Alkali Method for Rapid Recovery of Yersinia enterocolitica and Yersinia pseudotuberculosis from Foods

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1980, p /80/ /06$02.00/0 Vol. 39, No. 1 Alkali Method for Rapid Recovery of Yersinia enterocolitica and Yersinia pseudotuberculosis from Foods C. C. G. AULISIO,* I. J. MEHLMAN, AND A. C. SANDERS Division ofmicrobiology, Food and Drug Administration, Washington, D.C A new culture method employing a potassium hydroxide treatment was compared with the conventional cold enrichment method for efficacy in recovering Yersinia sp. from naturally and artificially contaminated food. The new method increased the yield of Yersinia sp. fourfold and the sensitivity 100-fold, shortened the incubation period, and appreciably decreased the growth of non- Yersinia bacteria from a variety of meats, shellfish, and vegetables. Yersinia foodborne infections in humans have been reported in Japan (1), Czechoslovakia (8), Canada (5), and the United States (2). As a result of an episode of food poisoning in the United States caused by ingestion of Yersiniacontaminated chocolate milk, a Yersinia monitoring program was established at the Food and Drug Administration in At the beginning of the program it was known that the two species of Yersinia associated with food poisoning (Y. enterocolitica and Y. pseudotuberculosis) were among the few enteric pathogens capable of growth at refrigeration temperature (4 C). It was also known that Yersinia grows slowly at 4 C, that at higher temperatures (22 to 30 C) Yersinia is overgrown by other bacteria frequently present in food samples, and that at 4 C, depending upon the initial contamination level of the food sample and the type of enrichment broth used, the recovery period of Yersinia from contaminated foods ranges from 2 to 4 weeks. Data are presented on the tolerance of Yersinia species to dilute alkali, and a rapid recovery method dependent upon this tolerance is given for the isolation of Yersinia from foods. MATERIALS AND METHODS Yersinia species and strains. Y. enterocolitica and Y. pseudotuberculosis used in this study were obtained as follows: Y. enterocolitica strains P-336, 1474, 867, and 48 and Y. pseudotuberculosis strain P- 281 were from D. J. Brenner, Center for Disease Control, Atlanta, Ga.; Y. pseudotuberculosis strains 1A and 1B were from D. Cavanaugh, Walter Reed Army Medical Center, Washington, D.C.; and Y. pseudotuberculosis strain DSWHF was from C. Ormes, Washington Hospital Center, Washington, D.C. Strain HOBS-b7 was recovered in our laboratory. Enriched media. Phosphate-buffered saline (PBS), sorbitol-bile broth, and hemoglobin-oxalatebile-sorbitol broth (HOBS) as described by I. J. Mehlman et al. (92nd Annu. Meet. Assoc. Off. Anal. Chem., 1978, abstr. no. 171), as well as standard preparations of Difco veal infusion broth and Difco brain heart infusion medium (BHI) were used as enriched media. Plating medium. MacConkey agar (BBL, Cockeysville, Md.) was prepared according to the recommendations of the manufacturer. Food samples. Celery, chicken, lettuce, oyster, pork, and spinach purchased from local supermarkets were selected at random from the fresh meat, shellfish, and vegetable counters and were used as test food samples. Weak alkali solution. Potassium hydroxide (KOH) pellets (J. T. Baker Chemical Co., Phillipsburg, N.J.) were weighed and dissolved in 0.5% NaCl to give a stock solution of 40% KOH. The stock solution was filter-sterilized, and it was stored at 4 C. A 1:80 dilution of the stock solution was made in 0.5% NaCl to give a solution of 0.5% KOH. Methodology. Foods were weighed (50 g) and mixed with enrichment medium (450 ml) in a Waring blender at approximately 8,000 rpm for 2 min. Portions of food suspension were distributed (100 ml) in Wheaton storage bottles (Wheaton Scientific, Millville, N.J.) and artificially contaminated with a Yersinia sp. culture grown at 26 C for 48 h (bacterial count approximately 109 cells/ml). Tenfold serial dilutions of the bacterial suspension were made in sterile 0.5% NaCl; 1 ml of the bacterial dilution was added to 100 ml of the food homogenate; 1 ml of sterile 0.5% NaCl solution was added to 100 ml of uncontaminated control food homogenate. Contaminated and control food homogenates were incubated at either 4 or 26 C; other contaminated and control homogenates were prepared in duplicate and simultaneously incubated at both temperatures. The samples were examined for recoverable Yersinia on days 1, 2, 3, 7, 10, and 13. Two loopsful from each enrichment were examined for Yersinia. One loopful (0.04 ml) of enrichment was gently mixed in 0.1 ml of 0.5% NaCl for a few seconds, then streaked on a MacConkey agar plate. The other loopful was treated similarly but mixed with 0.1 ml of 0.5% KOH in 0.5% NaCl and streaked to another MacConkey agar plate. To reduce the sampling error, especially with shellfish and vegetables, the procedure was repeated by diluting 0.5 ml of enrichment in 4.5 ml of 0.5% NaCl or in 4.5 ml of 0.5% KOH in 0.5% L35

2 136 AULISIO, MEHLMAN, AND SANDERS NaCl before streaking on MacConkey agar. The MacConkey agar plates were incubated at 26 C for 48 h. Usually two or three small, colorless colonies were selected and characterized biochemically in accordance with procedures described by I. J. Mehlman et al. (92nd Annu. Meet. Assoc. Off. Anal. Chem., 1978, abstr. no. 171). RESULTS Data on relative resistance of Enterobacteriaceae to weak alkali are presented in Table 1. The viable counts per milliliter determination was performed with veal infusion cultures, incubated at 220C for 48 h, on Trypticase soy agar. A 0.5-ml portion of veal infusion culture was added to 4.5 ml of 0.5% KOH in 0.5% NaCl and incubated at 220C. After 1 min of exposure to alkali, the Yersinia sp. viable count dropped 2 to 3 logs. The viable count for the other Enterobacteriaceae dropped 7 to 8 logs. Y. enterocolitica was recoverable even after a 5-min exposure to alkali. Figure 1 illustrates a MacConkey agar plate after 48 h of incubation at 260C. One-half of the plate was streaked with alkali-treated inoculum, the other half with saline-treated inoculum. Many small, distinct, colorless colonies characteristic of Yersinia were seen on the alkalitreated inoculum; numerous large, pigmented, mucous colonies characteristic of non-yersinia colonies were seen on the saline-treated inoculum. Table 2 presents results obtained in tests with chicken meat artificially contaminated with Y. enterocolitica and suspended in several enriched media. After 3 days of incubation at 40C, the percent recovery of Yersinia was greater in the NaCl control preparations than in KOH test material (33 versus 6%); after 7 days, the percent recovery was the same for both NaCl and KOH preparations (46%). After 10 days, however, the percent recovery was strikingly different: about TABLE 1. Tolerance ofenterobacteriaceae to KOHa Viable count/ml' Cultureb 0 min 1 min 3 min 5 min Y. enterocolitica x x x x 103 Y. pseudotuberculosis 1.0 x x 103 ND ND P-281 E. coli 1.0 x 108 <10 S. typhimurium 5.0 x 107 <10 S. sonnei 6.0 x 107 <10 S. flexneri 5.0 x 107 <10 A 0.5-rnl amount of veal infusion culture added to 4.5 ml of 0.5% KOH (in 0.5% saline) incubated at 22 C. b48-h culture in veal infusion, 22 C. 'Titer, Trypticase soy agar for total viable count. ND, Not done. - I.P; 11 IF APPL. ENVIRON. MICROBIOL. / I..:, 11!I-II I" FIG. 1. MacConkey agar plate after 48 h of incubation at 26 C. Streaked with artificially contaminated pork homogenate treated with saline (top section) and alkali (bottom section). 53% for the NaCl material and 93% for the KOHtreated preparation. The test strain was recovered from four of five media at all levels of contamination and at two of three levels of contamination in BHI medium by the alkali method. The 100-level contaminated BHI culture was retested on day 13. Numerous Yersinia colonies were observed on the alkali-treated plate but not on the saline-treated plate. Although the HOBS enrichment medium appeared to be more suitable than PBS for Yersinia growth, it facilitated growth of many non- Yersinia bacteria, some of which were resistant to alkali. For this reason, PBS was used as the enrichment medium in this study. In 1960, Li et al. (6) reported bactericidal action by shellfish extracts; in 1967, Liu and Cipola (7) found that shellfish liver (digestive diverticula) contained most of the bactericidal factor. In our experiment, therefore, we compared whole oyster and oyster minus stomach and digestive diverticula as media for Yersinia growth. Results of our study confirmed the findings of Li et al. (6) and Liu and Cipola (7). A low level (100 cells/ml) of Yersinia contamination of oyster was detected 3 to 6 days earlier in oyster minus stomach and digestive diverticula than in the whole oyster. Our experiment also showed that after 3 days of incubation at 4 C the percent recovery of Yersinia was greater in the NaCl control preparations than in the KOH test material (66 versus 0%) (Table 3). After 7 days, the percent recovery was the same for both NaCl and KOH preparations (66%). The oyster

3 VOL. 39, 1980 TABLE 2. a ALKALI METHOD FOR YERSINIA RECOVERY 137 Recovery of Y. enterocolitica P-336 from chicken: comparison of alkali and saline treatment of enriched media incubated at 4 C Presence of Y. enterocolitica on day:b Medium' Initial inoculation (cells/ ml) NaCl KOH NaCl KOH NaCl KOH PBS Uninoculated , , SB Uninoculated , , HOBS Uninoculated , , VIB Uninoculated , , BHI Uninoculated , , % Recovery SB, Sorbitol-bile broth; VIB, veal infusion broth. beach symbol indicates presence (+) or absence (0) of Y. enterocolitica. Final value in each column represents cumulative percent recovery from inoculated enrichments. TABLE 3. Recovery of Y. enterocolitica P-336: comparison of alkali and saline treatment on oyster enrichmenta incubated at 4 C Presence of Y. enterocolitica on day:b Food Initial inoculation (cells/ml) NaCl KOH NaCl KOH NaCl KOH NaCl KOH Oyster (whole) Uninoculated , ,000, Oyster (minus stomach Uninoculated and digestive divertic ula) 10, ,000, % Recovery a Oyster homogenized to 10% in PBS (ph 7.6). b See footnote b to Table 2. minus stomach and digestive diverticula preparation accounted for five of eight recoveries. After 10 and 13 days, the percent recovery was remarkably different for the NaCl and KOH preparations, that of the control being approximately one-half that of the KOH-treated preparations (50 versus 83 and 100%). All levels of contamination were detected in the oyster minus stomach and digestive diverticula preparation by the KOH method on days 7, 10, and 13, and only on day 13 in the whole oyster preparation. The alkali method was better than the saline method for retrieving Y. enterocolitica (P-336) from cold enrichments (Tables 2 and 3). To determine whether the alkali method was useful in recovering other strains of Yersinia, an experiment employing representative strains of Y. enterocolitica and Y. pseudotuberculosis was

4 138 AULISIO, MEHLMAN, AND SANDERS carried out. The biochemical profiles for these to 100 cells/ml) of Yersinia. One set, containing strains are given in Table 4. both contaminated and control media, was in- Pork homogenized in PBS served as the en- cubated at 40C, and the other set was incubated riched medium. Enrichments were prepared in at 260C. Results are shown in Table 5. After 1 duplicate sets and inoculated with low levels (10 day of incubation at 260C, all but one strain of Biochemical profile of Y. enterocolitica and Y. pseudotuberculosis strains resistant to weak alkali solution Biochemical reaction' Species Strain' Source TSIb Orni- Gela- Raffi- Sor- Rham- In- VP Citthine tin nose bitol nose dole rate TABLE 4. APPL. ENVIRON. MICROBIOL. Y. enterocolitica P-336 Human A/A Water K/A Human A/A Human A/A HOBS-b7 Celery A/A Y. pseudotuberculosis P-281 Swallow K/A DSWHC Human K/A A Unknown K/A B Guineapig K/A 'All strains were motile at 26 C but not at 370C. b TSI, Triple sugar iron. Slant/butt. K, Alkaline; A, acid. c Performed at 26 C, except gelatin hydrolysis held at room temperature. All strains had positive reactions for urease and methyl red. All strains had negative reactions for lysine decarboxylase, phenylalanine, and KCN. VP, Voges-Proskauer. TABLE 5. Effect of temperature on the retrieval of representative Yersinia strains from pork by enrichment in PBSa Presence of Yersinia on day:' Initial inoc- Strainb ulation (cells/ 1 (260C) 2 (260C) 7 (40C) 13 (40C) ml) NaCl KOH NaCl KOH NaCl KOH NaCl KOH Y. enterocolitica P HOBS-b Y. pseudotuberculosis P DSWHC A B Uninoculated control % Recovery a Pork homogenized to 10% in PBS (ph 7.6). 'Bacterial inoculum grown 48 h in veal infusion broth at 26 C. 'See footnote b to Table 2.

5 VOL. 39, 1980 Y. enterocolitica (1474) and Y. pseudotuberculosis (DSWHC) were recovered by the alkali method; none of the test strains were recovered by the saline method. The percent recovery of Yersinia in the KOH test material increased from 55% on day 1 to 94% on day 2. Further incubation at 260C made Yersinia recovery difficult and erratic. After 7 days of incubation at 40C, only one strain (Y. enterocolitica 48) was recovered, and that was by the alkali method. After 13 days at 40C, the percent recovery by the alkali method increased to 33%, whereas recovery by the saline method remained at 0%. However, after only 2 days of incubation at 260C the percent recovery of test Yersinia by the KOH method was 94% as compared with 33% recovery after 13 days of incubation at 40C. None of the Y. pseudotuberculosis strains were recovered from any of the 40C enrichments. Fifty-four Yersinia isolates, biochemically different from any of the other test strains used, were isolated from six foods. Table 6 shows the biochemical reactions of these Yersinia and the number of times each was isolated from the various foods. Of 54 isolates, 43 (80%) were recovered by the alkali method, and 11 (20%) were recovered by the saline method. Biochemical reactions for one-half of the naturally contaminated food isolates were similar to those of isolates recovered from humans, as described by Bottone et al. (3), and to those of isolates from foods, as described by Hanna et al. (4). DISCUSSION This study shows that recovery of Yersinia from food is complicated by a technical difficulty ALKALI METHOD FOR YERSINIA RECOVERY 139 rather than by a nutritional requirement of Yersinia sp. Because Yersinia grows more slowly than non-yersinia organisms, its population is quickly overgrown and easily masked when streaked on a weakly selective isolatory agar. By treating the inoculum with 0.5% KOH in 0.5% NaCl, the difficulty is largely overcome. A number of recommended enrichments (PBS, sorbitol-bile broth, HOBS, veal infusion broth, and BHI) were found to be suitable for Yersinia recovery by the alkali method of preparing the plating inoculum; the alkali treatment killed a larger number of contaminating non- Yersinia and thus facilitated the isolation of Yersinia. The recovery of Yersinia from artificially contaminated chicken and oyster after 3 days of incubation at 40C by the saline control method was probably due to the large level (10,000 cells/ ml) of Yersinia used to contaminate the food rather than to an increase in the Yersinia population. Low levels (10 to 100 cells/ml) of Y. enterocolitica and Y. pseudotuberculosis contamination in food can be detected by the alkali method after 1 to 2 days of incubation at 260C, but only Y. enterocolitica can be detected after 7 to 10 days of incubation at 40C. This observation strongly suggests the superiority of the alkali method over the conventional method for retrieving Yersinia from food. These data also suggest that retrieval of Yersinia from lightly contaminated food is rapidly accomplished after 1 to 2 days of incubation at 260C. However, if heavy non- Yersinia contamination is suspected, the food enrichment should be incubated at 40C and tested for Yersinia on day 7 or 10. TABLE 6. Recovery of Yersinia indigenous in foods by NaCI or KOH method Cul- Pork Chicken Spinach Lettuce Celery Oyster Biochemical reactiona no. NaCl KOH NaCl KOH NaCl KOH NaCl KOH NaCl KOH NaC1 GeKa- Raff Sor- Rham- KCN rate ~~~+ b arb [ + 31ice-a b ture 7 inlbohmca 8 ecin:alsrin a oiieraton o ra antl+ oritin dcartolse 8 1oiiya26C 3 2l6tantan noaieratin o2 yin eabxlse, - + bitoylanose, + + roiiyate ~ 21 _b 4I { I+ I+ I+ + I+ a Additional biochemical reactions: all strains had positive reactions for urea, mannitol, ornithine decarboxylase, motility at 260C; all strains had negative reactions for lysine decarboxylase, phenylalanine, motility at 370C. All biochemical reactions were performned at 260C, except gelatin hydrolysis at 220C. b Biochemical reaction similar to that of isolates described by Bottone et al. (3) and Hanna et al. (4).

6 140 AULISIO, MEHLMAN, AND SANDERS Fifty-four indigenous Yersinia were found in six foods, and 43 (80%) were found by the alkali method. Like our test strains, the indigenous Yersinia were motile at 26 but not at 37 C, positive to urease and mannitol, and negative to lysine and arginine. However, they differed from the test strains in one or more sugars (raffmose, sorbitol, and rhamnose) and in KCN, gelatin, and citrate reactions. Some of the indigenous Yersinia were biochemically similar to Yersinia isolated from humans (3) and from vacuumpackaged beef and lamb (4). Alkali treatment of 48-h veal infusion cultures of Y. enterocolitica and Y. pseudotuberculosis resulted in a 3 to 5 logio loss of Yersinia, whereas Escherichia coli, Salmonella typhimurium, S. sonnei, and S. flexneri suffered a 7 to 8 log1o loss. The alkali method provides a simple, sensitive, and rapid technique for the recovery of Yersinia from mixed cultures. It has potential value in characterizing bacteria on the basis of resistance or sensitivity to weak alkali and in quickly establishing the etiology of Yersinia food poisoning, hence the more rapid removal of Yersinia-contaminated food from the marketplace. ACKNOWLEDGMENTS We thank Ralston B. Read, Jr., Acting Director, Division APPL. ENVIRON. MICROBIOL. of Microbiology, Bureau of Foods, Food and Drug Administration, and J. Anthony Morris, Department of Microbiology, University of Maryland, College Park, for their advice in the preparation of this paper. LITERATURE CITED 1. Asakawa, Y., S. Akahane, N. Kagata, M. Noguchi, R. Sakazak, and K. Tamura Two community outbreaks of human infection with Yersinia enterocolitica. J. Hyg. 71: Black, R. E., R. J. Jackson, T. Tsai, M. Medvesky, M. Shayegani, J. C. Feeley, K. I. E. Macleod, and A. M. Wakelee Epidemic Yersinia enterocolitica infection due to contaminated chocolate milk. N. Engl. J. Med. 298: Bottone, E. J., B. Chester, M. S. Malowany, and J. Allerhand Unusual Yersinia enterocolitica isolates not associated with mesenteric lymphadenitis. Appl. Microbiol. 27: Hanna, M. O., D. L. Zink, Z. L Carpenter, and C. Vanderzant Yersinia enterocolitica-like organisms from vacuum-packaged beef and lamb. J. Food Sci. 41: Health and Welfare Canada Yersinia enterocolitica gastroenteritis outbreak-montreal. Can. Dis. Weekly Rep. 2: Li, C. P Antimicrobial activity of certain marine fauna. Proc. Soc. Exp. Biol. Med. 104: Liu, 0. C., and R. J. Cipola Study of antiviral factor from shellfish. Bacteriol. Proc. 164: Olsousky, Z Mass occurrence of Yersinia enterocolitica in two establishments of collective care of children. J. Hyg. Epidemiol. Microbiol. Immunol. 19: Downloaded from on February 21, 2019 by guest