of Anaerobic Gram-Negative Bacilli and Clostridium Species

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1 JOURNAL OF CLINICAL MICROBIOLOGY, July 1979, p /79/ /05$02.00/0 Vol. 10, No. 1 API and Minitek Systems in Identification of Clinical Isolates of Anaerobic Gram-Negative Bacilli and Clostridium Species CHARLES W. HANSON,t* RODRIGO CASSORLA,' AND WILLIAM J. MARTIN"2 Departments of Pathology,1 and Microbiology and Immunology2, Clinical Laboratory, Microbiology Section, UCLA Center for the Health Sciences, Los Angeles, California Received for publication 5 April 1979 A comparison of the API and Minitek methods of biochemical testing was made on a variety of anaerobic bacteria. Although API and Minitek results were not compared to more standardized or conventional procedures of identification, multiple repeat testing of the two systems was done on routine clinical isolates and known organisms to determine (i) whether the reactions were reliably consistent, (ii) the ease of reading the two systems with respect to the frequency of questionable results, and (iii) the percentage of routine clinical isolates for which each system yielded an identification. The Minitek system gave a much lower incidence of difficult to interpret reactions. The two systems were comparable in terms of reproducibility and capability of yielding an identification of the anaerobic gram-negative bacilli and Clostridium species, but were unsatisfactory for routine use on most of the other anaerobic bacteria isolated. The awareness of the potential severity of anaerobic bacterial infections (4, 5) has dictated the necessity for rapid and correct identification of the organisms involved so that relevant antimicrobial treatment can be initiated at the earliest possible time. Conventional biochemical testing (3, 9) of anaerobic isolates for purposes of identification is not only time consuming and expensive, but is beyond the capabilities of many clinical laboratories. The necessity for a simplified system has led to the development of a number of miniaturized test procedures. In an attempt to select one of the more readily available micromethods, an evaluation of the API (Analytab Products, Inc., Plainview, N.Y.) and Minitek (BBL Microbiology Systems, Cockeysville, Md.) systems was initiated to determine their overall usefulness for the identification of anaerobic bacteria. More specifically, multiple repeat testing of the two procedures was done on routine clinical isolates to determine (i) whether the reactions were reliably consistent, (ii) the ease of reading, with respect to the frequency of questionable results, and (iii) the percentage of routine clinical isolates for which each system was capable of yielding an identification. It was not within the scope of this investigation to make a comparison of the API and Minitek systems to conventional procedures. This t Present address: Antibiotic Screening Laboratory, Abbott Laboratories, N. Chicago, IL approach has been adequately covered in other investigations (6, 14, 18, 20). Our concern was to focus on the dilemma of conflicting and ambiguous results which have been encountered in the everyday use of the two procedures in clinical microbiology laboratories. MATERIALS AND METHODS Organisms and treatment of cultures. The organisms used were those routinely isolated from a variety of clinical specimens. In addition, anaerobic bacteria from the University of California at Los Angeles stock culture collection were used in some experiments. Treatment of cultures (9, 12, 13) and plating media (7) have been previously described. Each isolate was heavily inoculated onto one-half of a specially prepared anaerobic blood agar plate (7) and incubated anaerobically at 35 C in a Gas-Pak jar (BBL Microbiology Systems). A commercial blood agar plate (Clinical Standards Laboratory, Carson, Calif.) was also inoculated and incubated aerobically at 35 C to serve as a contamination control. After 48 h of incubation, a sterile cotton swab was used to harvest the growth from the anaerobic plate. Inoculation, incubation, and reading of API test strips. The bacteria were suspended by swirling the cotton swab in an ampoule of API inoculation medium to a density of about 1 x 109 cells per ml, and then inoculated on the API test strips according to the instructions of the manufacturer. The strips were incubated anaerobically at 35 C in Gas-Pak jars. After 48 h, the strips were removed from the jars, and fresh 0.02% bromocresol purple indicator was added to all carbohydrate and alcohol substrates immediately before reading. A purple color was recorded as negative,

2 VOL. 10, 1979 and yellow was recorded as positive. Equivocal reactions judged not to be definitely yellow or purple were recorded as ±. All other tests were run and interpreted according to the directions in the test kit. Inoculation, incubation, and reading of Minitek test trays. The organisms were suspended by swirling the cotton swab in a vial of Minitek inoculation medium to a density of approximately 5 x 109 cells per ml. The Minitek pipetter was used to inoculate filter paper disks containing the test substrates. The plates were incubated in Gas-Pak jars at 35 C. After 48 h, the trays were removed from the jars, and fresh 0.025% phenol red indicator was added to all carbohydrate and alcohol substrates immediately before reading. A red color was recorded as negative, and yellow was recorded as positive. Equivocal reactions judged not to be definitely red or yellow were recorded as ±. All other tests were interpreted according to the directions given in the test kit. With both test systems, esculin hydrolysis was determined by noting the absence of characteristic fluorescence when exposed to 360-nm wavelength ultraviolet light. Viability and contamination controls were run simultaneously on every organism tested by inoculating two blood agar plates and incubating one aerobically and the other anaerobically. Unless otherwise indicated, the API and Minitek test results were interpreted from the reactions given in the respective identification table supplied with each system. In situations where a genus-species identification could not be made with a given microsystem, or when there was disagreement between the two systems, or both, the correct genus was determined by using the Gram stain, cell morphology from thioglycolate culture, presence or absence of spores, gas-liquid chromatography (9), and biochemical test procedures similar to those sug-,ested by Porschen and Stalons (17). Subsequent to the completion of this investigation, API made available an expanded analytical profile index, and our data have accordingly been reevaluated in light of this more recent information. RESULTS A preliminary evaluation of the API and Minitek test systems was made on 114 known organisms, and the results were compared to those in the Virginia Polytechnic Institute Anaerobe Laboratory Manual (9). This effort was an attempt to find an expanded identification data source, since both the API and Minitek identification tables were considered to be inadequate at that time. Results obtained with the Minitek and especially the API system employing stock cultures of anaerobic bacteria most commonly encountered in clinical specimens were found generally to agree with the Virginia Polytechnic Institute data. At the outset, however, it was realized that the comparison of results from one system to the identification tables of another system was not strictly valid, due to the inherent differences between systems as to the use of API AND MINITEK IDENTIFICATION OF ANAEROBES buffers, ph indicators, inoculum size, and total volume under which each procedure was run (19). In spite of these considerations, Minitek employs for its identification tables data taken directly from the Center for Disease Control manual (3). Furthermore, using this approach on a limited number of unknown organisms, the identification frequency was found to be no greater than when the results obtained from each test system were analyzed with their respective identification tables. It became apparent early in the study that in a significant number of instances repeat testing of the same organism with either procedure did not consistently give the same results, due to variability within the individual substrate reactions. To gain support for this contention, 197 clinical isolates were tested with both API and Minitek, and evaluated for ease of reading and reproducibility of repeat testing on the same organism. The substrates used with each procedure were as follows: indole, urea, glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, H2S, esculin, glycerol, cellobiose, mannose, raffinose, sorbitol, rhamnose, and trehalose. Gelatin and melezitose were not available from Minitek, and nitrate reductase was not available from API. Table 1 records the percentage of final identification reproducibility obtained from repeat testing of the same organism a minimum of three times on different days. Little difference was noted between the two systems. Reproducibility was shown to be good with Bacteroides species but less so with other genera of anaerobic bacteria. The substrates most often found to give variable results with the API system were indole, xylose, esculin, and trehalose. Salicin, H2S, esculin, cellobiose, raffinose, and trehalose most TABLE 1. Reproducibility of individual substrate reactions affecting final identification to genusspecies level % Agreement of multiple testing within Organism No. of orga- the complete nisms panel API Minitek Clostridium species Bacteroides species Fusobacterium species Gram-positive cocci Gram-positive non sporeforming bacilli Overall average

3 16 HANSON, CASSORLA, AND MARTIN TABLE 2. Percentage of questionable readings observed within each of the two systems for the groups of anaerobic bacteria listed % of total tests giving a questionable read- Organism of orga- ing API Minitek Clostridium species Bacteroides species Fusobacterium species Gram-positive cocci Antinomyces species Eubacterium lentum Overall average often yielded variable results with the Minitek system. In both cases, variability in these reactions affected the final identification. Table 2 shows the percentage of questionable readings observed with each test system. These data indicate that the API system had a greater degree of variability, requiring the observer to exercise a certain amount of judgment as to whether the reaction in question was positive or negative. In this regard, virtually all substrates were implicated with the API system, whereas with Minitek the substrates most often involved were indole, mannitol, maltose, salicin, xylose, arabinose, glycerol, cellobiose, sorbitol, rhamnose, and trehalose. Relatively few attempts were made during this investigation to use API and Minitek in the identification of anaerobic cocci and nonsporeforming, Gram-positive bacilli. Indeed, our laboratory has eliminated testing these groups of anaerobic bacteria, except in a relatively small number of cases, because of an extremely low success rate. Although a limited amount of data J. CLIN. MICROBIOL. are given to document this deficiency, our efforts were limited mainly to the anaerobic gram-negative bacilli and Clostridium species. Attempts were made to try to identify an additional 272 clinical isolates of anaerobic bacteria, using each of the two systems for every isolate. Results of each evaluation were interpreted from the respective identification schemes supplied with the test system used, and the data presented in Table 3 record the number of strains attempted for each genus and the percent for which an identification was obtained. API gave a slightly higher rate of identification, except for the genus Fusobacterium, in which case the Minitek system was far superior, due primarily to the reproducibility of its indole reaction. Table 4 shows the amount of agreement or disagreement, or the inability of the two procedures to identify anaerobic bacteria. The percentages of isolates identified by either method for the various genera are given in the righthand column. It should be stressed that the figures in this column are not intended to suggest that one or the other system gave the correct identification, but only that within the framework of a given procedure, an identification was made. Subsequent to the completion of this investigation, API has made available a greatly expanded data base with its analytical profile index. In light of this new information, our API quick index results from 140 recent isolates of anaerobic gram-negative bacilli and Clostridium species were reevaluated. Table 5 shows that the identification success rate was increased by approximately 18% over what was achieved using the quick index, i.e., a 13% increase for Clostridium species, a 22% increase for Bacteroides species, and an apparent 20% increase with the very few Fusobacterium species tested. TABLE 3. Anaerobic isolates identified by API and Minitek systemsa Total no. attempted No. of isolates identi- % identified Organism fled API Minitek API Minitek API Minitek Bacteroides species Clostridium species Fusobacterium species Peptococcus species Peptostreptococcus species Anaerobic nonspore-forming gram-positive bacilli Gram-negative bacilli, uni dentifiable Mean percent identified a Regardless of whether the two systems agreed.

4 VOL. 10, 1979 API AND MINITEK IDENTIFICATION OF ANAEROBES 17 TABLE 4. Ability ofapi and Minitek to yield an identification of anaerobic bacteria isolated from clinical specimens' No. of isolates Ttestetendoare. tek diogree m unable to tek agree in by either sys- Organism API and Mini- API and Mini- API and Mini- % Identified tested tek identification but identify identification tem Bacteroides species Fusobacterium species Clostridium species Peptococcus species Peptostreptococcus species Anaerobic, gram-positive, non spore-forming bacilli Mean % identified 54 a Two gram-negative bacilli, unidentifiable by any means, are not included in the table. Each test system was evaluated independently; i.e., API results were evaluated with the API 20A quick index, and Minitek results were evaluted with the Minitek identification charts supplied with the test kit. TABLE 5. Percent of anaerobic gram-negative bacilli and Clostridium species identified with API No. identified with: Genus No. attempted Quick Aalytiidx cal Analyti- profile index Clostridium species (54.8) 21 (67.6) Bacteroides species (51.0) 70 (72.9) Fusobacterium species 5 2 (40.0) 3 (60) Other' 8 2 (25.0) 2 (25.0) Totals (50) 96 (68) a Values in parentheses indicate percent. ' Other strains included Eubacterium species, two Veillonella species, one Propionibacterium species, and one Peptococcus species. DISCUSSION A number of investigators have evaluated various miniaturized test systems currently available for the identification of anaerobic bacteria (6, 14, 18, 20). Many of the organisms used in these studies were fairly well characterized anaerobic bacteria obtained from stock culture collections of the laboratories involved (14, 18, 20). Comparison of API, Minitek, and conventional procedures (3, 9) sometimes resulted in better than 90% identification agreement among the various methods. The above, investigations notwithstanding, it has been our experience that a much lower rate of identification is actually achieved in attempts to identify to species level all anaerobic bacteria isolated from well collected specimens routinely submitted to the laboratory. A preliminary review of our API results from 568 clinical isolates of anaerobic gram-negative bacilli and Clostridium species showed an overall identification efficiency of only about 50%. The increased data base of the API analytical profile index seems to offer some relief in this regard, since it appears to provide a greater percentage of species identifications even though some of these isolates show an inherent degree of reaction variability. Reproducibility evaluations have been performed with miniaturized test systems designed primarily for use with facultative bacteria (2, 8, 11). These evaluations also encountered variable results upon repeat testing of the same isolate. For example, in one study (2) only 55% of the isolates gave identical reactions on repeat testing with API, but a 97.3% genus-species identification was achieved, presumably due to the larger size of the Enterobacteriaceae data base. In this light, the 90% level of reproducibility (Table 1) achieved with anaerobic bacteria is not as much a problem as having to rely on the use of the abbreviated identification tables supplied with the test kits. Many disagreements in species identification were due to the questionable readings of the biochemical substrate reactions. Minitek fared better than API in this regard, which is probably attributable to the higher bacterial density (5 x 109 cells/ml) Minitek requires for their inoculum. In support of this contention, anaerobic incubation of either API or Minitek is not required to achieve identification of a known strain of B. fragilis, suggesting that at a sufficient bacterial density, residual enzyme activity may be more important than bacterial growth for substrate utilization (16; unpublished data). When results from API or Minitek, or both, disagreed, or indeed, even when the two procedures were found to agree in the identification of a given isolate, we did not feel compelled to pursue an unequivocal identification by more

5 18 HANSON, CASSORLA, AND MARTIN standardized or conventional (3, 9) procedures. Considering the exigencies of a busy clinical laboratory, the objective was simply to determine which of the two procedures most often yielded an identification. Only about 50% of the anaerobic bacteria were identified by either system (Tables 3 and 4), and in 16% of these cases API and Minitek disagreed in their identification (Table 4). It is unrealistic to expect to achieve total agreement between API and Minitek or any other test system, since the assay conditions in any given procedure may differ from those used in another procedure (19). Therefore, identical biochemical reactions obtained from a given organism by using different systems should not be anticipated. However, these same systems should be internally reproducible and their identification tables sufficiently complete to enable the user to satisfactorily identify the majority of bacteria encountered from suitable specimens. Disagreement among various systems is of major concern in interlaboratory comparisons but may not be as serious on an intralaboratory basis as long as the same procedure is used consistently and the results are reproducible. These criteria have yet to be fully realized; however, the API analytical profile index with its expanded data base has significantly improved the frequency of identification to a more acceptable 68% with the API 20A strip. Some investigators have argued that it is not necessary to completely identify all of the anaerobic bacteria isolated (1, 15, 17). However, this philosophy is contrary to the instincts of well trained and conscientious microbiologists (10). Since sufficient tests are available and can be used with a certain degree of flexibility, as with the Minitek system, the argument against complete identification to species level loses some of its meaning. In general, our results show that with some of the more commonly isolated and rapidly growing anaerobic bacteria, both test procedures are satisfactory, with the edge being given to API because of the availability of its new analytical profile index. However, each system showed a less than satisfactory capacity to identify the anaerobes that were slow growing or difficult to cultivate, or both. This deficiency is probably directly related to the inability of those slowgrowing organisms to reach an inoculum size needed for optimal utilization of the substrates for each test system. Fortunately, the anaerobic bacteria for which these systems are best suited comprise approximately 50% of the clinically significant bacteria isolated in the anaerobic laboratory (13). ACKNOWLEDGMENTS We are grateful for the technical assistance of William J. CLIN. MICROBIOL. Welch in some of these experiments. Paul Rhode, Myrna Maher, and Brian Rampsch of Bio-Quest generously provided most of the Minitek equipment and substrates used in this investigation. LITERATURE CITED 1. Blank, F. C Identification of anaerobic bacteria. Letter to the editor. Am. Soc. Microbiol. News 42: Butler, D. A., C. M. Lobregat, and T. L. Gavan Reproducibility of the Analytab (API 20E) system. J. Clin. Microbiol. 2: Dowell, V. R., and T. M. Hawkins Laboratory methods in anaerobic bacteriology. Center for Disease Control, Atlanta, Ga. 4. Finegold, S. W., J. Bartlett, A. W. Chow, 0. J. Flora, S. L. Gorbach, E. J. Harder, and F. P. Tally UCLA Conference. Management of anaerobic infections. Ann. Intern. Med. 83: Gorbach, S. L., and J. G. Bartlett Medical progress: anaerobic infections. N. Engl. J. Med. 290: , , Hansen, S. L., and B. J. Stewart Comparison of API and Minitek to Center for Disease Control methods for the biochemical characterization of anaerobes. J. Clin. Microbiol. 4: Hanson, C. W., and W. J. Martin Evaluation of enrichment, storage, and age of blood agar medium in relation to its ability to support growth of anaerobic bacteria. J. Clin. Microbiol. 4: Hanson, C. W., E. Marso, and W. J. Martin Comparison of the Minitek test system with a conventional screening procedure for identification of Enterobacteriaceae. Health Lab. Science. 15: Holdeman, L. V., E. P. Cato, and W. E. C. Moore Anaerobic bacteriology manual. Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Va. 10. Holdeman, L. V Identification of clinical bacteria. Letter to the editor. Am. Soc. Microbiol. News 42: Holmes, B., W. R. Wilcox, S. P. LaPage, and H. Malnick Test reproducibility of the API (20 E), Enterotube, and Pathotek systems. J. Clin. Pathol. 30: Martin, W. J Practical method for isolation of anaerobic bacteria in the clinical laboratory. Appl. Microbiol. 22: Martin, W. J Isolation and identification of anaerobic bacteria in the clinical laboratory. Mayo Clin. Proc. 49: Moore, H. B., V. L. Sutter, and S. M. Finegold Comparison of three methods for biochemical testing of anaerobic bacteria. J. Clin. Microbiol. 1: Porschen, R. K., and D. R. Slalons Evaluation of simplified dichotomous schemata for the identification of anaerobic bacteria from clinical material. J. Clin. Microbiol. 3: Schreckenberger, P. C., and D. J. Blazevic Rapid fermentation testing of anaerobic bacteria. J. Clin. Microbiol. 3: Stalons, D. R., and R. K. Porschen Identification of anaerobic bacteria. Letter to the editor. Am. Soc. Microbiol. News 42: Stargel, M. D., F. S. Thompson, S. E. Phillips, G. L. Lombard, and V. R. Dowell, Jr Modification of the Minitek miniaturized differentiation system for characterization of anaerobic bacteria. J. Clin. Microbiol. 3: Stargel, M. D., G. L. Lombard, and V. R. Dowell Alternative procedures for identification of anaerobic bacteria. Am. J. Med. Technol. 44: Starr, S. E., F. S. Thompson, V. R. Dowell, Jr., and A. Balows Micromethod system for identification of anaerobic bacteria. Appl. Microbiol. 25: