APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1990,

p.

1166-1168

Vol. 56, No. 4

0099-2240/90/041166-03$02.00/0 Copyright © 1990, American Society for Microbiology

Same-Day Identification Scheme for Colonies of Listeria monocytogenes R. VICTOR LACHICA U.S. Army Natick Research, Development, and Engineering Center, Natick, Massachusetts 01760 Received 2 November 1989/Accepted 23 January 1990

A diagnostic scheme is described for the same-day identification of food-borne cells of Listeria monocytogenes that emerge in 40 h at 30°C as large colonies, representatives of which are used to advantage as heavy inocula on agar plates for the rapid determination of hemolytic activity and acidification of rhamnose and xylose. Additional tests consisting of phase-contrast microscopy for cell morphology and motility, the catalase production test, and the KOH viscosity test in place of Gram staining complete the rapid identification scheme.

(0.1 ml) in duplicate onto LCA plates, which were subsequently incubated for 40 h at 30°C. SHOT technique. The initial recognition of Listeria colonies on LCA plates was facilitated by using a simplified Henry oblique transillumination (SHOT) technique (7). RAP-ID scheme. Each well-isolated representative large colony that emerged from an LCA plate and appeared bluish by the SHOT technique was directly subjected to a battery of tests without the prior purification step required for the conventional procedure. With a sterile inoculation loop, a colony was harvested as a viscid cell mass for inoculation of three diagnostic agar plates that were divided into quadrants. (i) Rapid hemolytic activity test. A sterile disk of ,-lysin (Remel) was aseptically placed at the center of a blood agar plate (5% sheep blood in Columbia blood agar base [Difco]) to allow the diffusion of the ,-lysin through the agar gel. As many as four heavy inocula were smeared as bands no larger than 3 by 4 mm at a distance of 2 to 3 mm perpendicular to the edge of the lysin disk. Each inoculum was massive enough to be visible to the naked eye. Observation of complete hemolytic reactions was made after 4, 5, 6, and 7 h at 35°C. (ii) Rapid sugar acidification. Acid production from rhamnose and xylose was detected by an agar plate method. Each fermentation test agar plate was prepared by adding an appropriate sample of a filter-sterilized sugar stock solution to an autoclave-sterilized purple agar base (Difco) medium. The final carbohydrate concentration in the agar medium was 1%. The plates were inoculated with the viscid cell mass and incubated in a fashion similar to that used in the rapid hemolytic activity test. (iii) Phase-contrast microscopy. The remaining cells on the inoculation loop were suspended in a loopful of distilled water on a microscope slide, covered with a glass cover slip, and examined by phase-contrast microscopy for cell morphology and motility. (iv) Catalase test. After phase-contrast microscopy, the cell suspension on the microscope slide was examined for catalase by adding 1 drop of 3% hydrogen peroxide to one side of the cover slip and observing for an immediate effervescence from one end of the cover slip to the other. (iv) KOH viscosity test. The convenient KOH viscosity test of Ryu (12) was used in place of the Gram stain. It involved using a heavy cell mass obtained from one of the diagnostic agar plates described above after an incubation period of 7 h. A heavy suspension in 1 drop of 3% KOH solution on a glass slide was stirred with an inoculation loop, and within a few

Recent outbreaks of listeriosis and the subsequent recognition of a significant lack of information about the behavior of Listeria monocytogenes have underscored the need for a more rapid and reliable detection procedure for the coldtolerant pathogen in food, clinical, and environmental samples (14). Currently, identification often takes weeks to complete by standard microbiological procedures (8). Consequently, other approaches such as DNA hybridization (4) and cell surface fatty acid composition analysis (M. Sasser and M. Roy, Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, P44, p. 281) have been explored. These approaches have reduced the completion time for L. monocytogenes identification to 48 to 72 h. Recent advances in the taxonomy of the genus Listeria have clarified the picture of this heterogeneous group of bacteria (13). The group includes not only L. monocytogenes but also L. seeligeri, L. welshimeri, L. ivanovii, L. innocua, L. grayi, and L. murrayi. The first five species are distinguished from each other on the basis of hemolytic activity and fermentation of certain sugars. Only L. monocytogenes strains are presently considered human pathogens, although L. seeligeri and L. welshimeri have been documented recently to have caused infections in humans (1, 10). The present study describes a rapid identification (RAPID) scheme for the identification of L. monocytogenes and four other Listeria species in less than 8 h after colony formation in 40 h at 30°C in lithium chloride-ceftazidime agar (LCA; 6). (Results of this study were presented at the 89th Annual Meeting of the American Society for Microbiology in New Orleans, La., 14 to 18 May 1989 [Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, P36, p. 325].) Bacterial strains and plating media. The same 59 listeriae and 14 nonlisterial strains described elsewhere (7) were used in this study. These were streak inoculated onto plates of LCA (6), a recently developed selective medium consisting of brain heart infusion agar (BHIA; Difco Laboratories) and the following selective agents: 0.5% LiCl, 1.0% glycine anhydride, and 50 ,ug of ceftazidime pentahydrate per ml (6). Well-isolated colonies were obtained after 40 h at 30°C. Those that were unable to grow on LCA plates were inoculated onto BHIA plates. Food samples. The same four naturally contaminated food samples described elsewhere (7) were used in this study. Enumeration procedure. Food slurries were serially diluted in sterile 0.1% peptone water and surface inoculated 1166

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seconds, a sticky slurry was observed with cells of gramnegative bacteria. Gram-positive bacteria such as the listeriae were impervious to the lytic action of alkali. Conventional microbiological procedures. After the diagnostic plates had been examined, each culture was streaked with a sterile inoculation loop to obtain isolated colonies after 24 h at 35°C. The plates were examined, and the tests recommended by McClain and Lee (9) were used for the complete identification of the Listeria species. Pure cultures (i) Recognition of the listeriae. Well-isolated cells of listeriae grew on LCA as white, finely textured, dense, dome-shaped colonies that attained a diameter of about 2.5 mm, with the exception of L. ivanovii colonies, which were smaller (diameter, 2.0 mm). The colonies that appeared bluish at the rim when viewed by the SHOT technique consisted predominantly of distinct short rods whose lengths were about three times their width. Many of these cells were in pairs, with a few single cells that were long. Consistent with the findings of Lovett (8), tumbling motility was observed among the short rods from cultures that were grown at 30°C. All showed vigorous catalase activity and, as expected, were negative in the KOH viscosity test. Some Listeria cultures showed a slight stickiness that should not be confused with the marked stickiness induced by the KOH solution in gram-negative bacteria. This composite of characteristics was highly efficient in screening for Listeria colonies. Moreover, LCA was observed to be highly selective for the listeriae; Corynebacterium aquaticum formed pin-point colonies on LCA. Among the nonlisterial cultures on BHIA plates, C. aquaticum and Jonesia denitrificans (previously designated Listeria denitrificans and now considered to be in a separate genus) exhibited similar characteristics, such as the bluish hue of colonies when viewed by the SHOT technique. However, the colonies of the former were tiny, while the colonies of the latter were smaller than those of the listeriae. The iridescence of the colonies of the gram-positive cocci and Kurthia zopfii was quite distinct from the bluish hue of the Listeria colonies. (ii) Identification of Listeria species. Five hours after transfer from LCA, colonies of L. monocytogenes and L. seeligeri were observed to be CAMP test positive, as indicated by the clearing on the area of the blood agar in proximity to the colony and the P-lysin disk. There was no clearing on the area next to the colony and distal to the disk until P-lysin had diffused into the area. L. ivanovii colonies exhibited rapid hemolysis (4 h) without potentiation by the staphylococcal ,-lysin disk. As expected, the rest of the Listeria species exhibited no hemolytic activity. In 6 h, L. monocytogenes colonies turned yellowish on rhamnose purple agar but not on xylose purple agar. The opposite was true with colonies of L. seeligeri. Only the colonies of L. seeligeri, L. welshimeri, and L. ivanovii turned yellowish on xylose purple agar. The yellowing of the colonies was best observed by indirect lighting. Table 1 summarizes the differentiation of all but three Listeria species by the abbreviated number of properties that constitute the RAP-ID scheme. Additional tests, such as mannitol fermentation and nitrate reduction, were required for the differentiation of the rhamnose-negative strains of L. innocua from L. gray, L. murrayi, and J. denitrificans. Naturally contaminated food samples. Advantage was taken of the availability of naturally contaminated food samples from a previous study (7) to assess the efficacy of the RAP-ID scheme. Suspect colonies recovered on LCA plates from raw oysters, brie cheese, raw pork sausage, and

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TABLE 1. Differentiation of most Listeria species by the RAP-ID scheme Species

L. L. L. L. L.

monocytogenes seeligeri ivanovii welshimeri

innocuad

Hemolytic activity determined by: CAMP testa Hemolysisb

Fermentation of: L-Rhamnose D-Xylose

+ + -

-

-

+

+

-

+

-

_ -

_

+

+

+

a Detection of hemolytic activity in 6 h with staphylococcal P-lysin potentiation. b Detection of hemolytic activity in 4 h without potentiation. c +, Positive and negative results. d Excluding rhamnose-negative strains.

sliced chicken luncheon meat were typical (white, dense, finely textured, and 2.5 mm in diameter) and readily recognized by their bluish hue when viewed by the SHOT technique. Examination of representative colonies by the RAP-ID scheme indicated that they were L. monocytogenes; all were positive for the CAMP test and rhamnose acidification within 6 h but negative for xylose acidification. Identical results were obtained after the conventional procedure of culture purification and prolonged incubation (18 to 24 h). The nonlisterial colonies that emerged on LCA were mostly gram-positive cocci (streptococci and staphylococci), whose red-blue iridescence was distinct from the bluish hue of Listeria colonies when viewed by the SHOT technique. The few large Bacillus and small yeast colonies appeared white. Reddish, pin-point colonies of lactobacilli were also observed. Further characterization with biochemical reactions were deemed unnecessary, since these organisms were distinctly different from the listeriae. The results of the present study demonstrate the efficacy of the RAP-ID scheme for the same-day identification of L. monocytogenes colonies recovered from foods. The approach involves subjecting each representative suspect colony directly to a battery of accelerated conventional tests without prior purification such that results are obtained within a matter of a few hours. The scheme is essentially a truncated version of the differentiation scheme of Rocourt et al. (11) in that only the most essential characters are included, except for the redundancy of rhamnose and xylose fermentation, which serves as a check for atypical strains of L. monocytogenes. The omission of tests such as mannitol fermentation, the CAMP test with Rhodococcus equi, and umbrella motility has no effect on the efficiency of the RAP-ID scheme in identifying various strains of L. monocytogenes. However, the scheme is still capable of identifying L. seeligeri, L. welshimeri, L. ivanovii, and strains of L. innocua that ferment rhamnose. Although L. monocytogenes is currently the only Listeria species recognized as pathogenic, L. seeligeri and L. Welshimeri have been documented in human infections (1, 10). As a primary plating medium, LCA plays a critical role in the RAP-ID scheme by selectively promoting the growth of listeriae in the form of large colonies of distinctly rod-shaped cells with high catalase activity. Consequently, the scheme obviates the practice of subculturing to a rich, nonselective medium for the purpose of eliminating the likelihood of mistakenly discarding isolates of listeriae that appear in their coccoid forms and exhibit very poor catalase activity that may be overlooked (13). Therefore, the possibility of confusing colonies of gram-positive cocci (streptococci and

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staphylococci) with colonies of listeriae is remote. Moreover, the robustness of the mass of cells which have also been doubling during this period on the test plates appears to contribute to the acceleration of the hemolytic and fermentative activities. The determination of these properties within a few hours of incubation indicates that LCA-grown cells may already have the necessary infrastructure to synthesize hemolysins and to catabolize sugars. Although LCA is quite inhibitory for gram-negative bacteria, the use of the KOH viscosity test as a substitute for the Gram stain provides a convenient means to ascertain that one is dealing only with gram-positive isolates. The test was described over 50 years ago (12), and its efficacy has been verified in recent years (2, 3, 5); however, the use of this convenient and cost-effective procedure does not appear to be widespread. The number of food samples that can be determined to contain significant levels of listeriae by one person in 48 h would depend on the level of contamination and the stringency in the number of suspect colonies examined. At levels of about 100 listeria cells per gram, one can handle 18 food samples by dividing them into three sets with the examination of two suspect colonies per sample. At higher levels of contamination, examination of food samples may be reduced to three if 10 representative suspect colonies are examined per sample. In conclusion, the RAP-ID scheme is to date the most rapid procedure that can be performed by a person trained in basic aseptic techniques for the identification of L. monocytogenes and four other listeriae. It does not require any special equipment or unstable reagents. Moreover, it is

cost-effective and convenient. LITERATURE CITED 1. Andre, P., and A. Genicot. 1987. First isolation of Listeria welshimeri from human beings. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 263:605-606. 2. Bourgault, A.-M., and F. Lamothe. 1988. Evaluation of the

3. 4.

5. 6. 7.

8. 9. 10.

11.

12.

13.

14.

KOH test and the antibiotic disk test in routine clinical anaerobic bacteriology. J. Clin. Microbiol. 26:2144-2146. Buck, J. D. 1982. Nonstaining (KOH) method for determination of Gram reactions of marine bacteria. Appl. Environ. Microbiol. 44:992-993. Datta, A. R., B. A. Wentz, D. Shook, and M. W. Trucksess. 1988. Synthetic oligodeoxyribonucleotide probes for detection of Listeria monocytogenes. Appl. Environ. Microbiol. 54:29332937. Gregersen, T. 1978. Rapid method for distinction of Gramnegative from Gram-positive bacteria. Eur. J. Appl. Microbiol. Biotechnol. 5:123-127. Lachica, R. V. 1990. Selective plating medium for quantitative recovery of food-borne Listeria monocytogenes. Appl. Environ. Microbiol. 56:167-169. Lachica, R. V. 1990. Simplified Henry technique for initial recognition of Listeria colonies. Appl. Environ. Microbiol. 56:1164-1165. Lovett, J. 1987. Listeria isolation, p. 29.01-29.12. In Bacteriological analytical manual (supplement). Association of Official Analytical Chemists, Arlington, Va. McClain, D., and W. H. Lee. 1988. Development of USDA-FSIS method for isolation of Listeria monocytogenes from raw meat and poultry. J. Assoc. Off. Anal. Chem. 71:660-664. Rocourt, J., H. Hof, A. Schrettenbrunner, R. Malinverli, and J. Brille. 1986. Meningite purulente aige a Listeria seeligeri chez un adulte immunocompetente. Schweiz. Med. Wochenschr. 116:248-251. Rocourt, J., A. Schrettenbrunner, and H. P. R. Seeliger. 1983. Differentiation biochimique des groupes genomiques de Listeria monocytogenes (sensu lato). Ann. Inst. Pasteur (Paris) 134: 65-71. Ryu, E. 1938. On the Gram-differentiation of bacteria by the simplest method. J. Jpn. Soc. Vet. Sci. 17:31. Seeliger, H. P. R., and D. Jones. 1986. Genus Listeria Pirie 1940, ALp. 1235-1245. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. Wehr, H. M. 1987. Listeria monocytogenes-a current dilemma. J. Assoc. Off. Anal. Chem. 70:769-772.

Same-day identification scheme for colonies of Listeria monocytogenes.

A diagnostic scheme is described for the same-day identification of food-borne cells of Listeria monocytogenes that emerge in 40 h at 30 degrees C as ...
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