ylococcus aureus and Rhodococcus equi CAMP tests-are required for identification to the species level. The profile index indicated which of these tests were ...
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1993, p. 749-750
Vol. 31, No. 3
0095-1137/93/030749-02$02.00/0 Copyright © 1993, American Society for Microbiology
Evaluation of the API Coryne System for Identification of Listenia Species K. G. KERR,* P. M. HAWKEY, AND R. W. LACEY Department of Microbiology, University of Leeds, Leeds, West Yorkshire LS2 9JT,
Received 26 June 1992/Accepted 23 November 1992
The API Coryne system, a commercially available system for the identification of coryneform bacteria, was used to identify 103 strains of Listeria spp. from clinical and environmental sources. All isolates were identified correctly to the genus or species level, although complete characterization also required tests for beta-hemolysis and CAMP reaction.
In recent years epidemiologic and microbiologic investigations of both epidemic and sporadic cases of human listeriosis have implicated contaminated food products as the primary means of transmission of the causative organism, Listeria monocytogenes (2). Although L. monocytogenes is readily isolated from clinical material (4), unfamiliarity with the morphological and biochemical characteristics of L. monocytogenes may result in initial misidentification as a coryneform or diphtheroid on the basis of its morphological appearance. The nonavailability of a simple reliable method for the identification of coryneforms, coupled with an opinion that many coryneforms are of no clinical significance, might conceivably lead to a missed microbiological diagnosis of listeriosis. A commercially available system for the identification of coryneform bacteria (API Coryne; Biomerieux, Marcy l'Etoile, France) has been introduced in an effort to overcome the problems of the characterization of these organisms by conventional methods (1). As Listeria spp. are taxonomically related to members of the genus Corynebacterium, Listeria spp. are included in the data base of the API Coryne system. Therefore, it is possible that this method can be used to identify Listena spp., either intentionally or unintentionally. A recently published evaluation of the API Coryne system employed only four strains of only two species of the genus Listena (2). We have extended the previous evaluation of the API Coryne system for the identification of Listeria spp. (1) by examining 103 strains of all seven currently recognized members of the genus Liste-
dase, ,-glucuronidase, and gelatinase. The remainder consists of eight substrates to test carbohydrate fermentation (glucose, ribose, xylose, mannitol, maltose, lactose, sucrose, and glycogen) and also a negative control for the fermentation reactions. Catalase activity was determined by adding a drop of hydrogen peroxide (3%) to the gelatinase cupule. Strips were stored at 4°C until used. Strains to be tested for beta-hemolysis were grown on horse blood agar for 18 h at 37°C in air. A dense suspension of the organism was made (greater than no. 6 McFarland standard) in 3 ml of sterile distilled water and used to inoculate the substrates. A 0.5-ml aliquot was added to GP medium (Analytab Products, Marcy L'Etoile, France) containing phenol red indicator and to the remaining cupules. Purity plates were also inoculated from this suspension. Purity plates and test strips were incubated in air at 37°C for 24 h. All enzymatic tests with the exception of the urease, ,-glucosidase, and gelatinase tests required the addition of further reagents before interpretation was possible. Carbohydrate fermentation was deemed to have occurred if the phenol red indicator had turned yellow. Results obtained were converted into a seven-digit profile, and the resulting profile was used to identify the isolates by using the analytical profile index supplied by the manufacturer. The system correctly identified all 103 isolates to the genus level (Table 1). As with virtually all other commercially available systems for the identification of Listeria spp., further tests-beta-hemolysis on blood agar and the Staphylococcus aureus and Rhodococcus equi CAMP tests-are required for identification to the species level. The profile index indicated which of these tests were appropriate for the subsequent identification to the species level. The system, despite the inclusion of a test for nitrate reduction, did not distinguish between L. murrayi and L. grayi; however, given the unlikelihood of isolation of those bacteria in the clinical setting, this is not a major disadvantage. Furthermore, Rocourt et al. recommend that these organisms be considered members of a single species: L. grayi (7). Several commercially available systems are now available for the identification of Listeria spp. However, these are primarily intended for this purpose only, and given the infrequency of isolation of L. monocytogenes from clinical specimens, it is doubtful whether it would be cost-effective for a clinical laboratory to include such a system in its test repertoire; furthermore, unfamiliarity with an infrequently used method may lead to problems with proficiency in performance and interpretation of test results. The API system, however, is likely to find use in many clinical
One hundred three isolates of Listeria spp.
(72 L. monocytogenes, 13 L. seeligeri, 7 L. innocua, 3 L. ivanovii, 3 L. welshimeri, 3 L. murrayi, and 2 L. grayi isolates). These isolates were obtained from our own laboratory and from the National Collection of Type Cultures, Colindale, United Kingdom; the Institut Pasteur, Paris, France; and Universitat Wurzburg, Wurzburg, Germany, and represented strains from clinical, veterinary, food, and environmental sources. All isolates had been identified by both conventional and commercially available systems (5, 6). Strains were stored on blood or nutrient agar at 4°C. The API Coryne kit consists of a plastic strip with 20 cupules containing dehydrated substrates for the reactions catalyzed by 10 enzymes: nitrate reductase, pyrazinamidase, pyrrolidonyl arylamidase, ,B-galactosidase, alkaline phosphatase, a-glucosidase, 3-glucosidase, N-acetylglucosamini*
Corresponding author. 749
J. CLIN. MICROBIOL.
NOTES TABLE 1. API Coryne profiles obtained with 103 isolates of Listeria spp. Species
monocytogenes seeligen innocua welshimeri ivanovii murrayi grayi
L. L. L. L. L. L. L. a
72 13 7 3 3 3 2
L. monocytogeneslinnocuaa Listeria spp.a L. monocyto eneslinnocuaa Listena spp. Listena spp.c L. grayilmurrayid L. grayilmurrayid
6 5 2 3 2 2
between pathogenic and nonpathogenic strains of Yersinia enterocolitica has been noted (3). Although 80% of the 72 L. monocytogenes strains were associated with 5 numerical profiles, a total of 12 profiles were observed. Further evaluation of the system should be undertaken to assess its potential usefulness in biotyping isolates of clinical and environmental origin during epidemiologic investigations of listeriosis. We thank J. Rocourt, Institut Pasteur, Paris, France, and H. P. R. Seeliger, University of Wurzburg, Wurzburg, Germany, for some of the isolates used in this study.
Final identification dependent on beta-hemolysis or CAMP reaction with
b Final identification dependent on lack of beta-hemolysis or negative CAMP reaction. c Final identification dependent on beta-hemolysis or CAMP reaction with R equi. d Further tests for identification to species level not given.
REFERENCES 1. Freney, J., M. T. Duperron, C. Courtier, W. Hansen, F. Allard, J. M. Bouefgrass, D. Monget, and J. Fleurette. 1991. Evaluation of API Coryne in comparison with conventional methods for identifying coryneform bacteria. J. Clin. Microbiol. 29:38-41. 2. Gellin, B. G., and C. V. Broome. 1989. Listeriosis. JAMA
laboratories, given both the increasing recognition of infections caused by coryneforms and the difficulties associated with conventional identification techniques for these bacteria, and its use for the identification of L. monocytogenes can be recommended. For L. monocytogenes isolates no correlation between serotype and profile was demonstrated; however, with strains whose source was known, it was noted that only 1 of 16 clinical isolates demonstrated pyrazinamidase activity, whereas 21 of 41 environmental strains were pyrazinamidase positive. Although highly statistically significant (P < 0.001, x2 with Yates' correction), the importance of this finding remains unclear, but it is of interest that a similar association
3. Kandolo, K., and G. Wauters. 1985. Pyrazinamidase activity in Yersinia enterocolitica and related organisms. J. Clin. Microbiol. 21:980-982. 4. Kerr, K. G., and R. W. Lacey. 1991. Isolation and identification of Listena monocytogenes. J. Clin. Pathol. 44:624-627. 5. Kerr, K. G., N. A. Rotowa, P. M. Hawkey, and R. W. Lacey. 1990. Evaluation of the Mast ID and APISOCH systems for the identification of Listeria spp. Appl. Environ. Microbiol. 56:652660. 6. McGlauchlin, J. 1988. The identification of Listeria species. DMRQC Newsl. 3:1-3. 7. Rocourt, J., P. Boerlin, F. Grimont, C. Jacquet, and J.-C. Piffaretti. 1992. Assignment of Listeriagrayi and Listeria murrayi to a single species, Listeria grayi, with a revised description of Listenia grayi. Int. J. Syst. Bacteriol. 42:171-174.