- Email: [email protected]
Isolation of Yersinia enterocolitica from foods
Intemalional Journal of Food Microbiology, 17 (1992) 75-84 © 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1605/92/$05.110
75
FOOD 00550
Isolation of Yersinia enterocolitica from foods E. de Boer lnspec,orate for Health Proreetio~m- - Food b~pection Service. Zatphen. The Netherlands
Many ~lective enrichment and plating media for the isolation of Yersima enterocolitiexz from foods are described. However. at pre~nt no single isolation procedttre is available for the recovery of all pathogenic strains of Yersinia emerocoliik'a. Cold enrichment in phosphate-buffered saline plus I% sorbitol and 0.15% bile sails (PBSSB) and two.step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth arc very efficient methods for the recffoery o [ a wide spectrum of serotyl:~es of lt'. enterocolilica. Enrichment in irgasan ticarciJlin chlorate (ITC) broth was found to be the most efficient method for the recovery of strains of scmtype 0: 3, which is the most common clinical serotype of E enterocolitica ill Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsutodin irgasan mwobiocin (CiN) agar anti Salmonetla-Shigella deoxycholale 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/scrotypes of Yersinia spp. sought and on the type of f(agl 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. (i) Enrichment in 1TC for 2 days at 24~, plating on SSDC agar (2 days at .~C). (2) Pre-enrichmem in TSB for I day at 24"C; enrichment in BOS for 5 days at 24°C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0,5% NaCI for 5 s); plating on CIN agar (2 days at 24°C), Key words: Yersinia enrerocolitica; Isolation:. Media
Introduction Yersinia enterocolitica is a c o c c o i d - s h a p o d G r a m - n e g a t i v e , facultativoly a n a e r o bic b a c t e r i u m b e l o n g i n g t o t h e g e n u s Yersinia in t h e E n t e r o b a c t o r i a c c a e family. T h e o r g a n i s m is r e c o g n i s e d as a f o o d b o r n e p a t h o g e n a n d s o m e l a r g e f o o d - a s s o c i a t e d o u t b r e a k s o f yersiniosis h a v e b e e n r e p o r t e d , i n d e v e l o p e d c o u n t r i e s , Y. enterocolitica c a n b e i s o l a t e d f r o m I - 2 % o f all h u m a n c a s e s o f a c u t e e n t e r i t i s ( K a p p e r u d , 1991). Yersinia enterocolitica a n d r e l a t e d s p e c i e s h a v e b e e n i s o l a t e d f r o m m a n y types o f f o o d . T h e m a j o r i t y o f t h e s e food i s o l a t e s d i f f e r in b i o c h e m i c a l a n d s e r o l o g i c a l c h a r a c t e r i s t i c s f r o m typical clinical s t r a i n s a n d a r e usually m e n t i o n e d ' n o n - p a t h o -
Ccrreslmndence address: E. de Boer. lnspectorate for Health Protection - - Food Inspection Service, P,O. Box 9012, 7200 GN Zutphen. The Netherlands.
76 genic' or 'environmental' Yersinia strains. These strains, which include the 'related species" 1 frederiksenii, Y. kristensenii, Y. intermedia, Y. aldocae, Y. rohdei, Y. mollaretff and E bercorieri, 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 E 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°C 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 E 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 aI., 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 Hopper, 1989). The long period required for cold enrichment is often unacceptable for quality assurance of foods. Schiemann and OIson (1984) showed that an incubation at 15°C for 2 days was as efficient as enrichment at 4°C for some weeks. Doyle and Hugdahl (1983) incuhated PBS for 1-3 days at 25°C, and Greenwood and Hopper (1989) incubated tris-buffered peptone water at 9~C for 11-14 days. Several other enrichment proced,Jres involving incubation at higher temperature for shorter periods and usin£ ~elective media have been pro0osed. 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 Z enterocolitica (Schiemann, 1983a; Walker and Gilmour, 1986). Carbenicillin in me,dified Rappaport broth was shown to inhibit the growth of certain serotype 0:3 strains (Schiemann, 1982). However, Wauters et al. (1988)
77 stated that serogroup 0:3 strains are not inhibited by carbenicillin and that deletion of this antibiotic results in decrease of selectivity of the enrichment medium. Lee et al. 0980) described two modified selenite media that were effective for recovery of certain strains of Z 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. emerocolitica would be overgrown by the faster growing normal bacterial flora of the meat. Sehiemann (1982) developed a two-step enrichment procedure for recovery of Y, enterocolitica from food. In this procedure pre-enrichment for 9 days at 4°C in yeast extract rose bengal broth is followed by selective enrichment with bile oxalate sorbose (BOS) broth at 22°C 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 irBasan, ticarcillin and potassium chlorate. In comparative studies ITC broth was espe,'ially 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 a|., 1988a; Kwaga et al, 1990; De Boer and Nouws, 1991). Aulisio et al, (1980) found that strains of E 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 strongly 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 E enterocolitica even when growing in the presence of numerous organisms. Direct KOH treatment of me.t 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 rain 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 I). Initially agar media like MacConkey agar,
78 TABLE 1 Selective agents in enrichment and plating medm for Yersinia emerocofitica Medium"
Seleclive asenls
Enrichment
MRB
magnesium chloride 12.8%), malachite green (O.t~)13e~), carbenieillin (0.011025¢~)
Selenite Media
s~nlium ~lenite (0,15 or (I.25q~).malachite green (11.11t!2%), earbenicillin (O,111H%)
PBSSB
bile salts (1LI5%)
BOS
sodium oxalate (0.5%). bile salts 111,2%),irGasan(O.IX~M%). sodium furadantin (l).tll)l¢~,)
ITC
magnesium chloride (6r)~),malachile Green (i).llOlr),:), irGasan ((},(H~XII~';"), licarcillin (O.IH)(XII~ )~ .r~tassium chlorate (0, i%)
Plaling CIN
sodium deo~cholate (().115%).crystal violet (t).O(XHr~,), irgasan 1(l.111114%).cefsuh)din (0. 1X115%),novobi(min (It. iX1025¢';)
VYE
sodium d¢oxycholate (0.1,%), crystal violet {(I.[~}t%), irgasan ((I,iXlt)4~),cefsuhxtin (().{ltXB%h oleandomycin {I}.(X)1%}.josamycin (0. 002%)
SSDC
sadium deoxycholate (0. 85%)
MacConkey
bile salts ((1.15~,). crysl:ll violet (i).O001~,)
" MRB. modified Rappaport broth (Wauters. 19731: Selenite Media (Lee et al., 19801: PBSSB, phosphate-buffered saline with sorbitol and bile salts (Mehlman el al., 19781: BOS, bile oxaiate sorbos¢ broth (Schiemann, 1982; Curtis and Baird. 19931: 1TC. irgasan ticarciliin chlorate (Wauters ¢t al., 1988a; Curtis and Baird, 19931;CIN, cefsulodi, irgasan novobiocin agar (Schicmann, 1979b: Baird et al., 19871: VYE, virulent Yer~iniaenterocolitica agar (Fukushima, 1087): SSDC, Salmone|la-Shigella deoxyeholate calcium chloride agar (Waulers, 1973: Curtis and Baird, 19931, MacConkey Agar ha. 3 (Oxoid CM 115}. S a l m o n e l l a - S h i g e l l a agar, desoxycholate c i t r a t e a g a r and bismuth sulphite agar, d e s t i n e d for t h e isolation o f e n t e r o p a t h o g e n s , were used for Y. emerocolitica. Lee 119771 modified M a c C o n k e y a g a r with T w e e n 80 to improve d i f f e r e n t i a t i o n o f Yersinia colonies from o t h e r lactose-negative colonies. However, lipolytic Yersinia strains which a r e easily r e c o g n i z e d on this ~,. "dium as white wrinkled colonies s u r r o u n d e d by a sheen, are usually n o n - p a t h o g e n i c ( D e B o e r a n d S e l d a m , 1987). S a l m o n e l l a - S h i g e l l a a g a r was m a d e m o r e selective for Z enterocolitica by a d d i t i o n o f sodium d e o x y c h o l a t e a n d CaCI 2 ( S S D C ) ( W a u t e r s , 1973, 1988a). C o l o n i e s of Y. enterocolitica on this m e d i u m a r e small, r o u n d a n d colourless. S o m e species of Morganella, Proteus, Serratia and Aeromonas can grow on S S D C a n d d i f f e r e n t i a t i o n o f Yersinia from t h e s e c o m p e t i n g o r g a n i s m s may be difficult, S c h i e m a n n (1979b) d e v e l o p e d cefsulodin-irgasan-novobiocin ( C I N ) agar, a selective and differential a g a r m e d i u m for Z enterocolitiea. O r g a n i s m s c a p a b l e o f
79 TABLE
II
Differential
characteristics
of
Yer.~inia a n d r e l a t e d g e n e r a
Y. entvrocolilita
Characteristics
ltafma
Serr~ztia ('imp. l-mere. h~*ctt'r hewtcr
Urcase
+
-
d
-
d
Motility at 25°C
+
+
+
+
+
+
-
+
Motility at 37°C
-
+
+
+
+
d
-
+
Atginine
-
-
-
+
d
-
-
-
-
+
~!
-
d
+
d
-
-
-
d
Lysine
dihydrolasc decatboxylam
Phenylalanine
deamil~as¢
H ,S prtntuetion '~ d . d i f f e r e n t
reactions
.
.
.
.
-
-
in d i f f e r e n t
,species.
d
.
.
¢-
.
d
Esche- Kleh. Pmteu~ ri~'hia siell~t
.
d
.
.
+
+
fermenting mannitol, like yersinia¢, produce red coloured 'bullseye" colonies on CIN agaL CIN medium was found to be inhibitory to Pseutlomonas aentginosa, Escherichia cell, Klehsiella pneumoniae and Proteus mirabilis, but ,~.~me Enterolmcter, Aeromonas and Prowus strains showed a colony appearance similar to Yersini,t (De Boer and Se!dam, 1987). The addition of 1 # g / m l of streptomycin improv,"s the selectivity of CIN near, but colonies of Y offerrx'olifit'a are smaller (Schiemann, 1987). As the recovery rate and the colony size of Y. enterocoliticu reducus 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 Vers#tia strains appear similar, Fukushima (1987) developed a selective agar medium for isolation of pathogenic (virulent) 1,'. atwmcolitica (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 'Fable 1] may be used. Table Ill shows the characteristics differentiating the foodlx~rne species within the genus Yersinia. Miniaturised identification kits like the AP! 20E (bioMdrieux) and the Minitek system (BBL) have proven to bc valuable for rapid identification of Y. enwrocolitica strains (Restaino et aL, 1979). Some biochemical activities (eellobiose, 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 3WC, rather than at 37°C. Serotyping, biotyping and virulence testing is essential for the dift,.rentiation between pathogenic and environmental Versinia strains. The serotyping scheme of E enterocolitica and related Yersinia species now involves 67 major O factors and 44 H factors (Wauters et al., 1991). Table IV shows the biotyping scheme of Y.
80 T A B L E Ill
Yersinia ( f r o m
B i o c h e m i c a l d i f f e r e n t i a t i o n wilhin t h e g e n u s
W a u l e r s el hi., ig88b)
Y. Y Y Y. Y. Y. Y. Y. Y entero, inter- frede- kristen- aldovae rl;odei mollaretii bercot,ieri pseudoeolirica media riksenii senii tuberculosis
Test
lndole d " V o g e s - P r o s k a u e r t; Citrate (Simmons) L-Ornithine +
+ + + +
+ d d +
d +
. + d +
M u c a t e , acid Pyrazinamidase Sucrose Cellobiose L- Rhamnose Melibiosc
d + + -
d + + + + +
d + + + + -
+ + -
d + -
L -- S o r b o s e L - Fu¢os¢
d d
+ d
+ +
+ d
.
. . + +
. .
. .
+
. +
-
+ -
+ + + d
+ + + + -
+ + + + --
+ +
d
ND ND
+ -
+
-
d, d i f f e r e n t reactions; N D , not d e t e r m i n e d .
enterocolitica
as
proposed
strainr, of serotypes are
the
most
0:3
frequently
by
Wauters
(biogroup isolated
et
4), 0:9 human
al. (1987). (biogroup pathogenic
In
Europe,
2) and strains.
Y.
0:5,
enterocolitica
27 (biogroup
In
North
2)
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 de.;cribed to determine the potential virulence of Yersinia isolates (Prpic et al., 1985). Tests for autoagglutination at 37"C (Laird and Cavanaugh, 1980), calcium dependency at 37°C 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.
T A B L E IV Biotypes o f
Yersinia enterocolitica
( W a u t e r s el al,, ItJ87) Biogroups IA
Lipase (Tween-esterase)
+
A e s c u l i n / S a l i c i n 24h lndole XyLose Trehalose/Nitrate Pyrazinamidase
:1: + + + +
IB ÷
2
3
4
m
~
m
5
i
+
(+)
-
_
-I-I-
-4-k
-6 -9-
q..
m
81 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 tryptonc 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 E emerocolitica were isolated. The enrichment medium ITC was found superior to enrichment in BOS for isolation of pathogenic Z enterocoliticu from pork products, though enrichment in BOS isolated more non-pathogenic strains (Wauters et al., 19883; De Bocr and Nouws, 1991; Kwaga et aL, 1990), In different studies cold enrichment in PBSSB gave high isolation rates of Yersinia spp., which mainly proved to be non-pathogenic environmental strains (De 8oer et al., 1982, 1986: De Boer and Seldam, 1987). In these studies MRB was !0und a suitable medium for the isolation of serogroup 0:3 and 0:9 strains, but much less efficient for the isol~ltion of environmental strains. Alkali treatment often resulted in a marked increase in Ihe number of Yersinia isolations. This occurred especially when high numbers of competing organisms were present, as was usually the case for P3SSB, and when a plating medium with low selectivity, such as MacConkey agar, was used (De Boer et ai., 1982; De Boer and Seldam, 1987). MacConkey agar has been demonstrated to be a very productive medium for Y. enterocolitica (Mehlman et ai., 1978; Schiemann, 1979b; De Boer and Seldam, 1987). However, Yersinia colonies arc 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. (AIdova ct al., i990~ Cox et al., 1990: Harmon ct al., 1983; Schiemann, 19833; Walker and Gilmour, 1986). Fukushima (1987) found that biotypc 3B, scrogroup 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 scrogroup 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 ctal., 1988a). Discussion
Addition of selective agents such as magnesium chloride, malachite green, bile salts, irgasan, and the antibiotics carbenicillin, tiearcillin and cefsulodin, to Yersinia
82 isolation media results in growth inhibition of the Gram-positive and part of the Gram-negative flora (Table I). With regard to the differential properties of the Yersinia isolation media r.ow 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 ~Jways necessary. At present, no single isolation procedure is available for the recovery of all pathogenic strains of Y. enterocolilica 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 a l ~ 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 Enterobaeteriaeeae. 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 15. 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°C; plating on SSDC agar (2 days at 300C). (2) Pre-enrichment in TSB for 1 day at 24°(:; enrichment in BOS for 5 days at 24°C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaCI for 5 s); plating on CIN agar (2 days at 24°(:). Monographs in the Pharmacopoeia of Culture Media for Food Microbiology are described for CIN (Baird et a1.,1987), BOS, ITC and SSDC (Curtis and Baird, 1993).
References Aldova, E., Svandova, E., Votypka. I. and Sourek J. (19901 Comparative study of culture methods to detect Yersinia enlertJcolitica serogroup 0:3 on swine tongues. Zbl. Bakleriol. 272, 3{~-312. Aulisio. C.C.G., Mehlman, I,J, and S:mders, C. (19801 Alkali metht~l for rapid recovery of Ycr~i,iu emerocolitiea and Yersinia p.~eudfmda'rculo.~'is from fi~ds. Appl, Environ. Mierobiol. 39, I3Y,-|JD. Baird, R.M., Corry, J.E.L. and Curtis, G,D.W. (Eds.) (1987) Pharmacopoeia of culture media for [~x~d microbiology. Int. J. Food Microbi!~l. 5. 208-209. Ikrcovicr. H. and Motlarel, H.H, (19841 Yersinia, In: Kricg. N,R, and Holt, J.G. (Eds.). Bcrgey's Manual of Systematic Bacteriology. Vol, I, Williams and Wilkins, Baltimore and I~mdon, Cox, N.A., Bailey, J.S., Del Corral, F., Shotts. E.B. (i99{11 Comparison of enrichment and plating media for i.'~dation of Yer~'iszia. Poultry Sci. 6t), 686-693, Cards, G.D,W, anti Baird, R.M. (Eds.) (It~131 Pharmacopoeia o! cahurc media for focal microbk~togy ', additional monographs (2). Int. J. FoLut Microbiol. 17, in press. De Boer, E.. Hartog, B.J. and Ooslerom, J. 0982) Occurrence of ~'rsinia etlter~a'~ditit'a in poultry products. J. Fot~l Prut. 45. 322-325. De Boer, E., Seldam, W.M. and Oosterom, J. (19~461 Characleri~,ation of Yersblia euterocolitiea and related species isolated from foo~s and porcine tonsils in the Netherlands. Int. J, Fot~l Microbiol, 3, 217-224. De Boer, E. and Scldam. W.M. (1987) Compari~m of methods for the istdathm of Yer,~iniri entenwoliiica from I~reine tonsils and pork. Int. J. Food Mierohiol. 5. 95-1ill, De Boer. E. and Nouws. J.F.M. (U~gl) Slaughter pigs and pork as a .q~urce of human pathogenic Yersiniu emerocolitica. Int. J. Food Microbiol. 12, 375-3781 Delmas, C.L and Vidon, D,J.M. (19851 Isolation of Yersinm emerl~=~d#i¢'a and related species from foods in France. Appl. Environ. Microbiol. 50, 767-771, Doyle, M.P. and Hugdahl. M,B. (1983) Improved pxoccdure for recovery ol Ver~hliu euter~'olilicu from meats. Appl. Environ. Microbh~t. 45, 127-135. Fukushima. H. (1985) Direct i~lation tff Yer.~iniu es~rerocrTlitic~ and Yersinia p,~t,ud~Jtttbt,rculosis from meat, Appl. Environ. Microbiol. 50. 71[)-712. Fukushima, Ii. ( 19871 New selective agar medium fin isolation of virulent Yersihia e,wrocolitica, J. Clin. Microbiol. Z~, 10~'-~-1073. Gemski, P., L,',zere. J.R. and Cascy, T, (19801 Plasmid a~sociatcd with pathogenicity and caleiunt dependency of Yer~'i,ia enter(n'¢~fitica. Infect. Immun. 27, 082-685. Grccnw~txl. M.H. and Htx)per. W.L. (1989) Improved m¢lh~ls |or the i~)lation of Yt'r~inia species from milk and fi~Js. F~nd Microbiol, 6, 99-104. ilarmon. M.C., Yu, C.L. and Swaminathan, B. (19831 An e'¢aluatkm of ,'~lective differential plating media for the i.,mlation of Yersinia e~twr~',titic, from cxperimcmally inoculated fresh g~ound pork homogenate. J. F t ~ Sci. 48. b-9. Kandolo, K, and Wimters. (]. (Iq~5) Pyrazinamidasc activity it~ Yt'rsi, iu cmeroeolitica and related organisms. J. Clio. Mic~biol. 2 I, q80-982. Kapperud. G. (1991) Yer.~inia em*'~a'olith'a ia fix,:l hygiene, Int. J. F~x~d Microbiol. 12, 53-66. Kwaga. J.. lvers,:n. J,O. and Saunders, J.R, (l~,JO) (\~mparison of t,.~'~ enrichment protocols for the detection of Yer~iniu in shmghtered pigs and ~ r k products. J. F*md Prol. 53, I(M7-1049. Laird. W J, and Cavanaugh. D.C. (19811l Correlation of auloagglutination and virulence of yersinae. J. Clio. Mierobi0l. I I, 4311-432. Lee, W.H. (19771 Two plating media modified with Tween 8(1 for isolating Yersinia enterocolilica. Appl. Enviroz'. Microbiol. 33. 215-21b. Lee, W.H,, Harris, M.E,, McClain, D,, Smith, R.E, and Johnston, R.W. 0980)Two modified Selenite media for the recovery of Vershffa emerocolitica from meats, Appl. Environ. Microbiol. 39, 2(15-209. Mehlman, ],J., Aulisio. C.C.G. and Sanders. A.C. (197S) Problems in the recovery and identification of Yer~inia from food. J. As.~c. Off. Anal Chem. 61. 761-771. Petersen, T. (19851 Keeping quality of cefsulodin-irgasan-novobiocin (CIN) medium for detection and enumcra'~ion of Yersinia emeroc~diliea. Int, J, Food Microbiol, 2, 49-54.
84
Prpic, J.K., Robins-Browne, R,M, and Davey. R.B, (1983) Differentiation between virulent and avirulcnt Yersi~da ~'nler<~'olitic~ isolatc~ by using Congo red agar. J, Clin. Microbial. 18, 48~-490. Prpic. J.K., Robins.Browne, R.M. and Davey, R.B. (1995) In vitro as~¢sment o[ virulence in Yerxinia euterm'oliti<'a and related species. ,I. Clin. Micr0biol, 22, 105-1 tO. Restaino, L., Grauman, G.S., McCall, W.A. and Hill, W.M. (1979) Evaluation of the Minilek and APl 20E systems for identification of Yer.sinia enterocofilica, J. Food Prol. 42, 120-123. Schicmann. D.A. (1979a) E~richment methods for recovery of Yer.v#da enterocolitica from foods and raw milk. Conlr. Microbial. immunol. 5, 212-227. Schicmann, D,A. (1979b) Synthesis of ~'k selective agar medium for Yeesinia enter¢u'olitiea. Can. J. Microbial. 25, 1298-13{)4, Schiemann. D.A. (1982) Development of a two-step enrichment procedure for recovery of Yersima emer¢~cr~litica from food. Appl. Environ. Microbial. 43, 14-27. Schicmann. D.A. (1983a) Cumparison of enrichment and plating media for recovery of virulent strains of Yersinia t,nterocofitk,¢ from inoculated beef stew..I. Foq~l Prot. 46, 957-%4. Schiemann. D.A. (1983b) AIkalotolerance of Yers&ia enlcrocolitica as a basis for selective i.~lation from food enrichments. Appl. Environ. Microbial 46, 22-27, Schicmann. D.A, (1987) Yersinia eltter~'~litica in milk and dairy products. J. Dairy Sci. 70, 383-391. Schiemann, D,A. and OIson, S.A. (1984) Antagonism by Oram-negative bacteria to growth of Yersiuia enter¢g'olitica in mixed cultures. Appl. Environ. Microbial. 48, 539-544. Schraft, It. and Unlcrnmnn, F. (19891 Use of KOH treatment fur direct detection of Ycr.smia etsterocolitica in foods. 10th WAVFH lnt, Syrup., Stockhelm, Abstracts, p, 31 Van Pe¢, W. and Stragier, J. (1979) Evaluation of .'~me cold enrichment and isolation media for the recovery of Yersinia enterocolitica. Antonio van L¢cuwcnhoek J. Microbial. Serol. 45, 465-477. Vidon, DJ.M. and Detmas, C.L. (19el) Ine[derice of Yersinia enter~n'olitica in raw milk in Eastern France. Appl. Environ. Microbial, 41,355-359, Walker, SJ, and Gilmour, A. (1986) A comparison of media and methods for the recovery of Y~w~iniu t,irterocolitica and Yer.sinia enterc~'olitica.like bacteria from milk containing simulated raw milk microfloras. J. Appl. Bacl. 60. 175-183. Wauters, G. (1973) Improved mcthl~ds for the isolnlion and the recognition of Ycrsinia elffert~ctditit'a. Co~llr. Microbial. Immunol. 2, 68-7{). Wauters, G., Kandolo, K. and Janssens, M. (1987) Revised biogrouping scheme tff Yer.~inia om, rocolit. ica. Contr. Microbial. lmmunol. 9, 14-21. Wauters, G.+ Goossens, V., Jansscns, M. and Vandepitle, J. (1988a) New enrichment method for isolation of pathogenic Y{~sitffa entero~'ofitica serogroup 0:3 from pork. Appl. Environ. Microbial. 54, 851-854. Wautcrs, G., Junssens, M., Sleigerwait, A.G. and Brenner, D.J. (191'lgb) Yersiuia molhm, tii sp.nov, and Ycr,~iJ;ia ta,r¢'m'icri sp. nov., formerly called Y~,rsinia cntcroe'oliti<'a bit)groups 3A and 3B, Int. J. Syst. Bacterial. 38, 424-429. Wauters, G., Aleksic, S., Charlicr, J. and Schulz¢, G, (1991) Somatic and flagellar :mtigens of Yer,~h&r ellterocofitic~ and related species. Contr, Microbial, Immunol. 12, 239-243, Weagant, S,D. and Kaysncr, C.A. (1983) M~xiified enrichment broth for isolation of Yersmia emerc~'olit. ica from nonfood sources. Appl. Environ. Microbial. 45, 468-471.
75
FOOD 00550
Isolation of Yersinia enterocolitica from foods E. de Boer lnspec,orate for Health Proreetio~m- - Food b~pection Service. Zatphen. The Netherlands
Many ~lective enrichment and plating media for the isolation of Yersima enterocolitiexz from foods are described. However. at pre~nt no single isolation procedttre is available for the recovery of all pathogenic strains of Yersinia emerocoliik'a. Cold enrichment in phosphate-buffered saline plus I% sorbitol and 0.15% bile sails (PBSSB) and two.step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth arc very efficient methods for the recffoery o [ a wide spectrum of serotyl:~es of lt'. enterocolilica. Enrichment in irgasan ticarciJlin chlorate (ITC) broth was found to be the most efficient method for the recovery of strains of scmtype 0: 3, which is the most common clinical serotype of E enterocolitica ill Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsutodin irgasan mwobiocin (CiN) agar anti Salmonetla-Shigella deoxycholale 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/scrotypes of Yersinia spp. sought and on the type of f(agl 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. (i) Enrichment in 1TC for 2 days at 24~, plating on SSDC agar (2 days at .~C). (2) Pre-enrichmem in TSB for I day at 24"C; enrichment in BOS for 5 days at 24°C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0,5% NaCI for 5 s); plating on CIN agar (2 days at 24°C), Key words: Yersinia enrerocolitica; Isolation:. Media
Introduction Yersinia enterocolitica is a c o c c o i d - s h a p o d G r a m - n e g a t i v e , facultativoly a n a e r o bic b a c t e r i u m b e l o n g i n g t o t h e g e n u s Yersinia in t h e E n t e r o b a c t o r i a c c a e family. T h e o r g a n i s m is r e c o g n i s e d as a f o o d b o r n e p a t h o g e n a n d s o m e l a r g e f o o d - a s s o c i a t e d o u t b r e a k s o f yersiniosis h a v e b e e n r e p o r t e d , i n d e v e l o p e d c o u n t r i e s , Y. enterocolitica c a n b e i s o l a t e d f r o m I - 2 % o f all h u m a n c a s e s o f a c u t e e n t e r i t i s ( K a p p e r u d , 1991). Yersinia enterocolitica a n d r e l a t e d s p e c i e s h a v e b e e n i s o l a t e d f r o m m a n y types o f f o o d . T h e m a j o r i t y o f t h e s e food i s o l a t e s d i f f e r in b i o c h e m i c a l a n d s e r o l o g i c a l c h a r a c t e r i s t i c s f r o m typical clinical s t r a i n s a n d a r e usually m e n t i o n e d ' n o n - p a t h o -
Ccrreslmndence address: E. de Boer. lnspectorate for Health Protection - - Food Inspection Service, P,O. Box 9012, 7200 GN Zutphen. The Netherlands.
76 genic' or 'environmental' Yersinia strains. These strains, which include the 'related species" 1 frederiksenii, Y. kristensenii, Y. intermedia, Y. aldocae, Y. rohdei, Y. mollaretff and E bercorieri, 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 E 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°C 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 E 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 aI., 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 Hopper, 1989). The long period required for cold enrichment is often unacceptable for quality assurance of foods. Schiemann and OIson (1984) showed that an incubation at 15°C for 2 days was as efficient as enrichment at 4°C for some weeks. Doyle and Hugdahl (1983) incuhated PBS for 1-3 days at 25°C, and Greenwood and Hopper (1989) incubated tris-buffered peptone water at 9~C for 11-14 days. Several other enrichment proced,Jres involving incubation at higher temperature for shorter periods and usin£ ~elective media have been pro0osed. 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 Z enterocolitica (Schiemann, 1983a; Walker and Gilmour, 1986). Carbenicillin in me,dified Rappaport broth was shown to inhibit the growth of certain serotype 0:3 strains (Schiemann, 1982). However, Wauters et al. (1988)
77 stated that serogroup 0:3 strains are not inhibited by carbenicillin and that deletion of this antibiotic results in decrease of selectivity of the enrichment medium. Lee et al. 0980) described two modified selenite media that were effective for recovery of certain strains of Z 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. emerocolitica would be overgrown by the faster growing normal bacterial flora of the meat. Sehiemann (1982) developed a two-step enrichment procedure for recovery of Y, enterocolitica from food. In this procedure pre-enrichment for 9 days at 4°C in yeast extract rose bengal broth is followed by selective enrichment with bile oxalate sorbose (BOS) broth at 22°C 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 irBasan, ticarcillin and potassium chlorate. In comparative studies ITC broth was espe,'ially 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 a|., 1988a; Kwaga et al, 1990; De Boer and Nouws, 1991). Aulisio et al, (1980) found that strains of E 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 strongly 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 E enterocolitica even when growing in the presence of numerous organisms. Direct KOH treatment of me.t 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 rain 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 I). Initially agar media like MacConkey agar,
78 TABLE 1 Selective agents in enrichment and plating medm for Yersinia emerocofitica Medium"
Seleclive asenls
Enrichment
MRB
magnesium chloride 12.8%), malachite green (O.t~)13e~), carbenieillin (0.011025¢~)
Selenite Media
s~nlium ~lenite (0,15 or (I.25q~).malachite green (11.11t!2%), earbenicillin (O,111H%)
PBSSB
bile salts (1LI5%)
BOS
sodium oxalate (0.5%). bile salts 111,2%),irGasan(O.IX~M%). sodium furadantin (l).tll)l¢~,)
ITC
magnesium chloride (6r)~),malachile Green (i).llOlr),:), irGasan ((},(H~XII~';"), licarcillin (O.IH)(XII~ )~ .r~tassium chlorate (0, i%)
Plaling CIN
sodium deo~cholate (().115%).crystal violet (t).O(XHr~,), irgasan 1(l.111114%).cefsuh)din (0. 1X115%),novobi(min (It. iX1025¢';)
VYE
sodium d¢oxycholate (0.1,%), crystal violet {(I.[~}t%), irgasan ((I,iXlt)4~),cefsuhxtin (().{ltXB%h oleandomycin {I}.(X)1%}.josamycin (0. 002%)
SSDC
sadium deoxycholate (0. 85%)
MacConkey
bile salts ((1.15~,). crysl:ll violet (i).O001~,)
" MRB. modified Rappaport broth (Wauters. 19731: Selenite Media (Lee et al., 19801: PBSSB, phosphate-buffered saline with sorbitol and bile salts (Mehlman el al., 19781: BOS, bile oxaiate sorbos¢ broth (Schiemann, 1982; Curtis and Baird. 19931: 1TC. irgasan ticarciliin chlorate (Wauters ¢t al., 1988a; Curtis and Baird, 19931;CIN, cefsulodi, irgasan novobiocin agar (Schicmann, 1979b: Baird et al., 19871: VYE, virulent Yer~iniaenterocolitica agar (Fukushima, 1087): SSDC, Salmone|la-Shigella deoxyeholate calcium chloride agar (Waulers, 1973: Curtis and Baird, 19931, MacConkey Agar ha. 3 (Oxoid CM 115}. S a l m o n e l l a - S h i g e l l a agar, desoxycholate c i t r a t e a g a r and bismuth sulphite agar, d e s t i n e d for t h e isolation o f e n t e r o p a t h o g e n s , were used for Y. emerocolitica. Lee 119771 modified M a c C o n k e y a g a r with T w e e n 80 to improve d i f f e r e n t i a t i o n o f Yersinia colonies from o t h e r lactose-negative colonies. However, lipolytic Yersinia strains which a r e easily r e c o g n i z e d on this ~,. "dium as white wrinkled colonies s u r r o u n d e d by a sheen, are usually n o n - p a t h o g e n i c ( D e B o e r a n d S e l d a m , 1987). S a l m o n e l l a - S h i g e l l a a g a r was m a d e m o r e selective for Z enterocolitica by a d d i t i o n o f sodium d e o x y c h o l a t e a n d CaCI 2 ( S S D C ) ( W a u t e r s , 1973, 1988a). C o l o n i e s of Y. enterocolitica on this m e d i u m a r e small, r o u n d a n d colourless. S o m e species of Morganella, Proteus, Serratia and Aeromonas can grow on S S D C a n d d i f f e r e n t i a t i o n o f Yersinia from t h e s e c o m p e t i n g o r g a n i s m s may be difficult, S c h i e m a n n (1979b) d e v e l o p e d cefsulodin-irgasan-novobiocin ( C I N ) agar, a selective and differential a g a r m e d i u m for Z enterocolitiea. O r g a n i s m s c a p a b l e o f
79 TABLE
II
Differential
characteristics
of
Yer.~inia a n d r e l a t e d g e n e r a
Y. entvrocolilita
Characteristics
ltafma
Serr~ztia ('imp. l-mere. h~*ctt'r hewtcr
Urcase
+
-
d
-
d
Motility at 25°C
+
+
+
+
+
+
-
+
Motility at 37°C
-
+
+
+
+
d
-
+
Atginine
-
-
-
+
d
-
-
-
-
+
~!
-
d
+
d
-
-
-
d
Lysine
dihydrolasc decatboxylam
Phenylalanine
deamil~as¢
H ,S prtntuetion '~ d . d i f f e r e n t
reactions
.
.
.
.
-
-
in d i f f e r e n t
,species.
d
.
.
¢-
.
d
Esche- Kleh. Pmteu~ ri~'hia siell~t
.
d
.
.
+
+
fermenting mannitol, like yersinia¢, produce red coloured 'bullseye" colonies on CIN agaL CIN medium was found to be inhibitory to Pseutlomonas aentginosa, Escherichia cell, Klehsiella pneumoniae and Proteus mirabilis, but ,~.~me Enterolmcter, Aeromonas and Prowus strains showed a colony appearance similar to Yersini,t (De Boer and Se!dam, 1987). The addition of 1 # g / m l of streptomycin improv,"s the selectivity of CIN near, but colonies of Y offerrx'olifit'a are smaller (Schiemann, 1987). As the recovery rate and the colony size of Y. enterocoliticu reducus 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 Vers#tia strains appear similar, Fukushima (1987) developed a selective agar medium for isolation of pathogenic (virulent) 1,'. atwmcolitica (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 'Fable 1] may be used. Table Ill shows the characteristics differentiating the foodlx~rne species within the genus Yersinia. Miniaturised identification kits like the AP! 20E (bioMdrieux) and the Minitek system (BBL) have proven to bc valuable for rapid identification of Y. enwrocolitica strains (Restaino et aL, 1979). Some biochemical activities (eellobiose, 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 3WC, rather than at 37°C. Serotyping, biotyping and virulence testing is essential for the dift,.rentiation between pathogenic and environmental Versinia strains. The serotyping scheme of E enterocolitica and related Yersinia species now involves 67 major O factors and 44 H factors (Wauters et al., 1991). Table IV shows the biotyping scheme of Y.
80 T A B L E Ill
Yersinia ( f r o m
B i o c h e m i c a l d i f f e r e n t i a t i o n wilhin t h e g e n u s
W a u l e r s el hi., ig88b)
Y. Y Y Y. Y. Y. Y. Y. Y entero, inter- frede- kristen- aldovae rl;odei mollaretii bercot,ieri pseudoeolirica media riksenii senii tuberculosis
Test
lndole d " V o g e s - P r o s k a u e r t; Citrate (Simmons) L-Ornithine +
+ + + +
+ d d +
d +
. + d +
M u c a t e , acid Pyrazinamidase Sucrose Cellobiose L- Rhamnose Melibiosc
d + + -
d + + + + +
d + + + + -
+ + -
d + -
L -- S o r b o s e L - Fu¢os¢
d d
+ d
+ +
+ d
.
. . + +
. .
. .
+
. +
-
+ -
+ + + d
+ + + + -
+ + + + --
+ +
d
ND ND
+ -
+
-
d, d i f f e r e n t reactions; N D , not d e t e r m i n e d .
enterocolitica
as
proposed
strainr, of serotypes are
the
most
0:3
frequently
by
Wauters
(biogroup isolated
et
4), 0:9 human
al. (1987). (biogroup pathogenic
In
Europe,
2) and strains.
Y.
0:5,
enterocolitica
27 (biogroup
In
North
2)
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 de.;cribed to determine the potential virulence of Yersinia isolates (Prpic et al., 1985). Tests for autoagglutination at 37"C (Laird and Cavanaugh, 1980), calcium dependency at 37°C 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.
T A B L E IV Biotypes o f
Yersinia enterocolitica
( W a u t e r s el al,, ItJ87) Biogroups IA
Lipase (Tween-esterase)
+
A e s c u l i n / S a l i c i n 24h lndole XyLose Trehalose/Nitrate Pyrazinamidase
:1: + + + +
IB ÷
2
3
4
m
~
m
5
i
+
(+)
-
_
-I-I-
-4-k
-6 -9-
q..
m
81 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 tryptonc 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 E emerocolitica were isolated. The enrichment medium ITC was found superior to enrichment in BOS for isolation of pathogenic Z enterocoliticu from pork products, though enrichment in BOS isolated more non-pathogenic strains (Wauters et al., 19883; De Bocr and Nouws, 1991; Kwaga et aL, 1990), In different studies cold enrichment in PBSSB gave high isolation rates of Yersinia spp., which mainly proved to be non-pathogenic environmental strains (De 8oer et al., 1982, 1986: De Boer and Seldam, 1987). In these studies MRB was !0und a suitable medium for the isolation of serogroup 0:3 and 0:9 strains, but much less efficient for the isol~ltion of environmental strains. Alkali treatment often resulted in a marked increase in Ihe number of Yersinia isolations. This occurred especially when high numbers of competing organisms were present, as was usually the case for P3SSB, and when a plating medium with low selectivity, such as MacConkey agar, was used (De Boer et ai., 1982; De Boer and Seldam, 1987). MacConkey agar has been demonstrated to be a very productive medium for Y. enterocolitica (Mehlman et ai., 1978; Schiemann, 1979b; De Boer and Seldam, 1987). However, Yersinia colonies arc 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. (AIdova ct al., i990~ Cox et al., 1990: Harmon ct al., 1983; Schiemann, 19833; Walker and Gilmour, 1986). Fukushima (1987) found that biotypc 3B, scrogroup 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 scrogroup 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 ctal., 1988a). Discussion
Addition of selective agents such as magnesium chloride, malachite green, bile salts, irgasan, and the antibiotics carbenicillin, tiearcillin and cefsulodin, to Yersinia
82 isolation media results in growth inhibition of the Gram-positive and part of the Gram-negative flora (Table I). With regard to the differential properties of the Yersinia isolation media r.ow 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 ~Jways necessary. At present, no single isolation procedure is available for the recovery of all pathogenic strains of Y. enterocolilica 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 a l ~ 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 Enterobaeteriaeeae. 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 15. 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°C; plating on SSDC agar (2 days at 300C). (2) Pre-enrichment in TSB for 1 day at 24°(:; enrichment in BOS for 5 days at 24°C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaCI for 5 s); plating on CIN agar (2 days at 24°(:). Monographs in the Pharmacopoeia of Culture Media for Food Microbiology are described for CIN (Baird et a1.,1987), BOS, ITC and SSDC (Curtis and Baird, 1993).
References Aldova, E., Svandova, E., Votypka. I. and Sourek J. (19901 Comparative study of culture methods to detect Yersinia enlertJcolitica serogroup 0:3 on swine tongues. Zbl. Bakleriol. 272, 3{~-312. Aulisio. C.C.G., Mehlman, I,J, and S:mders, C. (19801 Alkali metht~l for rapid recovery of Ycr~i,iu emerocolitiea and Yersinia p.~eudfmda'rculo.~'is from fi~ds. Appl, Environ. Mierobiol. 39, I3Y,-|JD. Baird, R.M., Corry, J.E.L. and Curtis, G,D.W. (Eds.) (1987) Pharmacopoeia of culture media for [~x~d microbiology. Int. J. Food Microbi!~l. 5. 208-209. Ikrcovicr. H. and Motlarel, H.H, (19841 Yersinia, In: Kricg. N,R, and Holt, J.G. (Eds.). Bcrgey's Manual of Systematic Bacteriology. Vol, I, Williams and Wilkins, Baltimore and I~mdon, Cox, N.A., Bailey, J.S., Del Corral, F., Shotts. E.B. (i99{11 Comparison of enrichment and plating media for i.'~dation of Yer~'iszia. Poultry Sci. 6t), 686-693, Cards, G.D,W, anti Baird, R.M. (Eds.) (It~131 Pharmacopoeia o! cahurc media for focal microbk~togy ', additional monographs (2). Int. J. FoLut Microbiol. 17, in press. De Boer, E.. Hartog, B.J. and Ooslerom, J. 0982) Occurrence of ~'rsinia etlter~a'~ditit'a in poultry products. J. Fot~l Prut. 45. 322-325. De Boer, E., Seldam, W.M. and Oosterom, J. (19~461 Characleri~,ation of Yersblia euterocolitiea and related species isolated from foo~s and porcine tonsils in the Netherlands. Int. J, Fot~l Microbiol, 3, 217-224. De Boer, E. and Scldam. W.M. (1987) Compari~m of methods for the istdathm of Yer,~iniri entenwoliiica from I~reine tonsils and pork. Int. J. Food Mierohiol. 5. 95-1ill, De Boer. E. and Nouws. J.F.M. (U~gl) Slaughter pigs and pork as a .q~urce of human pathogenic Yersiniu emerocolitica. Int. J. Food Microbiol. 12, 375-3781 Delmas, C.L and Vidon, D,J.M. (19851 Isolation of Yersinm emerl~=~d#i¢'a and related species from foods in France. Appl. Environ. Microbiol. 50, 767-771, Doyle, M.P. and Hugdahl. M,B. (1983) Improved pxoccdure for recovery ol Ver~hliu euter~'olilicu from meats. Appl. Environ. Microbh~t. 45, 127-135. Fukushima. H. (1985) Direct i~lation tff Yer.~iniu es~rerocrTlitic~ and Yersinia p,~t,ud~Jtttbt,rculosis from meat, Appl. Environ. Microbiol. 50. 71[)-712. Fukushima, Ii. ( 19871 New selective agar medium fin isolation of virulent Yersihia e,wrocolitica, J. Clin. Microbiol. Z~, 10~'-~-1073. Gemski, P., L,',zere. J.R. and Cascy, T, (19801 Plasmid a~sociatcd with pathogenicity and caleiunt dependency of Yer~'i,ia enter(n'¢~fitica. Infect. Immun. 27, 082-685. Grccnw~txl. M.H. and Htx)per. W.L. (1989) Improved m¢lh~ls |or the i~)lation of Yt'r~inia species from milk and fi~Js. F~nd Microbiol, 6, 99-104. ilarmon. M.C., Yu, C.L. and Swaminathan, B. (19831 An e'¢aluatkm of ,'~lective differential plating media for the i.,mlation of Yersinia e~twr~',titic, from cxperimcmally inoculated fresh g~ound pork homogenate. J. F t ~ Sci. 48. b-9. Kandolo, K, and Wimters. (]. (Iq~5) Pyrazinamidasc activity it~ Yt'rsi, iu cmeroeolitica and related organisms. J. Clio. Mic~biol. 2 I, q80-982. Kapperud. G. (1991) Yer.~inia em*'~a'olith'a ia fix,:l hygiene, Int. J. F~x~d Microbiol. 12, 53-66. Kwaga. J.. lvers,:n. J,O. and Saunders, J.R, (l~,JO) (\~mparison of t,.~'~ enrichment protocols for the detection of Yer~iniu in shmghtered pigs and ~ r k products. J. F*md Prol. 53, I(M7-1049. Laird. W J, and Cavanaugh. D.C. (19811l Correlation of auloagglutination and virulence of yersinae. J. Clio. Mierobi0l. I I, 4311-432. Lee, W.H. (19771 Two plating media modified with Tween 8(1 for isolating Yersinia enterocolilica. Appl. Enviroz'. Microbiol. 33. 215-21b. Lee, W.H,, Harris, M.E,, McClain, D,, Smith, R.E, and Johnston, R.W. 0980)Two modified Selenite media for the recovery of Vershffa emerocolitica from meats, Appl. Environ. Microbiol. 39, 2(15-209. Mehlman, ],J., Aulisio. C.C.G. and Sanders. A.C. (197S) Problems in the recovery and identification of Yer~inia from food. J. As.~c. Off. Anal Chem. 61. 761-771. Petersen, T. (19851 Keeping quality of cefsulodin-irgasan-novobiocin (CIN) medium for detection and enumcra'~ion of Yersinia emeroc~diliea. Int, J, Food Microbiol, 2, 49-54.
84
Prpic, J.K., Robins-Browne, R,M, and Davey. R.B, (1983) Differentiation between virulent and avirulcnt Yersi~da ~'nler<~'olitic~ isolatc~ by using Congo red agar. J, Clin. Microbial. 18, 48~-490. Prpic. J.K., Robins.Browne, R.M. and Davey, R.B. (1995) In vitro as~¢sment o[ virulence in Yerxinia euterm'oliti<'a and related species. ,I. Clin. Micr0biol, 22, 105-1 tO. Restaino, L., Grauman, G.S., McCall, W.A. and Hill, W.M. (1979) Evaluation of the Minilek and APl 20E systems for identification of Yer.sinia enterocofilica, J. Food Prol. 42, 120-123. Schicmann. D.A. (1979a) E~richment methods for recovery of Yer.v#da enterocolitica from foods and raw milk. Conlr. Microbial. immunol. 5, 212-227. Schicmann, D,A. (1979b) Synthesis of ~'k selective agar medium for Yeesinia enter¢u'olitiea. Can. J. Microbial. 25, 1298-13{)4, Schiemann. D.A. (1982) Development of a two-step enrichment procedure for recovery of Yersima emer¢~cr~litica from food. Appl. Environ. Microbial. 43, 14-27. Schicmann. D.A. (1983a) Cumparison of enrichment and plating media for recovery of virulent strains of Yersinia t,nterocofitk,¢ from inoculated beef stew..I. Foq~l Prot. 46, 957-%4. Schiemann. D.A. (1983b) AIkalotolerance of Yers&ia enlcrocolitica as a basis for selective i.~lation from food enrichments. Appl. Environ. Microbial 46, 22-27, Schicmann. D.A, (1987) Yersinia eltter~'~litica in milk and dairy products. J. Dairy Sci. 70, 383-391. Schiemann, D,A. and OIson, S.A. (1984) Antagonism by Oram-negative bacteria to growth of Yersiuia enter¢g'olitica in mixed cultures. Appl. Environ. Microbial. 48, 539-544. Schraft, It. and Unlcrnmnn, F. (19891 Use of KOH treatment fur direct detection of Ycr.smia etsterocolitica in foods. 10th WAVFH lnt, Syrup., Stockhelm, Abstracts, p, 31 Van Pe¢, W. and Stragier, J. (1979) Evaluation of .'~me cold enrichment and isolation media for the recovery of Yersinia enterocolitica. Antonio van L¢cuwcnhoek J. Microbial. Serol. 45, 465-477. Vidon, DJ.M. and Detmas, C.L. (19el) Ine[derice of Yersinia enter~n'olitica in raw milk in Eastern France. Appl. Environ. Microbial, 41,355-359, Walker, SJ, and Gilmour, A. (1986) A comparison of media and methods for the recovery of Y~w~iniu t,irterocolitica and Yer.sinia enterc~'olitica.like bacteria from milk containing simulated raw milk microfloras. J. Appl. Bacl. 60. 175-183. Wauters, G. (1973) Improved mcthl~ds for the isolnlion and the recognition of Ycrsinia elffert~ctditit'a. Co~llr. Microbial. Immunol. 2, 68-7{). Wauters, G., Kandolo, K. and Janssens, M. (1987) Revised biogrouping scheme tff Yer.~inia om, rocolit. ica. Contr. Microbial. lmmunol. 9, 14-21. Wauters, G.+ Goossens, V., Jansscns, M. and Vandepitle, J. (1988a) New enrichment method for isolation of pathogenic Y{~sitffa entero~'ofitica serogroup 0:3 from pork. Appl. Environ. Microbial. 54, 851-854. Wautcrs, G., Junssens, M., Sleigerwait, A.G. and Brenner, D.J. (191'lgb) Yersiuia molhm, tii sp.nov, and Ycr,~iJ;ia ta,r¢'m'icri sp. nov., formerly called Y~,rsinia cntcroe'oliti<'a bit)groups 3A and 3B, Int. J. Syst. Bacterial. 38, 424-429. Wauters, G., Aleksic, S., Charlicr, J. and Schulz¢, G, (1991) Somatic and flagellar :mtigens of Yer,~h&r ellterocofitic~ and related species. Contr, Microbial, Immunol. 12, 239-243, Weagant, S,D. and Kaysncr, C.A. (1983) M~xiified enrichment broth for isolation of Yersmia emerc~'olit. ica from nonfood sources. Appl. Environ. Microbial. 45, 468-471.