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Chapter 13 Isolation of yersinia enterocolitica from foods
Culture Media for Food Microbiology, J.E.L. Corry et al. (Eds.) 9 1995 Elsevier Science B.V. All rights reserved
219
Chapter 13 Isolation of Yersinia enterocolitica from foods E. de Boer Inspectorate for Health Protection - Food Inspection Service, PO Box 9012, 7200 GN Zutphen, The Netherlands
Many selective enrichment and plating media for the isolation of Yersinia enterocolitica from foods are described. However, at present no single isolation procedure is available for the recovery of all pathogenic strains of Yersinia enterocolitica. Cold enrichment in phosphate-buffered saline plus 1% sorbitol and 0.15% bile salts (PBSSB) and two-step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth are very efficient methods for the recovery of a wide spectrum of serotypes of Y. enterocolitica. Enrichment in irgasan ticarcillin chlorate (ITC) broth was found to be the most efficient method for the recovery of strains of serotype 0:3, which is the most common clinical serotype of Y. enterocol#ica in Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsulodin irgasan novobiocin (CIN) agar and Salmonella-Shigella deoxycholate calcium chloride (SSDC) agar are the most commonly used plating media. For the recovery of serotype 0:8 strains, the common clinical isolates in North America, enrichment in BOS and plating on CIN seems the most efficient procedure. Selection of the proper enrichment procedure will depend on the bio/serotypes of Yersinia spp. sought and on the type of food to be examined. The use of more than one medium for both enrichment and plating will result in higher recovery rates of Yersinia spp. from foods. Parallel use of the following two isolation procedures is recommended. (1) Enrichment in ITC for 2 days at 24~ plating on SSDC agar (2 days at 30~ (2) Pre-enrichment in TSB for 1 day at 24~ enrichment in BOS for 5 days at 24~ alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% K O H in 0.5% NaC1 for 5 s); plating on CIN agar (2 days at 24~
Introduction
Yersinia enterocolitica is a coccoid-shaped Gram-negative, facultatively anaerobic bacterium belonging to the genus Yersinia in the Enterobacteriaceae family. The organism is recognised as a foodborne pathogen and some large food-associated outbreaks of yersiniosis have been reported. In developed countries, Y. enterocolitica can be isolated from 1-2% of all human cases of acute enteritis (Kapperud, 1991). Yersinia enterocolitica and related species have been isolated from many types of food. The majority of these food isolates differ in biochemical and serological characteristics from typical clinical strains and are usually classified as 'non-patho-
220 genic' or 'environmental' Yersinia strains. These strains, which include the 'related species' Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. aldovae, Y. rohdei, Y. mollaretii and Y. bercovieri, are ubiquitous and lack clinical significance with the exception of a few atypical cases (Kapperud, 1991). The strains associated with human disease mainly belong to the serogroups 0:3, 0:5, 27, 0:8 and 0:9 of Y. enterocolitica sensu stricto. The epidemiology of Y. enterocolitica infections is, for the greater part, not understood. There is an association with consumption of contaminated foods, especially pork (De Boer and Nouws, 1991). The increasing interest in Y. enterocolitica infections and the role of foods in some outbreaks of yersiniosis has led to the development of improved procedures for the isolation of this organism from foods during the last 10-15 years. As the numbers of Y. enterocolitica organisms in foods are usually low and there is often a great variety of background flora, direct isolation on selective plating media is seldom successful. Isolation methods usually involve enrichment of the sample followed by plating onto selective agar media and confirmation of typical colonies.
Enrichment
As a psychrotrophic organism Y. enterocolitica is able to multiply at 4~ and enrichment at this temperature for 2-4 weeks is widely used. At this low temperature, the growth rate of competitive bacteria is slowed sufficiently to enable Y. enterocolitica to multiply to numbers necessary for isolation on plating media. Media used for this 'cold enrichment' include simple buffers like phosphatebuffered saline (PBS), PBS modified by addition of 1% sorbitol and 0.15% bile salts (PBSSB) (Mehlman et al., 1978), PBS supplemented with 1% mannitol (Schiemann, 1979a), PBS with 0.5% peptone (Weagant and Kaysner, 1983), PBS with peptone and cycloheximide (Vidon and Delmas, 1981), tryptone soy broth (Van Pee and Stragier, 1979), trypticase soy broth (Schiemann, 1983a) and trisbuffered peptone water, pH 8.0 (Greenwood and Hooper, 1989). The long period required for cold enrichment is often unacceptable for quality assurance of foods. Schiemann and Olson (1984)showed that an incubation at 15~ for 2 days was as efficient as enrichment at 4~ for some weeks. Doyle and Hugdahl (1983) incubated PBS for 1-3 days at 25~ and Greenwood and Hooper (1989) incubated tris-buffered peptone water at 9~ for 11-14 days. Several other enrichment procedures involving incubation at higher temperature for shorter periods and using selective media have been proposed. Modified Rappaport broth (Wauters, 1973) has been used for many years as the first-choice medium for the isolation of the major pathogenic serotypes of Y. enterocolitica in Europe. This medium has been shown to inhibit the common North American serotype 0:8 strains of Y. enterocolitica (Schiemann, 1983a; Walker and Gilmour, 1986). Carbenicillin in modified Rappaport broth was shown to inhibit the growth of certain serotype 0:3 strains (Schiemann, 1982). However, Wauters et al. (1988)
221
stated that serogroup 0:3 strains are not inhibited by carbenicillin and that omission of this antibiotic results in decrease of selectivity of the enrichment medium. Lee et al. (1980) described two modified selenite media that were effective for recovery of certain strains of Y. enterocolitica from meats. They found it critical to limit the sample size of the blended meat suspension to 0.2 g per 100 ml enrichment medium to restrict the growth of competitive bacteria. Otherwise the slower growing Y. enterocolitica would be overgrown by the faster growing normal bacterial flora of the meat. Schiemann (1982) developed a two-step enrichment procedure for recovery of Y. enterocolitica from food. In this procedure pre-enrichment for 9 days at 4~ in yeast extract rose bengal broth is followed by selective enrichment with bile oxalate sorbose (BOS) broth at 22~ for 5 days. As the pre-enrichment medium is less selective, it will allow multiplication of small inocula and repair of injured cells. BOS broth was found especially useful for the isolation of serotype 0:8 strains, but strains of serotype 5, 27 were more difficult to recover (Schiemann, 1983a). Recently Wauters et al. (1988) developed a new enrichment broth, named ITC, derived from modified Rappaport broth and based on the selective agents irgasan, ticarcillin and potassium chlorate. In comparative studies ITC broth was especially effective for the recovery of Y. enterocolitica 0:3 from pork and porcine tonsils, while cold and two-step enrichments yielded better results for non-pathogenic strains (Wauters et al., 1988a; Kwaga et al., 1990; De Boer and Nouws, 1991). Aulisio et al. (1980) found that strains of Y. enterocolitica are more tolerant of alkaline solutions than other Gram-negative bacteria. By treating food enrichments with potassium hydroxide (KOH) solutions before plating, the background flora is markedly reduced, making selection of Yersinia colonies from similar colonies on the isolation medium easier. However, Schiemann (1983b) found that various factors, including medium, temperature and growth phase, influence alkalotolerance and reduce the effectiveness of alkali treatment. Weagant and Kaysner (1983) concluded that a specific treatment time cannot be recommended. They found that streaking three to four successive plates from the KOH rinse at 10-s intervals enhanced the probability of obtaining isolated colonies of Y. enterocolitica even when growing in the presence of numerous organisms. Direct KOH treatment of meat samples proved to be a valuable rapid method for direct isolation of Yersinia from meat contaminated with more than 102 cells per g (Fukushima, 1985). Based on results of comparative experiments the use of 0.125% KOH with an exposure time of 5 min was recommended for the direct detection of Y. enterocolitica in foods (Schraft and Untermann, 1989). Direct plating after KOH treatment is only suitable for strongly positive material (Wauters et al., 1988a).
Plating media Different agar plating media have been used to isolate Y. enterocolitica from clinical specimens and food (Table 1). Initially media like MacConkey, Salmonella-
222 TABLE 1 Selective agents in enrichment and plating media for Yersinia enterocolitica Medium a
Selective agents
Enrichment
MRB
magnesium chloride (2.8%), malachite green (0.0013%), carbenicillin (0.00025%)
Selenite Media
sodium selenite (0.15 or 0.25%), malachite green (0.002%), carbenicillin (0.001%)
PBSSB
bile salts (0.15%)
BOS
sodium oxalate (0.5%), bile salts (0.2%), irgasan (0.0004%), sodium furadantin (0.001%)
ITC
magnesium chloride (6%), malachite green (0.001%), irgasan (0.00001%), ticarcillin (0.00001%), potassium chlorate (0.1%)
Plating
CIN
sodium deoxycholate (0.05%), crystal violet (0.0001%), irgasan (0.0004%), cefsulodin (0. 0015%), novobiocin (0. 00025%)
VYE
sodium deoxycholate (0.1%), crystal violet (0.0001%), irgasan (0.0004%), cefsulodin (0.0004%), oleandomycin (0.001%), josamycin (0. 002%)
SSDC
sodium deoxycholate (0. 85%)
MacConkey
bile salts (0.15%), crystal violet (0.0001%)
a MRB, modified Rappaport broth (Wauters, 1973); Selenite Media (Lee et al., 1980); PBSSB, phosphate-buffered saline with sorbitol and bile salts (Mehlman et al., 1978); BOS, bile oxalate sorbose broth (Schiemann, 1982; this volume); ITC, irgasan ticarcillin chlorate (Wauters et al., 1988a; this volume); CIN, cefsulodin irgasan novobiocin agar (Schiemann, 1979b; this volume); VYE, virulent Yersinia enterocolitica agar (Fukushima, 1987); SSDC, Salmonella-Shigella deoxycholate calcium chloride agar (Wauters, 1973; this volume), MacConkey Agar no. 3 (Oxoid CMll5).
Shigella, desoxycholate citrate and bismuth sulphite agars, designed for the isolation of enteropathogens, were used. Lee (1977) modified MacConkey agar with Tween 80 to improve differentiation of Yersinia colonies from other lactose-negative colonies. However, lipolytic Yersinia strains which are easily recognized on this medium as white wrinkled colonies surrounded by a sheen, are usually non-pathogenic (De Boer and Seldam, 1987). Salmonella-Shigella agar was made more selective for Y. enterocolitica by addition of sodium deoxycholate and CaC12 (SSDC)(Wauters, 1973, 1988a). Colonies of Y. enterocolitica on this medium are small, round and colourless. Some species of Morganella, Proteus, Serratia and Aeromonas can grow on SSDC and differentiation of Yersinia from these competing organisms may be difficult. Schiemann (1979b) developed cefsulodin-irgasan-novobiocin (CIN) agar, a selective and differential agar medium for Y. enterocolitica. Organisms capable of
223
TABLE
2
Differential
characteristics
of
Yersinia
Characteristics
Y. enterocolitica
Urease
and
related
genera
Hafnia
Serratia
Citrobacter
Enterobacter
Escherichia
Klebsiella
Proteus
+
-
d
d
d
-
d
+
Motility
at 25~
+
+
+
+
+
+
-
+
Motility
at 37~
-
+
+
+
+
d
-
+
dihydrolase
-
-
-
+
d
-
-
-
decarboxylase
-
+
d
-
-
-
d
Arginine Lysine
Phenylalanine
deaminase
H 2 S production a
d, d i f f e r e n t
. -
reactions
in different
+ .
d .
-
. +
.
d .
d
. -
+
species.
fermenting mannitol, like yersiniae, produce red coloured 'bullseye' colonies on CIN agar. CIN medium was found to be inhibitory to Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis, but some Enterobacter, Aeromonas and Proteus strains showed a colony appearance similar to Yersinia (De Boer and Seldam, 1987). The addition of 1 /xg/ml of streptomycin improves the selectivity of CIN agar, but colonies of Y. enterocolitica are smaller (Schiemann, 1987). As the recovery rate and the colony size of Y. enterocolitica diminishes during storage of the medium, it is recommended to use CIN medium within 14 days of preparation (Petersen, 1985). On CIN agar colonies of pathogenic and environmental Yersinia strains appear similar. Fukushima (1987) developed a selective agar medium for isolation of pathogenic (virulent) Y. enterocolitica (VYE agar). Pathogenic strains form red colonies on this medium, while most environmental Yersinia strains form dark colonies with a dark peripheral zone as a result of aesculin hydrolysis.
Identification
For the differentiation of Yersinia from related genera the tests listed in Table 2 may be used. Table 3 shows the characteristics differentiating the foodborne species within the genus Yersinia. Miniaturised identification kits like the API 20E (bioMdrieux) and the Minitek system (BBL) have proven to be valuable for rapid identification of Y. enterocolitica strains (Restaino et al., 1979). Some biochemical activities (cellobiose, raffinose, indole, ONPG hydrolysis, ornithine decarboxylase, Voges-Proskauer) of Yersinia strains are temperature-dependent (Bercovier and Mollaret, 1984). These tests are preferably incubated at 25 or 30~ rather than at 37~ Serotyping, biotyping and virulence testing is essential for differentiation between pathogenic and environmental Yersinia strains. The serotyping scheme of Y. enterocolitica and related Yersinia species now involves 67 major O factors and 44 H factors (Wauters et al., 1991). Table 4 shows the biotyping scheme of Y.
224
TABLE
3
Biochemical
differentiation
Y.
Test
Yersinia
within the genus
Y.
Y.
Y.
(from Wauters
Y
et al., 1 9 8 8 b )
Y.
Y.
Y.
Y.
entero- inter- frede- kristen- aldovae rhodei mollaretii bercovieri pseudocolitica media riksenii senii tuberculosis Indole
d a
+
+
d
.
Proskauer
d
+
d
-
+
.
Citrate (Simmons)
-
+
d
-
d
+
-
-
-
L-- Ornithine
+
+
+
+
+
+
+
+
-
Mucate,
-
d
d
-
d
-
+
+
-
d
+
+
+
+
+
+
+
-
Voges-
acid
Pyrazinamidase
.
.
. .
. .
.
Sucrose
+
+
+
-
-
+
+
+
Cellobiose
+
+
+
+
-
+
+
+
-
L-- Rhamnose
-
+
+
-
+
-
-
-
+
Melibiose
-
+
-
-
-
d
-
-
+
L-- Sorbose
d
+
+
+
-
ND
+
-
-
L-- Fucose
d
d
+
d
d
ND
-
+
-
a
d, d i f f e r e n t
reactions;
ND,
.
-
not determined.
enterocolitica as proposed by Wauters et al. (1987). In Europe, Y. enterocolitica
strains of serotypes 0:3 (biogroup 4), 0:9 (biogroup 2) and 0:5, 27 (biogroup 2) are the most frequently isolated human pathogenic strains. In North America, strains causing human yersiniosis usually belong to biogroup 1B (serotypes 0:4, 0: 8, 013a,13b, 0: 18, 0: 20) Several in vitro tests have been described to determine the potential virulence of Yersinia isolates (Prpic et al., 1985). Tests for autoagglutination at 37~ (Laird and Cavanaugh, 1980), calcium dependency at 37~ on magnesium oxalate agar (Gemski et al., 1980), uptake of Congo red (Prpic et al., 1983) and pyrazinamidase activity (Kandolo and Wauters, 1985) are easy to perform in routine laboratories.
TABLE Biotypes
4 of
Yersinia enterocolitica
(Wauters
e t al., 1987)
Biogroups 1A Lipase (Tween-esterase)
+
Aesculin/Salicin
_+
24h
Indole
+
Xylose
+
Trehalose/Nitrate
+
Pyrazinamidase
+
1B
2
3
4
+
(+)
-
_
-I+
+ +
+ +
-+
5
225
Comparative studies From a comparison of pre-enrichment media, Schiemann (1983a) proposed tryptone soy broth as the medium of choice. Comparison of the two-step enrichment against cold enrichment and modified Rappaport broth showed improved recovery of human pathogenic strains of Y. enterocolitica from inoculated foods using pre-enrichment in tryptone soy broth followed by enrichment in BOS (Schiemann, 1982, 1983a; Walker and Gilmour, 1986). In a study on the occurrence of Y. enterocolitica in foods Delmas and Vidon (1985) evaluated seven enrichment procedures. Most positive samples obtained within the shortest time were found using enrichment in PBSSB and BOS, with alkali treatment before plating onto CIN agar. However, only environmental serogroups of Y. enterocolitica were isolated. The enrichment medium ITC was found superior to enrichment in BOS for isolation of pathogenic Y. enterocolitica from pork products, though enrichment in BOS isolated more non-pathogenic strains (Wauters et al., 1988a; Kwaga et al., 1990; De Boer and Nouws, 1991). In different studies cold enrichment in PBSSB gave high isolation rates of Yersinia spp., which mainly proved to be non-pathogenic environmental strains (De Boer et al., 1982, 1986; De Boer and Seldam, 1987). In these studies MRB was found a suitable medium for the isolation of serogroup 0:3 and 0:9 strains, but much less efficient for the isolation of environmental strains. Alkali treatment often resulted in a marked increase in the number of Yersinia isolations. This occurred especially when high numbers of competing organisms were present, as was usually the case for PBSSB, and when a plating medium with low selectivity, such as MacConkey agar, was used (De Boer et al., 1982; De Boer and Seldam, 1987). MacConkey agar has been demonstrated to be a very productive medium for Y. enterocolitica (Mehlman et al., 1978; Schiemann, 1979b; De Boer and Seldam, 1987). However, Yersinia colonies are hard to recognize on this medium because of its low selectivity. In several comparative studies CIN agar was found to be the most selective plating medium for Yersinia spp. (Aldova et al., 1990; Cox et al., 1990; Harmon et al., 1983; Schiemann, 1983a; Walker and Gilmour, 1986). Fukushima (1987)found that biotype 3B, serogroup 0:3 strains were inhibited on CIN agar. Differentiation of Yersinia colonies from other colonies on CIN agar is not always easy (Fukushima, 1987; De Boer and Seldam, 1987). Plating ITC enrichments onto SSDC isolated more serogroup 0:3 strains than plating onto CIN agar (Wauters et al., 1988; De Boer and Nouws, 1991). SSDC proved to be unsuitable after alkali treatment (Wauters et al., 1988a).
Discussion Addition of selective agents such as magnesium chloride, malachite green, bile salts, irgasan, and the antibiotics carbenicillin, ticarcillin and cefsulodin, to Yersinia
226
isolation media results in growth inhibition of the Gram-positive and part of the Gram-negative flora (Table 1). With regard to the differential properties of the Yersinia isolation media now in use, it is evident that the colony appearance of Yersinia on these media is not very characteristic and confirmation tests of presumptive colonies are always necessary. At present, no single isolation procedure is available for the recovery of all pathogenic strains of Y. enterocolitica from foods. Cold enrichment in PBSSB, two-step enrichment with TSB and BOS, and enrichment in ITC are the most commonly used enrichment procedures. CIN and SSDC agars are the most commonly used plating media and are also commercially available. These enrichment and plating media are not very selective for Y. enterocolitica as they support the growth of several other members of the family of Enterobacteriaceae. This makes the isolation of low numbers of Yersinia in products containing many other contaminants rather difficult. Moreover, non-pathogenic environmental Y. enterocolitica strains are very common in many raw foods and may greatly hinder the isolation of pathogenic Yersinia strains from these products. Cold enrichment and two-step enrichment are very efficient methods for the recovery of a wide spectrum of serotypes of Y. enterocolitica. However, usually one is only interested in the pathogenic serotypes and as the colony appearance of pathogenic and environmental strains on CIN and SSDC agar is similar, it requires much work to select pathogenic strains from these media. This highlights the need for the development and evaluation of agar media selecting for pathogenic serotypes, like VYE agar (Fukushima, 1985). Enrichment in ITC was found to be the most efficient method for the recovery of strains of serotype 0:3, which is the most common clinical serotype of Y. enterocolitica in Europe. The usefulness of this enrichment broth for the isolation of other pathogenic serotypes remains to be determined. This also holds for SSDC agar, which is probably less suitable for pathogenic strains other than serotype 0:3. For the recovery of the North American serotype 0:8 strains, enrichment in BOS and plating on CIN seems the most efficient procedure. Selection of the proper enrichment procedure will depend on the bio/serotypes of Yersinia spp. sought and on the type of food to be examined. The use of more than one medium for both enrichment and plating will obviously result in higher recovery rates of Yersinia spp. from foods. Parallel use of the following two isolation procedures is recommended. (1) Enrichment in ITC for 2 days at 24~
plating on SSDC agar (2 days at 30~
(2) Pre-enrichment in TSB for 1 day at 24~ enrichment in BOS for 5 days at 24~ alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaC1 for 5 s); plating on CIN agar (2 days at 24~ M o n o g r a p h s are described for CIN, BOS, ITC and SSDC in this volume.
227
References Aldova, E., Svandova, E., Votypka, J. and Sourek J. (1990) Comparative study of culture methods to detect Yersinia enterocolitica serogroup 0:3 on swine tongues. Zbl. Bakteriol. 272, 306-312. Aulisio, C.C.G., Mehlman, I.J. and Sanders, C. (1980) Alkali method for rapid recovery of Yersinia enterocolitica and Yersinia pseudotuberculosis from foods. Appl. Environ. Microbiol. 39, 135-140. Baird, R.M., Corry, J.E.L. and Curtis, G.D.W. (Eds.) (1987) Pharmacopoeia of culture media for food microbiology. Int. J. Food Microbiol. 5, 208-209. Bercovier, H. and Mollaret, H.H. (1984) Yersinia. In: Krieg, N.R. and Holt, J.G. (Eds.), Bergey's Manual of Systematic Bacteriology, Vol. 1, Williams and Wilkins, Baltimore and London. Cox, N.A., Bailey, J.S., Del Corral, F., Shotts, E.B. (1990) Comparison of enrichment and plating media for isolation of Yersinia. Poultry Sci. 69, 686-693. Curtis, G.D.W. and Baird, R.M. (Eds.) (1993) Pharmacopoeia of culture media for food microbiology: additional monographs (2). Int. J. Food Microbiol. 17, in press. De Boer, E., Hartog, B.J. and Oosterom, J. (1982) Occurrence of Yersinia enterocolitica in poultry products. J. Food Prot. 45, 322-325. De Boer, E., Seldam, W.M. and Oosterom, J. (1986) Characterization of Yersinia enterocolitica and related species isolated from foods and porcine tonsils in the Netherlands. Int. J. Food Microbiol. 3, 217-224. De Boer, E. and Seldam, W.M. (1987) Comparison of methods for the isolation of Yersinia enterocolitica from porcine tonsils and pork. Int. J. Food Microbiol. 5, 95-101. De Boer, E. and Nouws, J.F.M. (1991) Slaughter pigs and pork as a source of human pathogenic Yersinia enterocolitica. Int. J. Food Microbiol. 12, 375-378. Delmas, C.L. and Vidon, D.J.M. (1985) Isolation of Yersinia enterocolitica and related species from foods in France. Appl. Environ. Microbiol. 50, 767-771. Doyle, M.P. and Hugdahl, M.B. (1983) Improved procedure for recovery of Yersinia enterocolitica from meats. Appl. Environ. Microbiol. 45, 127-135. Fukushima, H. (1985) Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat. Appl. Environ. Microbiol. 50, 710-712. Fukushima, H. (1987) New selective agar medium for isolation of virulent Yersinia enterocolitica. J. Clin. Microbiol. 25, 1068-1073. Gemski, P., Lazere, J.R. and Casey, T. (1980) Plasmid associated with pathogenicity and calcium dependency of Yersinia enterocolitica. Infect. Immun. 27, 682-685. Greenwood, M.H. and Hooper, W.L. (1989) Improved methods for the isolation of Yersinia species from milk and foods. Food Microbiol. 6, 99-104. Harmon, M.C., Yu, C.L. and Swaminathan, B. (1983) An evaluation of selective differential plating media for the isolation of Yersinia enterocolitica from experimentally inoculated fresh ground pork homogenate. J. Food Sci. 48, 6-9. Kandolo, K. and Wauters, G. (1985) Pyrazinamidase activity in Yersinia enterocolitica and related organisms. J. Clin. Microbiol. 21,980-982. Kapperud, G. (1991) Yersinia enterocolitica in food hygiene. Int. J. Food Microbiol. 12, 53-66. Kwaga, J., Iversen, J.O. and Saunders, J.R. (1990) Comparison of two enrichment protocols for the detection of Yersinia in slaughtered pigs and pork products. J. Food Prot. 53, 1047-1049. Laird, W.J. and Cavanaugh, D.C. (1980) Correlation of autoagglutination and virulence of yersinae. J. Clin. Microbiol. 11, 430-432. Lee, W.H. (1977) Two plating media modified with Tween 80 for isolating Yersinia enterocolitica. Appl. Environ. Microbiol. 33, 215-216. Lee, W.H., Harris, M.E., McClain, D., Smith, R.E. and Johnston, R.W. (1980) Two modified Selenite media for the recovery of Yersinia enterocolitica from meats. Appl. Environ. Microbiol. 39, 205-209. Mehlman, I.J., Aulisio, C.C.G. and Sanders, A.C. (1978) Problems in the recovery and identification of Yersinia from food. J. Assoc. Off. Anal. Chem. 61,761-771. Petersen, T. (1985) Keeping quality of cefsulodin-irgasan-novobiocin (CIN) medium for detection and enumeration of Yersinia enterocolitica. Int. J. Food Microbiol. 2, 49-54.
228 Prpic, J.K., Robins-Browne, R.M. and Davey, R.B. (1983) Differentiation between virulent and avirulent Yersinia enterocolitica isolates by using Congo red agar. J. Clin. Microbiol. 18, 486-490. Prpic, J.K., Robins-Browne, R.M. and Davey, R.B. (1985) In vitro assessment of virulence in Yersinia enterocolitica and related species. J. Clin. Microbiol. 22, 105-110. Restaino, L., Grauman, G.S., McCall, W.A. and Hill, W.M. (1979) Evaluation of the Minitek and API 20E systems for identification of Yersinia enterocolitica. J. Food Prot. 42, 120-123. Schiemann, D.A. (1979a) Enrichment methods for recovery of Yersinia enterocolitica from foods and raw milk. Contr. Microbiol. Immunol. 5, 212-227. Schiemann, D.A. (1979b) Synthesis of a selective agar medium for Yersinia enterocolitica. Can. J. Microbiol. 25, 1298-1304. Schiemann, D.A. (1982) Development of a two-step enrichment procedure for recovery of Yersinia enterocolitica from food. Appl. Environ. Microbiol. 43, 14-27. Schiemann, D.A. (1983a) Comparison of enrichment and plating media for recovery of virulent strains of Yersinia enterocolitica from inoculated beef stew. J. Food Prot. 46, 957-964. Schiemann, D.A. (1983b) Alkalotolerance of Yersinia enterocolitica as a basis for selective isolation from food enrichments. Appl. Environ. Microbiol. 46, 22-27. Schiemann, D.A. (1987) Yersinia enterocolitica in milk and dairy products. J. Dairy Sci. 70, 383-391. Schiemann, D.A. and Olson, S.A. (1984) Antagonism by Gram-negative bacteria to growth of Yersinia enterocolitica in mixed cultures. Appl. Environ. Microbiol. 48, 539-544. Schraft, H. and Untermann, F. (1989) Use of KOH treatment for direct detection of Yersinia enterocolitica in foods. 10th WAVFH Int. Symp., Stockholm, Abstracts, p. 31. Van Pee, W. and Stragier, J. (1979) Evaluation of some cold enrichment and isolation media for the recovery of Yersinia enterocolitica. Antonie van Leeuwenhoek J. Microbiol. Serol. 45, 465-477. Vidon, D.J.M. and Delmas, C.L. (1981) Incidence of Yersinia enterocolitica in raw milk in Eastern France. Appl. Environ. Microbiol. 41, 355-359. Walker, S.J. and Gilmour, A. (1986) A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras. J. Appl. Bact. 60, '175-183. Wauters, G. (1973) Improved methods for the isolation and the recognition of Yersinia enterocolitica. Contr. Microbiol. Immunol. 2, 68-70. Wauters, G., Kandolo, K. and Janssens, M. (1987) Revised biogrouping scheme of Yersinia enterocolitica. Contr. Microbiol. Immunol. 9, 14-21. Wauters, G., Goossens, V., Janssens, M. and Vandepitte, J. (1988a) New enrichment method for isolation of pathogenic Yersinia enterocolitica serogroup 0:3 from pork. Appl. Environ. Microbiol. 54, 851-854. Wauters, G., Janssens, M., Steigerwalt, A.G. and Brenner, D.J. (1988b) Yersinia mollaretii sp.nov, and Yersinia bercovieri sp. nov., formerly called Yersinia enterocolitica biogroups 3A and 3B. Int. J. Syst. Bacteriol. 38, 424-429. Wauters, G., Aleksic, S., Charlier, J. and Schulze, G. (1991) Somatic and flagellar antigens of Yersinia enterocolitica and related species. Contr. Microbiol. Immunol. 12, 239-243. Weagant, S.D. and Kaysner, C.A. (1983) Modified enrichment broth for isolation of Yersinia enterocolitica from nonfood sources. Appl. Environ. Microbiol. 45, 468-471.
219
Chapter 13 Isolation of Yersinia enterocolitica from foods E. de Boer Inspectorate for Health Protection - Food Inspection Service, PO Box 9012, 7200 GN Zutphen, The Netherlands
Many selective enrichment and plating media for the isolation of Yersinia enterocolitica from foods are described. However, at present no single isolation procedure is available for the recovery of all pathogenic strains of Yersinia enterocolitica. Cold enrichment in phosphate-buffered saline plus 1% sorbitol and 0.15% bile salts (PBSSB) and two-step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth are very efficient methods for the recovery of a wide spectrum of serotypes of Y. enterocolitica. Enrichment in irgasan ticarcillin chlorate (ITC) broth was found to be the most efficient method for the recovery of strains of serotype 0:3, which is the most common clinical serotype of Y. enterocol#ica in Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsulodin irgasan novobiocin (CIN) agar and Salmonella-Shigella deoxycholate calcium chloride (SSDC) agar are the most commonly used plating media. For the recovery of serotype 0:8 strains, the common clinical isolates in North America, enrichment in BOS and plating on CIN seems the most efficient procedure. Selection of the proper enrichment procedure will depend on the bio/serotypes of Yersinia spp. sought and on the type of food to be examined. The use of more than one medium for both enrichment and plating will result in higher recovery rates of Yersinia spp. from foods. Parallel use of the following two isolation procedures is recommended. (1) Enrichment in ITC for 2 days at 24~ plating on SSDC agar (2 days at 30~ (2) Pre-enrichment in TSB for 1 day at 24~ enrichment in BOS for 5 days at 24~ alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% K O H in 0.5% NaC1 for 5 s); plating on CIN agar (2 days at 24~
Introduction
Yersinia enterocolitica is a coccoid-shaped Gram-negative, facultatively anaerobic bacterium belonging to the genus Yersinia in the Enterobacteriaceae family. The organism is recognised as a foodborne pathogen and some large food-associated outbreaks of yersiniosis have been reported. In developed countries, Y. enterocolitica can be isolated from 1-2% of all human cases of acute enteritis (Kapperud, 1991). Yersinia enterocolitica and related species have been isolated from many types of food. The majority of these food isolates differ in biochemical and serological characteristics from typical clinical strains and are usually classified as 'non-patho-
220 genic' or 'environmental' Yersinia strains. These strains, which include the 'related species' Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. aldovae, Y. rohdei, Y. mollaretii and Y. bercovieri, are ubiquitous and lack clinical significance with the exception of a few atypical cases (Kapperud, 1991). The strains associated with human disease mainly belong to the serogroups 0:3, 0:5, 27, 0:8 and 0:9 of Y. enterocolitica sensu stricto. The epidemiology of Y. enterocolitica infections is, for the greater part, not understood. There is an association with consumption of contaminated foods, especially pork (De Boer and Nouws, 1991). The increasing interest in Y. enterocolitica infections and the role of foods in some outbreaks of yersiniosis has led to the development of improved procedures for the isolation of this organism from foods during the last 10-15 years. As the numbers of Y. enterocolitica organisms in foods are usually low and there is often a great variety of background flora, direct isolation on selective plating media is seldom successful. Isolation methods usually involve enrichment of the sample followed by plating onto selective agar media and confirmation of typical colonies.
Enrichment
As a psychrotrophic organism Y. enterocolitica is able to multiply at 4~ and enrichment at this temperature for 2-4 weeks is widely used. At this low temperature, the growth rate of competitive bacteria is slowed sufficiently to enable Y. enterocolitica to multiply to numbers necessary for isolation on plating media. Media used for this 'cold enrichment' include simple buffers like phosphatebuffered saline (PBS), PBS modified by addition of 1% sorbitol and 0.15% bile salts (PBSSB) (Mehlman et al., 1978), PBS supplemented with 1% mannitol (Schiemann, 1979a), PBS with 0.5% peptone (Weagant and Kaysner, 1983), PBS with peptone and cycloheximide (Vidon and Delmas, 1981), tryptone soy broth (Van Pee and Stragier, 1979), trypticase soy broth (Schiemann, 1983a) and trisbuffered peptone water, pH 8.0 (Greenwood and Hooper, 1989). The long period required for cold enrichment is often unacceptable for quality assurance of foods. Schiemann and Olson (1984)showed that an incubation at 15~ for 2 days was as efficient as enrichment at 4~ for some weeks. Doyle and Hugdahl (1983) incubated PBS for 1-3 days at 25~ and Greenwood and Hooper (1989) incubated tris-buffered peptone water at 9~ for 11-14 days. Several other enrichment procedures involving incubation at higher temperature for shorter periods and using selective media have been proposed. Modified Rappaport broth (Wauters, 1973) has been used for many years as the first-choice medium for the isolation of the major pathogenic serotypes of Y. enterocolitica in Europe. This medium has been shown to inhibit the common North American serotype 0:8 strains of Y. enterocolitica (Schiemann, 1983a; Walker and Gilmour, 1986). Carbenicillin in modified Rappaport broth was shown to inhibit the growth of certain serotype 0:3 strains (Schiemann, 1982). However, Wauters et al. (1988)
221
stated that serogroup 0:3 strains are not inhibited by carbenicillin and that omission of this antibiotic results in decrease of selectivity of the enrichment medium. Lee et al. (1980) described two modified selenite media that were effective for recovery of certain strains of Y. enterocolitica from meats. They found it critical to limit the sample size of the blended meat suspension to 0.2 g per 100 ml enrichment medium to restrict the growth of competitive bacteria. Otherwise the slower growing Y. enterocolitica would be overgrown by the faster growing normal bacterial flora of the meat. Schiemann (1982) developed a two-step enrichment procedure for recovery of Y. enterocolitica from food. In this procedure pre-enrichment for 9 days at 4~ in yeast extract rose bengal broth is followed by selective enrichment with bile oxalate sorbose (BOS) broth at 22~ for 5 days. As the pre-enrichment medium is less selective, it will allow multiplication of small inocula and repair of injured cells. BOS broth was found especially useful for the isolation of serotype 0:8 strains, but strains of serotype 5, 27 were more difficult to recover (Schiemann, 1983a). Recently Wauters et al. (1988) developed a new enrichment broth, named ITC, derived from modified Rappaport broth and based on the selective agents irgasan, ticarcillin and potassium chlorate. In comparative studies ITC broth was especially effective for the recovery of Y. enterocolitica 0:3 from pork and porcine tonsils, while cold and two-step enrichments yielded better results for non-pathogenic strains (Wauters et al., 1988a; Kwaga et al., 1990; De Boer and Nouws, 1991). Aulisio et al. (1980) found that strains of Y. enterocolitica are more tolerant of alkaline solutions than other Gram-negative bacteria. By treating food enrichments with potassium hydroxide (KOH) solutions before plating, the background flora is markedly reduced, making selection of Yersinia colonies from similar colonies on the isolation medium easier. However, Schiemann (1983b) found that various factors, including medium, temperature and growth phase, influence alkalotolerance and reduce the effectiveness of alkali treatment. Weagant and Kaysner (1983) concluded that a specific treatment time cannot be recommended. They found that streaking three to four successive plates from the KOH rinse at 10-s intervals enhanced the probability of obtaining isolated colonies of Y. enterocolitica even when growing in the presence of numerous organisms. Direct KOH treatment of meat samples proved to be a valuable rapid method for direct isolation of Yersinia from meat contaminated with more than 102 cells per g (Fukushima, 1985). Based on results of comparative experiments the use of 0.125% KOH with an exposure time of 5 min was recommended for the direct detection of Y. enterocolitica in foods (Schraft and Untermann, 1989). Direct plating after KOH treatment is only suitable for strongly positive material (Wauters et al., 1988a).
Plating media Different agar plating media have been used to isolate Y. enterocolitica from clinical specimens and food (Table 1). Initially media like MacConkey, Salmonella-
222 TABLE 1 Selective agents in enrichment and plating media for Yersinia enterocolitica Medium a
Selective agents
Enrichment
MRB
magnesium chloride (2.8%), malachite green (0.0013%), carbenicillin (0.00025%)
Selenite Media
sodium selenite (0.15 or 0.25%), malachite green (0.002%), carbenicillin (0.001%)
PBSSB
bile salts (0.15%)
BOS
sodium oxalate (0.5%), bile salts (0.2%), irgasan (0.0004%), sodium furadantin (0.001%)
ITC
magnesium chloride (6%), malachite green (0.001%), irgasan (0.00001%), ticarcillin (0.00001%), potassium chlorate (0.1%)
Plating
CIN
sodium deoxycholate (0.05%), crystal violet (0.0001%), irgasan (0.0004%), cefsulodin (0. 0015%), novobiocin (0. 00025%)
VYE
sodium deoxycholate (0.1%), crystal violet (0.0001%), irgasan (0.0004%), cefsulodin (0.0004%), oleandomycin (0.001%), josamycin (0. 002%)
SSDC
sodium deoxycholate (0. 85%)
MacConkey
bile salts (0.15%), crystal violet (0.0001%)
a MRB, modified Rappaport broth (Wauters, 1973); Selenite Media (Lee et al., 1980); PBSSB, phosphate-buffered saline with sorbitol and bile salts (Mehlman et al., 1978); BOS, bile oxalate sorbose broth (Schiemann, 1982; this volume); ITC, irgasan ticarcillin chlorate (Wauters et al., 1988a; this volume); CIN, cefsulodin irgasan novobiocin agar (Schiemann, 1979b; this volume); VYE, virulent Yersinia enterocolitica agar (Fukushima, 1987); SSDC, Salmonella-Shigella deoxycholate calcium chloride agar (Wauters, 1973; this volume), MacConkey Agar no. 3 (Oxoid CMll5).
Shigella, desoxycholate citrate and bismuth sulphite agars, designed for the isolation of enteropathogens, were used. Lee (1977) modified MacConkey agar with Tween 80 to improve differentiation of Yersinia colonies from other lactose-negative colonies. However, lipolytic Yersinia strains which are easily recognized on this medium as white wrinkled colonies surrounded by a sheen, are usually non-pathogenic (De Boer and Seldam, 1987). Salmonella-Shigella agar was made more selective for Y. enterocolitica by addition of sodium deoxycholate and CaC12 (SSDC)(Wauters, 1973, 1988a). Colonies of Y. enterocolitica on this medium are small, round and colourless. Some species of Morganella, Proteus, Serratia and Aeromonas can grow on SSDC and differentiation of Yersinia from these competing organisms may be difficult. Schiemann (1979b) developed cefsulodin-irgasan-novobiocin (CIN) agar, a selective and differential agar medium for Y. enterocolitica. Organisms capable of
223
TABLE
2
Differential
characteristics
of
Yersinia
Characteristics
Y. enterocolitica
Urease
and
related
genera
Hafnia
Serratia
Citrobacter
Enterobacter
Escherichia
Klebsiella
Proteus
+
-
d
d
d
-
d
+
Motility
at 25~
+
+
+
+
+
+
-
+
Motility
at 37~
-
+
+
+
+
d
-
+
dihydrolase
-
-
-
+
d
-
-
-
decarboxylase
-
+
d
-
-
-
d
Arginine Lysine
Phenylalanine
deaminase
H 2 S production a
d, d i f f e r e n t
. -
reactions
in different
+ .
d .
-
. +
.
d .
d
. -
+
species.
fermenting mannitol, like yersiniae, produce red coloured 'bullseye' colonies on CIN agar. CIN medium was found to be inhibitory to Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis, but some Enterobacter, Aeromonas and Proteus strains showed a colony appearance similar to Yersinia (De Boer and Seldam, 1987). The addition of 1 /xg/ml of streptomycin improves the selectivity of CIN agar, but colonies of Y. enterocolitica are smaller (Schiemann, 1987). As the recovery rate and the colony size of Y. enterocolitica diminishes during storage of the medium, it is recommended to use CIN medium within 14 days of preparation (Petersen, 1985). On CIN agar colonies of pathogenic and environmental Yersinia strains appear similar. Fukushima (1987) developed a selective agar medium for isolation of pathogenic (virulent) Y. enterocolitica (VYE agar). Pathogenic strains form red colonies on this medium, while most environmental Yersinia strains form dark colonies with a dark peripheral zone as a result of aesculin hydrolysis.
Identification
For the differentiation of Yersinia from related genera the tests listed in Table 2 may be used. Table 3 shows the characteristics differentiating the foodborne species within the genus Yersinia. Miniaturised identification kits like the API 20E (bioMdrieux) and the Minitek system (BBL) have proven to be valuable for rapid identification of Y. enterocolitica strains (Restaino et al., 1979). Some biochemical activities (cellobiose, raffinose, indole, ONPG hydrolysis, ornithine decarboxylase, Voges-Proskauer) of Yersinia strains are temperature-dependent (Bercovier and Mollaret, 1984). These tests are preferably incubated at 25 or 30~ rather than at 37~ Serotyping, biotyping and virulence testing is essential for differentiation between pathogenic and environmental Yersinia strains. The serotyping scheme of Y. enterocolitica and related Yersinia species now involves 67 major O factors and 44 H factors (Wauters et al., 1991). Table 4 shows the biotyping scheme of Y.
224
TABLE
3
Biochemical
differentiation
Y.
Test
Yersinia
within the genus
Y.
Y.
Y.
(from Wauters
Y
et al., 1 9 8 8 b )
Y.
Y.
Y.
Y.
entero- inter- frede- kristen- aldovae rhodei mollaretii bercovieri pseudocolitica media riksenii senii tuberculosis Indole
d a
+
+
d
.
Proskauer
d
+
d
-
+
.
Citrate (Simmons)
-
+
d
-
d
+
-
-
-
L-- Ornithine
+
+
+
+
+
+
+
+
-
Mucate,
-
d
d
-
d
-
+
+
-
d
+
+
+
+
+
+
+
-
Voges-
acid
Pyrazinamidase
.
.
. .
. .
.
Sucrose
+
+
+
-
-
+
+
+
Cellobiose
+
+
+
+
-
+
+
+
-
L-- Rhamnose
-
+
+
-
+
-
-
-
+
Melibiose
-
+
-
-
-
d
-
-
+
L-- Sorbose
d
+
+
+
-
ND
+
-
-
L-- Fucose
d
d
+
d
d
ND
-
+
-
a
d, d i f f e r e n t
reactions;
ND,
.
-
not determined.
enterocolitica as proposed by Wauters et al. (1987). In Europe, Y. enterocolitica
strains of serotypes 0:3 (biogroup 4), 0:9 (biogroup 2) and 0:5, 27 (biogroup 2) are the most frequently isolated human pathogenic strains. In North America, strains causing human yersiniosis usually belong to biogroup 1B (serotypes 0:4, 0: 8, 013a,13b, 0: 18, 0: 20) Several in vitro tests have been described to determine the potential virulence of Yersinia isolates (Prpic et al., 1985). Tests for autoagglutination at 37~ (Laird and Cavanaugh, 1980), calcium dependency at 37~ on magnesium oxalate agar (Gemski et al., 1980), uptake of Congo red (Prpic et al., 1983) and pyrazinamidase activity (Kandolo and Wauters, 1985) are easy to perform in routine laboratories.
TABLE Biotypes
4 of
Yersinia enterocolitica
(Wauters
e t al., 1987)
Biogroups 1A Lipase (Tween-esterase)
+
Aesculin/Salicin
_+
24h
Indole
+
Xylose
+
Trehalose/Nitrate
+
Pyrazinamidase
+
1B
2
3
4
+
(+)
-
_
-I+
+ +
+ +
-+
5
225
Comparative studies From a comparison of pre-enrichment media, Schiemann (1983a) proposed tryptone soy broth as the medium of choice. Comparison of the two-step enrichment against cold enrichment and modified Rappaport broth showed improved recovery of human pathogenic strains of Y. enterocolitica from inoculated foods using pre-enrichment in tryptone soy broth followed by enrichment in BOS (Schiemann, 1982, 1983a; Walker and Gilmour, 1986). In a study on the occurrence of Y. enterocolitica in foods Delmas and Vidon (1985) evaluated seven enrichment procedures. Most positive samples obtained within the shortest time were found using enrichment in PBSSB and BOS, with alkali treatment before plating onto CIN agar. However, only environmental serogroups of Y. enterocolitica were isolated. The enrichment medium ITC was found superior to enrichment in BOS for isolation of pathogenic Y. enterocolitica from pork products, though enrichment in BOS isolated more non-pathogenic strains (Wauters et al., 1988a; Kwaga et al., 1990; De Boer and Nouws, 1991). In different studies cold enrichment in PBSSB gave high isolation rates of Yersinia spp., which mainly proved to be non-pathogenic environmental strains (De Boer et al., 1982, 1986; De Boer and Seldam, 1987). In these studies MRB was found a suitable medium for the isolation of serogroup 0:3 and 0:9 strains, but much less efficient for the isolation of environmental strains. Alkali treatment often resulted in a marked increase in the number of Yersinia isolations. This occurred especially when high numbers of competing organisms were present, as was usually the case for PBSSB, and when a plating medium with low selectivity, such as MacConkey agar, was used (De Boer et al., 1982; De Boer and Seldam, 1987). MacConkey agar has been demonstrated to be a very productive medium for Y. enterocolitica (Mehlman et al., 1978; Schiemann, 1979b; De Boer and Seldam, 1987). However, Yersinia colonies are hard to recognize on this medium because of its low selectivity. In several comparative studies CIN agar was found to be the most selective plating medium for Yersinia spp. (Aldova et al., 1990; Cox et al., 1990; Harmon et al., 1983; Schiemann, 1983a; Walker and Gilmour, 1986). Fukushima (1987)found that biotype 3B, serogroup 0:3 strains were inhibited on CIN agar. Differentiation of Yersinia colonies from other colonies on CIN agar is not always easy (Fukushima, 1987; De Boer and Seldam, 1987). Plating ITC enrichments onto SSDC isolated more serogroup 0:3 strains than plating onto CIN agar (Wauters et al., 1988; De Boer and Nouws, 1991). SSDC proved to be unsuitable after alkali treatment (Wauters et al., 1988a).
Discussion Addition of selective agents such as magnesium chloride, malachite green, bile salts, irgasan, and the antibiotics carbenicillin, ticarcillin and cefsulodin, to Yersinia
226
isolation media results in growth inhibition of the Gram-positive and part of the Gram-negative flora (Table 1). With regard to the differential properties of the Yersinia isolation media now in use, it is evident that the colony appearance of Yersinia on these media is not very characteristic and confirmation tests of presumptive colonies are always necessary. At present, no single isolation procedure is available for the recovery of all pathogenic strains of Y. enterocolitica from foods. Cold enrichment in PBSSB, two-step enrichment with TSB and BOS, and enrichment in ITC are the most commonly used enrichment procedures. CIN and SSDC agars are the most commonly used plating media and are also commercially available. These enrichment and plating media are not very selective for Y. enterocolitica as they support the growth of several other members of the family of Enterobacteriaceae. This makes the isolation of low numbers of Yersinia in products containing many other contaminants rather difficult. Moreover, non-pathogenic environmental Y. enterocolitica strains are very common in many raw foods and may greatly hinder the isolation of pathogenic Yersinia strains from these products. Cold enrichment and two-step enrichment are very efficient methods for the recovery of a wide spectrum of serotypes of Y. enterocolitica. However, usually one is only interested in the pathogenic serotypes and as the colony appearance of pathogenic and environmental strains on CIN and SSDC agar is similar, it requires much work to select pathogenic strains from these media. This highlights the need for the development and evaluation of agar media selecting for pathogenic serotypes, like VYE agar (Fukushima, 1985). Enrichment in ITC was found to be the most efficient method for the recovery of strains of serotype 0:3, which is the most common clinical serotype of Y. enterocolitica in Europe. The usefulness of this enrichment broth for the isolation of other pathogenic serotypes remains to be determined. This also holds for SSDC agar, which is probably less suitable for pathogenic strains other than serotype 0:3. For the recovery of the North American serotype 0:8 strains, enrichment in BOS and plating on CIN seems the most efficient procedure. Selection of the proper enrichment procedure will depend on the bio/serotypes of Yersinia spp. sought and on the type of food to be examined. The use of more than one medium for both enrichment and plating will obviously result in higher recovery rates of Yersinia spp. from foods. Parallel use of the following two isolation procedures is recommended. (1) Enrichment in ITC for 2 days at 24~
plating on SSDC agar (2 days at 30~
(2) Pre-enrichment in TSB for 1 day at 24~ enrichment in BOS for 5 days at 24~ alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaC1 for 5 s); plating on CIN agar (2 days at 24~ M o n o g r a p h s are described for CIN, BOS, ITC and SSDC in this volume.
227
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