel for identification of Enterobacteriaceae and some common gram-negative nonfermenters

The MicroScan Rapid Neg ID3 panel (Dade Behring, Inc., West Sacramento, Calif.) is designed for the identification of gram-negative bacilli. We evaluated its ability to accurately identify Enterobacteriaceae that are routinely encountered in a clinical laboratory and glucose nonfermenting gram-negative bacilli. Using 511 stock cultures that were maintained at -70 degrees C and passaged three times before use, we inoculated panels according to the manufacturer's instructions and processed them in a Walk/Away instrument using version 22.01 software. The time to identification was 2 h and 30 min. All panel identifications were compared to reference identifications previously determined by conventional tube biochemicals. At the end of the initial 2.5-h incubation period, 405 (79.3%) identifications were correct. An additional 49 (9.6%) isolates were correctly identified after required additional off-line biochemical tests were performed. Thus, at 24 h, 88.8% of the 511 strains tested were correctly identified. Twenty-two (4.3%) were identified to the genus level only. Twenty-six (5.1%) strains were misidentified. Because the system is based on fluorogenics, there are no conventional tests readily available with which to compare possibly incorrect reactions. Of the 28 Salmonella strains that were tested, 5 were incorrectly reported. The 21 remaining errors were scattered among the genera tested. Testing on nine strains gave a result of "no identification" (very rare biotype). The Rapid Neg ID3 panel in this study approached 89% accuracy for the identification of gram-negative organisms encountered in the hospital laboratory.     

Multicenter evaluation of the MicroScan Rapid Gram-Negative Identification Type 3 Panel.

The accuracy and performance of the revised MicroScan Rapid Gram-Negative Identification Type 3 Panel (Dade MicroScan Inc., West Sacramento, Calif.) were examined in a multicenter evaluation. The revised panel database includes data for 119 taxa covering a total of 150 species, with data for 12 new species added. Testing was performed in three phases: the efficacy, challenge, and reproducibility testing phases. A total of 405 fresh and stock gram-negative isolates comprising 54 species were tested in the efficacy phase; 96.8% of these species were identified correctly in comparison to the identification obtained either with the API 20E system (bioMérieux Vitek, Hazelwood, Mo.) or by the conventional tube method. The number of correctly identified isolates in the challenge phase, including new species added to the database, was 221 of 247, or 89.5%, in comparison to the number correctly identified by the conventional tube method. A total of 465 isolates were examined for intra- and interlaboratory identification reproducibility and gave an agreement of 464 of 465, or 99.8%. The overall reproducibility of each individual identification test or substrate was 14,373 of 14,384, or 99.9%. The new Rapid Gram-Negative Identification Type 3 Panel gave accurate and highly reproducible results in this multiple-laboratory evaluation.     

Premarket evaluation of IDS RapID SS/u system for identification of urine isolates.

A total of 170 fresh clinical urine isolates were tested with a premarket configuration of the RapID SS/u system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.), a qualitative micromethod for the identification of selected organisms commonly isolated from urine specimens. Results were compared with those obtained with conventional methods of identifying gram-positive isolates and with the AutoMicrobic system (Vitek Systems, Inc., Hazelwood, Mo.), utilizing Gram-Negative Identification cards for the identification of gram-negative rods. Organisms representing 12 taxa were included in the study. Of the 170 isolates, 163 (95.9%) were correctly identified. A total of 144 strains (84.7%) were correctly identified without additional testing, whereas 19 isolates (11.2%) required further testing. Seven isolates (4.1%) were incorrectly identified. The SS/u system required minimal hands-on time inoculate and interpret reactions. Discrepancies most often occurred with regard to misinterpretation of Escherichia coli and Enterobacter sp. as Citrobacter sp. The IDS RapID SS/u system may indeed prove valuable for the rapid manual identification of urine isolates.     

Evaluation of the API 20E system for identification of nonfermentative Gram-negative bacteria.

The API 20E system for Enterobacteriaceae, recently broadened to include identification of nonfermentative gram-negative bacteria, was evaluated and compared with the conventional method for complete identification of 221 nonfermenters, which were well distributed into 48 species or biotypes and included organisms not listed in the API 20E data base. The results of 16 tests common to both systems were in close agreement. The API 20E system correctly identified 71 (43%) of the 165 organisms included in the API 20E data base. However, almost 90% of Acinetobacter calcoaceticus, three species of Pseudomonas, and Bordetella bronchiseptica were correctly identified to species.     

Comparison of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometer to BD Phoenix automated microbiology system for identification of gram-negative bacilli

We compared the BD Phoenix automated microbiology system to the Bruker Biotyper (version 2.0) matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) system for identification of gram-negative bacilli, using biochemical testing and/or genetic sequencing to resolve discordant results. The BD Phoenix correctly identified 363 (83%) and 330 (75%) isolates to the genus and species level, respectively. The Bruker Biotyper correctly identified 408 (93%) and 360 (82%) isolates to the genus and species level, respectively. The 440 isolates were grouped into common (308) and infrequent (132) isolates in the clinical laboratory. For the 308 common isolates, the BD Phoenix and Bruker Biotyper correctly identified 294 (95%) and 296 (96%) of the isolates to the genus level, respectively. For species identification, the BD Phoenix and Bruker Biotyper correctly identified 93% of the common isolates (285 and 286, respectively). In contrast, for the 132 infrequent isolates, the Bruker Biotyper correctly identified 112 (85%) and 74 (56%) isolates to the genus and species level, respectively, compared to the BD Phoenix, which identified only 69 (52%) and 45 (34%) isolates to the genus and species level, respectively. Statistically, the Bruker Biotyper overall outperformed the BD Phoenix for identification of gram-negative bacilli to the genus (P < 0.0001) and species (P = 0.0005) level in this sample set. When isolates were categorized as common or infrequent isolates, there was statistically no difference between the instruments for identification of common gram-negative bacilli (P > 0.05). However, the Bruker Biotyper outperformed the BD Phoenix for identification of infrequently isolated gram-negative bacilli (P < 0.0001).     

Comparison of the automicrobic system with API, enterotube, micro-ID, micro-media systems, and conventional methods for identification of Enterobacteriaceae.

Identification of Enterobacteriaceae by the AutoMicrobic System Enterobacteriaceae Biochemical Card was evaluated. Recent clinical isolates of enteric gram-negative bacilli (192) and glucose nonfermenters (3) were identified by the AutoMicrobic System, Micro-Media Systems, Micro-ID, API, and Enterotube II in comparison with conventional methods. The AutoMicrobic System and Micro-Media Systems correctly identified 97% of the organisms tested. Micro-ID, API, and Enterotube II correctly identified 94, 92, and 84% of the organisms, respectively. In addition to a high degree of identification accuracy, the AutoMicrobic System was convenient to operate and produced identification results in 8 h. Operation of the AutoMicrobic System also required minimal personnel time because it automatically monitored and interpreted the biochemical reactions and reported organism identifications. The AutoMicrobic System appears to be an efficient and accurate system for the identification of Enterobacteriaceae.