Evaluation of the Modified Micro-ID System for Identification

JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1979, p. 454-458 0095-1 137/79/10-0454/05$02.00/0 Vol. 10, No. 4 Evaluation of the Modified Micro-ID System for Identification of Enterobacteriaceae WILLIAM J. BUESCHING,' DWANE L. RHODEN,2 ANN 0. ESAIAS,2 PETER B. SMITH,2 AND JOHN A. WASHINGTON II'* Section of Clinical Microbiology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901,1 and Center for Disease Control, Atlanta, Georgia 303332 Received for publication 30 July 1979 Micro-ID is a system designed to identify the Enterobacteriaceae by utilizing reagent-impregnated disks for 15 biochemical tests. Since its initial evaluations, the system has undergone modification in formulation and in its computer data base. In a dual-center evaluation, 306 isolates of Enterobacteriaceae were tested: 145 common and typical isolates at the Mayo Clinic and 161 unusual or atypical isolates at the Center for Disease Control. Each laboratory also exchanged 50 cultures to test the system's reproducibility. Micro-ID correctly identified 142 (98%) of the common clinical isolates and 123 (76%) of the unusual or atypical organisms. However, in this latter group, three species tested were not in the system's data base. When these organisms were deleted from the analysis, 138 of 146 (95%) of the unusual or atypical isolates were correctly identified. Analysis of the 100 isolates identified in duplicate revealed 93% reproducibility of genus and species identification and 62% reproducibility of octal code numbers. Of the 31 strains with the same identification but different code numbers, 74% differed in only one biochemical test. In many laboratories, a majority of the work load involves the isolation and identification of members of the family Enterobacteriaceae. These organisms are often the causative agents of severe and life-threatening infections. Thus, the rapid and accurate identification of enteric pathogens by the clinical microbiology laboratory is of some importance. A new biochemical identification kit, the Micro-ID system (General Diagnostics, Morris Plains, N.J.), provides identification of the Enterobacteriaceae 4 h after inoculation. This system utilizes reagent-impregnated filter paper disks for 15 biochemical tests. A five-digit octal code number is generated from each set of biochemical reactions, and an identification is derived from a code book. Since its initial evaluation (1), the system has undergone extensive modification. The 15 biochemical tests are the same, but selected changes in substrate/indicator ratios have been made. Further, the system's computer data base has been modified. It was the purpose of this work to determine the ability of the modified Micro-ID system to identify both common and unusual isolates of Enterobacteriaceae and to obtain an estimate of the reproducibility of the system. MATERIALS AND METHODS Cultures tested. A total of 306 stock cultures representing 34 species of Enterobacteriaceae were 454 tested: 145 common and typical isolates at the Mayo Clinic and 161 atypical or unusual isolates at the Center for Disease Control. Cultures were maintained at room temperature on tryptic soy agar slants overlaid with sterile mineral oil or in tryptic soy agar stabs. To test the system's reproducibility, each laboratory exchanged 50 cultures. Thus, 100 isolates were identified in duplicate. All organisms were assigned a number, and the identity of no isolate was known until testing was complete. Identification methods. The stock cultures tested at the Mayo Clinic had been previously identified by means of classical tests (3, 5) and the API 20E system (Analytab Products Inc.). The API 20E strips were inoculated, incubated, and read according to the manufacturer's instructions. Since the performance of this system has been well characterized (2, 6, 7, 8), further evaluation in this study was not attempted. Those isolates tested at the Center for Disease Control had been previously identified by using conventional biochemical tubed media (4, 6). The nomenclature used in this study was based on the taxonomic systems of Edwards and Ewing (4), Ewing and Martin (6), and Brenner et al. (2). Micro-ID system. The Micro-ID system consists of a molded styrene tray containing 15 reaction wells and a hinged cover. The first five wells contain a substrate disk and an indicator disk; the remaining 10 wells contain a single combination substrate/indicator disk. The 15 biochemical tests used by the system include: Voges-Proskauer, nitrate, phenylalanine deaminase, hydrogen sulfide (H2S), indole, ornithine decarboxylase, lysine decarboxylase, malonate, urea, es-

VOL. 10, 1979 culin, o-nitrophenyl-fi-d-galactopyranoside, arabinose, adonitol, inositol, and sorbitol. Organisms tested were taken from MacConkey or eosin-methylene blue agar plates after overnight incubation at 35 C. Each organism was suspended in isotonic saline to a turbidity of a no. 2 McFarland standard. Each Micro-ID strip was labeled, and 0.2 ml of the organism suspension was pipetted into each inoculation well at the top of the unit. The cover was closed, and the strip was placed in an upright position and gently tapped, ensuring that the organism suspension was in contact with all substrate disks. Each strip was incubated in an upright position at 35 C for 4 h in an atmosphere without CO2. After incubation, two drops (0.1 ml) of 20% KOH were added to the inoculation well of the Voges-Proskauer test chamber, and the strip was set upright to allow the KOH to flow down into the test solution. The strip was then rotated about 90 so that the upper indicator disks in the first five wells became wet. The strip was again set upright and the reactions were read according to the color guide provided with the kit. The reactions were recorded on data sheets, and the fivedigit octal number was calculated. Each organism was identified by using the Micro-ID identification manual, edition 09178. When identification discrepancies occurred, the organism was tested again with the Micro-ID system. With occasional isolates, the biochemical reaction patterns were insufficient to provide a species identification. In such cases, additional biochemical tests, as suggested in the Micro-ID identification manual, were performed with conventional biochemical media. Organisms identified as species of Salnonella or Shigella were confirmed by serology (4), as suggested in the identification manual. Certain biochemical test results as determined by Micro-ID and other systems differ because of differences in formulation (1). Since the revised Micro-ID computer data base was developed independently of any other system, it is consistent within itself. Therefore, a test-by-test comparison of individual reactions was not attempted in this study. This evaluation was designed to determine the overall accuracy of identification and reproducibility of the Micro-ID system. RESULTS Overall identification. A total of 306 isolates of Enterobacteriaceae were tested: 145 common and typical isolates at the Mayo Clinic and 161 atypical or unusual isolates at the Center for Disease Control. The Micro-ID system correctly identified 142 (97.9%) of the common clinical isolates and 123 (76.4%) of the atypical or unusual organisms (Table 1). Of these atypical or unusual isolates, the biochemical reaction patterns of three species, Citrobacter amalonaticus (malonate- and H2S-negative C. freundii), Enterobacter gergoviae (sp. nov.; 2), and Pectobacterium (Erwinia-like), were not included in the system's data base. When these organisms were deleted from the analysis, 138 of 146 (94.5%) of the unusual organisms were correctly identified. Deletion of these organisms from the EVALUATION OF MODIFIED MICRO-ID SYSTEM 455 analysis also increased the overall identification rate from 86.6 to 96.2%. Thirty-two of the isolates tested were identified only to the genus level, and additional biochemical tests were required for identification to species level. The performance of Micro-ID was comparable to that of API 20E for the identification of common clinical isolates (Table 2). The former system correctly identified 97.9% of the organisms tested, the latter system correctly identified 99.3%. Of the 161 unusual or atypical organisms tested, 154 (95.6%) were correctly identified with conventional biochemical media as compared to 123 (77%) with the Micro-ID sys- TABLE 1. Summary of organism identification by Micro-ID No. requiring No. No. cor- additional Organism classification tested recta biochemical testsa Common and typical 145 142 (97.9) 6 (4.1) Unusual and atypical 161 123 (76.4) 26 (16.1) Total.... 306 265 (86.6) 32 (10.4) Common and typical 145 142 (97.9) 6 (4.1) Unusual and atypicalrevised. 146 138 (94.5) 25 (17.1) Total.291 280 (96.2) 31 (10.6) b Analysis excluding Citrobacter amnalonaticus, Enterobacter gergoviae, and Pectobacterium. TABLE 2. Comparison of identifications by Micro- ID and API 20E No. No. conrect No. correct Organism tested by API 20E" by micro-id' Citrobacter diversus 9 9 (100) 9 (100) Citrobacter freundii 9 9 (100) 9 (100) Enterobacter aerogenes... 13 13 (100) 12 (92.3) Enterobacter cloacae. 12 12 (100) 12 (100) Escherichia coli... 15 15 (100) 15 (100) Klebsiella pneumoniae... 9 9 (100) 9 (100) Klebsiella oxytoca (K. pneumoniae) 4 3 (75) 4 (100) Morganella morganii.. 12 12 (100) 11(91.7) Proteus mirabilis 12 12 (100) 12 (100) Proteus vulgaris... 13 13 (100) 12 (92.3) Salmonella... 13 13 (100) 13 (100) Salmonella typhi 1 1 (100) 1 (100) Serratia marcescens 12 12 (100) 12 (100) Shigella sonnei... 3 3 (100) 3 (100) Shigella species 8 8 (100) 8 (100) Avg (99.3) (97.9)..

456 BUESCHING ET AL. tem. However, deletion of the species not included in the Micro-ID data base resulted in correct identification of 94.5% of these organisms (Table 3). Misidentifications. A list of organisms misidentified by the Micro-ID system is presented in Table 4. Identification errors involved a variety of organisms with no single genus or species predominating. In several instances, identification errors appeared to be due to the insensitivity of certain of the Micro-ID biochemical tests. For example, two of three strains of Citrobacter freundii (H2S negative) were incorrectly identified as Citrobacter diversus (C. intermedius). The main source of error appeared to be the inability of Micro-ID to detect the production of acid from adonitol by C. divetsus. Further, two strains of Serratia (Enterobacter) liquefaciens TABLE 3. Rate of correct identifications by Micro- ID No. No. correct by Organism tested Micro-ID' Arizona hinshawii (Salmonella arizonae)... 16 15 (93.8) Citrobacter amalonaticusb... 5 0 (0) Citrobacter freundii (H2S negative)... 3 1 (33.3) Edwardsiella tarda... 10 10 (100) Enterobacter agglomerans (Erwinia)... 6 6 (100) -b Enterobacter gergoviae... 5 0 (0) Escherichia coli (H2S positive) 5 4 (80) E. coli (atypical)... 5 4 (80) Hafnia alvei...... 15 15 (100) Klebsiella ozaenae... 5 5 (100) Klebsiella rhinoscleromatis... 5 3 (60) Pectobacterium. 5 1 (25)c Providencia alcalifaciens (Proteus inconstans)... 6 6 (100) Providencia rettgeri (Proteus rettgeri)... 13 11(84.6) Providencia stuartii (Proteus inconstans)... 11 10 (91) Salmonella cholerae-suis... 4 3 (75) Salmonella paratyphi A... 4 2 (50) Salmonella (atypical).... 5 5 (100) Serratia liquefaciens... 11 8 (72.7) Serratia rubidaea... 4 3 (75) Yersinia enterocolitica... 10 7 (70) Yersinia pseudotuberculosis.. 4 3 (75) Avg (77) Avg excluding C. amalonaticus, E. gergoviae, and Pectobacteriumrm... (94.5) b Biochemical reaction patterns for this organism are not included in the Micro-ID data base. ' Four additional strains classified as Enterobacter agglomerans. were incorrectly identified as Serratia marcescens because of false-negative arabinose reactions. The small number of strains tested within each species, however, precluded an in-depth analysis of identification errors. TABLE 4. Errors in identification by the Micro-ID system Organism (no.) Incorrectly identified Citrobacter amalonaticusa (5)... Citrobacter freundii (H2S negative) (2) Edwardsiella tarda Enterobacter aerogenes (1)... Enterobacter gergoviaea (3)... Enterobacter gergoviaea (2) Escherichia coli (1) E. coli (1)... Klebsiella rhinoscleromatis (1)... K. rhinoscleromatis (1).. Morganella morganii (1)... Pectobacterium a (1)... Proteus vulgaris Providencia rettgeri (1)... P. rettgeri (1)... Salmonella choleraesuis (1)... Salmonella paratyphia (1)... S.:paratyphi A (1).. Salmonella typhi (1) Serratia liquefaciens (2)... S. liquefaciens (1) Serratia rubidaea Yersinia enterocolitica (1)... Y. enterocolitica (2)... J. CLIN. MICROBIOL. as: Citrobacter diversus C. diversus Salmonella cholerae-suis Serratia liquefaciens Klebsiella pneumoniae Hafnia alvei Citrobacter freundii Serratia marcescens Klebsiella ozaenae Enterobacter agglomerans Proteus rettgeri Yersinia pestis Proteus mirabilis Not on file Providencia alcalifaciens Citrobacter diversus Escherichia coli Hafnia alvei H. alvei Serratia marcescens Serratia rubidaea Enterobacter agglomerans Escherichia coli Yersinia pseudotuberculosis Yersinia pseudotuberculosis (1)... Yersinia pestis a Biochemical reaction patterns for this organism are not included in the Micro-ID data base.

VOL. 10, 1979 Reproducibility. To test the reproducibility of the Micro-ID system, each laboratory exchanged 50 cultures. Thus, both laboratories independently identified 100 isolates. Of the 100 isolates tested in duplicate, 93 were identified as the same genus and species. Sixty-two of the organisms produced identical profile numbers. Of the 38 organisms with different profile numbers, 69% varied in one biochemical test per Micro-ID profile, 21% varied in two, 5% varied in three, 0% varied in four, and 5% varied in five. In 31 isolates, the same identification was obtained but with differing profile numbers. Of these, 23 (74%) differed in only one of the 15 biochemical tests, seven (23%) showed variation in two biochemical tests, and one (3%) exhibited discrepancies in three biochemical reactions. All 50 common clinical isolates were correctly identified by both laboratories, whereas 7 of the 50 unusual or atypical organisms were identified as a different genus or species or both. Of these seven strains, three showed a single variable biochemical reaction, one varied in two reactions, one differed in three reactions, and two showed variation in five biochemical tests. These discrepancies in identification were: Escherichia coli versus C. diversus (2); E. coli versus S. marcescens (1); Salmonella paratyphi A versus E. coli (1); Salmonella typhi versus Hafnia alvei (1); S. liquefaciens versus S. marcescens (1); and S. marcescens versus Serratia rubidaea (sp. nov.; reference 6) (1). DISCUSSION The Micro-ID system utilizes reagent-impregnated filter paper disks to provide a 4-h identification of the Enterobacteriaceae. Inoculation of the unit with an organism-saline suspension is convenient. Inoculation does not require the use of sterile saline or sterile pipettes or pipette tips; however, the use of saline preparations containing preservatives, such as sodium azide, must be avoided. Since stock cultures were used in this evaluation, we could not determine the frequency with which the required 0.5 Mc- Farland inoculum density could be achieved without overnight incubation in a clinical setting. A previous report (1) indicated that sufficient growth to bring the inoculum to the required turbidity was achieved from the initial isolation plate for 74% of the clinical isolates tested. The performance of the Micro-ID system was comparable to those of the API 20E and classical tubed media. As expected, unusual or atypical organisms presented the greatest challenge for the system. Of this group of organisms, three species tested in this study, C. amalonaticus, E. EVALUATION OF MODIFIED MICRO-ID SYSTEM 457 gergoviae, and Pectobacterium, were not included in the Micro-ID data base and thus could not be identified by the system. It was noted in a previous study (1) that several misidentifications were due to "book errors" in the identification manual. These appear to have been eliminated in the current edition of the manual. The original identification manual was based on the data of Edwards and Ewing (4). The current edition of the manual is based on tests of numerous Enterobacteriaceae with Micro-ID. Although it was not the purpose of this evaluation to compare the results of individual Micro-ID biochemical tests with those of other systems, it should be pointed out that the relative insensitivity of certain of the Micro-ID tests (e.g., adonitol and arabinose) may be a possible source of error in the system. Of the 100 isolates identified in duplicate, 93% of genus and species identifications and 62% of profile numbers were in perfect agreement. Of the 31 strains with the same identification but different code numbers, 74% differed in only one biochemical reaction. These results are comparable to those reported for the reproducibility of the API 20E system (2). In several instances, the color reactions of various biochemical tests were difficult to read even with the expanded descriptions provided by the manufacturer. Some of the tests may have been misinterpreted, thus contributing to the variation observed. The use of a color chart with the kit would aid analysts in determining whether a given reaction is positive or negative. In conclusion, results of this study indicate that the Micro-ID system compared favorably to other conventional systems for the identification of Enterobacteriaceae. Since this is a 4-h system, it may prove advantageous in situations where a "same-day" identification is required. This study was designed only to determine the ability of Micro-ID to identify enteric organisms and to estimate its reproducibility. The performance of the system in a clinical setting requires additional evaluation. ACKNOWLEDGMENT This work was supported in part by General Diagnostics, Division of Warner-Lambert Company, Morris Plains, N.J. LITERATURE CITED 1. Aldridge, K. E., B. B. Gardner, S. J. Clark, and J. M. Matsen. 1978. Comparison of Micro-ID, API 20E and conventional media systems in identification of Enterobacteriaceae. J. Clin. Microbiol. 7:507-513. 2. Brenner, D. J., J. J. Farmer III, F. W. Hickman, M. A. Asbury, and A. G. Steigerwalt. 1977. Taxonomic and nomenclature changes in Enterobacteriaceae. Center for Disease Control, Atlanta. 3. Butler, D. A., C. M. Lobregat, and T. L. Gavan. 1975. Reproducibility of the Analytab (API 20E) system. J.

458 BUESCHING ET AL. Clin. Microbiol. 2:322-326. 4. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis. 5. Ewing, W. H. 1974. Differentiation of Enterobacteriaceae by biochemical reactions. Center for Disease Control, Atlanta. 6. Ewing, W. H., and W. J. Martin. 1974. Enterobacteriaceae, p. 189-221. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, J. CLIN. MICROBIOL. 2nd ed. American Society for Microbiology, Washington, D.C. 7. Nord, C. E., A. A. Lindberg, and A. Dahlback. 1974. Evaluation of five test-kits-api, Auxotab, Enterotube, PathoTec, and R/B-for identification of Enterobacteriaceae. Med. Microbiol. Immunol. (Berlin) 159:211-220, 1974. 8. Washington, J. A. II. 1976. Laboratory approaches to the identification of Enterobacteriaceae. Human Pathol. 7:151-159.