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Rapid urease screening of Yersinia on CIN agar plates
International Journal of Food Microbiology 84 (2003) 111 – 115 www.elsevier.com/locate/ijfoodmicro
Short communication
Rapid urease screening of Yersinia on CIN agar plates Friederike Hilbert *, Sigrid Mayrhofer, Frans J.M. Smulders Institute of Meat Hygiene, Meat Technology and Food Science, University of Veterinary Medicine Vienna, Veterinaerplatz 1, A-1210 Vienna, Austria Received 1 May 2002; accepted 18 July 2002
Abstract Yersinia enterocolitica is an important foodborne pathogen, but isolation of virulent Yersinia from food sources is still time consuming and requires skills. In this article, we describe a rapid urease screening on cefsulodin – irgasan – novobiocin (CIN) agar plates with an agar overlay assay. This test is simple to perform, all colonies on a plate can be checked simultaneously, it only takes minutes for detection of urease-positive colonies and the colonies survive for transfer, further characterisation, and storage. Additionally, this method is useful to isolate virulent (urease-positive and pYV harbouring) Y. enterocolitica from foodstuffs. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Yersinia; Food; Urease; Virulence
1. Introduction Infections with Yersinia enterocolitica are acquired by ingestion of contaminated food or water. Alongside to foodborne infections caused by Salmonella and Campylobacter, Yersinia is the third frequent cause of foodborne diseases in Austria (Anonymous, 2001). As an invasive enteric pathogen, Y. enterocolitica causes a variety of symptoms in humans such as gastroenteritis, terminal ileitis, mesenterical lymphadenitis (pseudoappendicitis), septicemia and reactive arthritis (Bottone, 1997). Especially in children and individuals with underlying disease, symptoms may
* Corresponding author. Tel.: +43-1-25077-3316; fax: +43-125077-3390. E-mail address: [email protected] (F. Hilbert).
be severe (Cornelis et al., 1987; Cover and Aber, 1989; Attwood et al., 1989). As swine are the only known reservoir for human pathogenic Y. enterocolitica, pork and contaminated sources represent special risk factors (Tauxe et al., 1987). Epidemiological studies in food microbiology revealed that refrigerated foods stored over a long period pose an additional risk, because Yersinia, as a psychrotrophic microbe, is able to grow at temperatures as low as 0 jC (Hanna et al., 1977; Bercovier and Mollaret, 1984; Goverde et al., 1998). To cause disease, Y. enterocolitica has to harbour certain virulence factors encoded on the chromosome as well as on the virulence plasmid, pYV (Cornelis and Wolf-Watz, 1997; Cornelis et al., 1998). Nevertheless, clinical isolates without the virulence plasmid have also been identified (Robins-Browne et al., 1989; Grant et al., 1998). However, all pathogenic strains isolated
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1605(02)00397-5
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F. Hilbert et al. / International Journal of Food Microbiology 84 (2003) 111–115
so far have the capacity to produce urease and the role of the urease enzyme in acid survival and the induction of arthritis has been demonstrated and discussed (Gripenberg-Lerche et al., 2000). The urease in Yersinia is most active at acidic pH and helps Yersinia to overcome the gastric barrier by developing extensive acid resistance (De Koning-Ward and Robins-Browne, 1995, 1996). By hydrolysing urea into carbonic acid and ammonia, the net pH of the medium is increasing. Different procedures exist for isolating Y. enterocolitica from food especially from fresh meat, meat products and fish (Wauters et al., 1988; Nesbakken et al., 1991; ISO, 1994; Jiang et al., 2000; Jourdan et al., 2000; Johannessen et al., 2000). Nevertheless, competing microflora in raw meat and fish makes direct isolation, as is relatively easy to achieve for clinical samples, extremely skilful. Moreover, cold enrichment enhances growth of various other Yersinia species. The most widely used method for culturing Yersinia from food includes enrichment in liquid media, alkali treatment, selective plating on cefsulodin – irgasan –novobiocin (CIN) agar plates followed by differentiation through morphology (bulls-eye colonies) and identification by biochemical testing, biotyping and occasionally virulence testing (Varnam and Evans, 1996). However, CIN agar is not totally selective and morphological differentiation of colonies requires experience. Generally only a number of presumptive colonies are selected for further discrimination, with the associated risk of isolating false-negatives as well as a large number of false-positives that need to be further tested biochemically. We developed a direct method for urease testing of colonies on CIN agar plates. This test is simple to perform, all colonies on a plate can be checked simultaneously, it only takes minutes for detection of ureasepositive colonies and the colonies survive for transfer, further characterisation, and storage. In addition, this method is useful for isolating virulent (urease-positive and pYV harbouring) Y. enterocolitica from foodstuffs.
1975), Y. enterocolitica DSM 11502, 11503, 11504 as reference strains. We isolated and defined 90 biochemical different Y. enterocolitica food isolates, food isolates of Citrobacter freundii, Serratia marcescens,
2. Materials and methods 2.1. Bacterial strains Strains tested in our experiment included Y. enterocolitica W22703 plasmid cured (Cornelis and Colson,
Fig. 1. (A) CIN agar plate after plating 0.1 ml of alkali-treated enrichment broth (25 g minced meat in 250 ml PSS broth) and an incubation time of 24 h at 28 jC. (B) Same plate as in (A) after 8min incubation with brom-cresol-red soft agar. Urease-positive colonies develop a bright violet-blue colour and urease-negative are yellow to reddish.
F. Hilbert et al. / International Journal of Food Microbiology 84 (2003) 111–115
Serratia fonticola, Serratia liquefaciens, and Morganella morganii (by API 20E BioMe´rieux) (this work) out of 120 different food samples (45 raw pork, 30 chicken, 30 beef, and 15 fish samples). 2.2. Food samples and isolation of Yersinia Food samples included raw pork, chicken, beef, and fish. These samples were analysed according to ISO-10273 with minor modification: in brief, 25 g of the food sample was inoculated in peptone, sorbitol and sodium pyrovate broth (PSS, Biermayer, 1994) for 24 h at 28 jC. The enrichment culture was diluted 1:10 in 0.5% and 0.25% KOH for 30 s and 2 min (alkali treatment; Aulisio et al., 1980) and 0.1 ml of the mixture was immediately plated on CIN agar plates (Oxoid CM 653 and SR109E). After an incubation period of 24 h at 28 jC, colonies were analysed for colony morphology and further discriminated biochemically (API 20E, BioMe´rieux). 2.3. Urease detection 2.3.1. Rapid method CIN agar plates were overlayed with 0.25% agarose at 42 jC in 20 mM sodium acetate, 1 mM EDTA, containing 1% w/v urea and 0.25% w/v brom-cresolred. After an incubation period of 5 – 10 min at room temperature, urease-positive colonies displayed a brightly violet blue colour, whereas, urease-negative colonies were yellow to reddish (Fig. 1). Overlay assays have already been described as being useful for diagnostic food microbiology (Weenk et al., 1995). 2.3.2. Conventional urease testing Based on colony morphology on CIN agar, suspective colonies were isolated and incubated in urea broth (Oxoid CM 71 and SR 20) for 24 – 48 h at 28 jC. Urease-positive colonies were picked and transferred for further identification from both methods. 2.4. Biochemical differentiation All urease-positive isolates were biochemically differentiated using the API 20E system (BioMe´rieux) according to the standard protocol.
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3. Results and discussion 3.1. Isolation of Y. enterocolitica from food The isolation of potential pathogenic Y. enterocolitica from different foods especially raw meat and meat products, fish, seafoods may cause difficulties, especially because of competing microflora. In contrast to clinical microbiological practice, direct isolation of Yersinia from unprocessed meat and fish requires skill. During the first enrichment step in peptone/sorbitol/bile salt (PSB) liquid media (according to ISO 10273—for the isolation of presumptively pathogenic Y. enterocolitica), the growth of Y. enterocolitica is enhanced provided sodium pyruvate is added as growth enhancer and bile salts are eliminated as enrichment component. From this enrichment media, direct plating on selective agar following enrichment is not advantageous because competing microflora will cover the plate and plating dilutions may result in a total disappearance of Y. enterocolitica colonies. Hence, alkali treatment prior to selective plating is required to avoid false-negative results. The cefsulodin – irgasan – novobiocin (CIN) agar is not totally selective and susceptive single colonies need to be further identified by biochemical differentiation. In our study C. freundii, S. marcescens, S. fonticola, S. liquefaciens, and M. morganii have been identified as the most important contaminants on CIN agar plates when the above procedure is followed. Some colonies of these species have a similar ‘bulls-eye’ appearance as Y. enterocolitica and hence further biochemical testing is essential. 3.2. Identification of presumptive Yersinia colonies Further biochemical testing is necessary to distinguish Yersinia from other species. Testing urease activity is widely used as initial screening reaction for colonies showing the typical morphology on CIN agar plates. Urease activity discriminates Yersinia from most other species and additionally, is required for assessing virulence. Urease-negative Y. enterocolitica have not been identified as pathogenic so far. Nevertheless, conventional methods for urease testing take 24 – 48 h. Single colonies have to be selected from CIN agar plates and must be grown in urea broth or agar. This is disadvantageous, not only the cultur-
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F. Hilbert et al. / International Journal of Food Microbiology 84 (2003) 111–115
ing for reasons of time efficiency but also because preselection of colonies requires considerable experience. Consequently, less experienced persons will have to screen a large number of colonies with the risk of obtaining false-negative results. The rapid and direct urease detection we developed is shown in Fig. 1. We first tested Y. enterocolitica reference strains W22703 plasmid cured (Cornelis and Colson, 1975), DSM 11502, 11503 and 11504 by streaking them on CIN agar plates. After an incubation period of 24 h at 28 jC, the overlay assay was performed. All colonies tested positive for urease. Colonies positive for urease production are identified within 1– 5 min and our method allows simultaneous monitoring of all colonies on a plate. Furthermore, the bacterial colonies survive for transfer and subsequent characterisation. Then 45 raw pork, 30 chicken, 30 beef and 15 fish samples were examined. In total, 90 urease-positive Y. enterocolitica were isolated and further biochemically differentiated. All of them tested positive for urease in the rapid overlay assay as well as with the conventional method. A small number of competitive species growing on CIN agar plates—such as M. morganii and C. freundii—produce similar urease activity but these microorganisms are easily distinguished by their larger colony diameters. Acknowledgements This work was partly supported by the Austrian National Bank Jubilaeumsfonds Project No. 8559. We thank Erich Schopf for technical assistance. References Anonymous, 2001. Mitt. Sanitaetsverwalt. 102 (2), 47. Attwood, S.E., Cafferkey, M.T., Keane, F.B., 1989. Yersinia infections in surgical practice. Br. J. Surg. 76, 499 – 504. Aulisio, C.C.G., Mehlman, I.J., Sanders, A.C., 1980. Alkali method for rapid recovery of Yersinia enterocolitica and Yersinia pseudotuberculosis from foods. Appl. Environ. Microbiol. 39, 135 – 140. Bercovier, H., Mollaret, H.H., 1984. Yersinia. In: Krieg, N.R., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 1. Williams and Wilkins, Baltimore, pp. 498 – 506. Biermayer, J., 1994. Incidence and detection of Yersinia in Austrian slaughter pigs. Thesis, Veterinary Medical University of Vienna. Bottone, E.J., 1997. Yersinia enterocolitica: the charisma continues. Clin. Microbiol. Rev. 10, 257 – 276.
Cornelis, G., Colson, C., 1975. Restriction of DNA in Yersinia enterocolitica detected by recipient ability for a R factor from Escherichia coli. J. Gen. Microbiol. 87, 285 – 291. Cornelis, G.R., Wolf-Watz, H., 1997. The Yersinia Yop virulon: a bacterial system for subverting eukaryotic cells. Mol. Microbiol. 23, 861 – 867. Cornelis, G., Laroche, Y., Balligand, G., Sory, M.P., Wauters, G., 1987. Yersinia enterocolitica, a primary model for bacterial invasiveness. Rev. Infect. Dis. 9, 64 – 87. Cornelis, G.R., Boland, A., Boyd, A.P., Geuijen, C., Iriarte, M., Neyt, C., Sory, M.P., Stainier, I., 1998. The virulence plasmid of Yersinia, an antihost genome. Microbiol. Mol. Biol. Rev. 62, 1315 – 1352. Cover, T.L., Aber, R.C., 1989. Yersinia enterocolitica. N. Engl. J. Med. 321, 16 – 24. De Koning-Ward, T.F., Robins-Browne, R.M., 1995. Contribution of urease to acid tolerance in Yersinia enterocolitica. Infect. Immun. 63, 3790 – 3795. De Koning-Ward, T.F., Robins-Browne, R.M., 1996. Analysis of the urease gene complex of members of the genus Yersinia. Gene 182, 225 – 228. Goverde, R.L., Huis in’t Veld, J.H., Kusters, J.G., Mooi, F.R., 1998. The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5 degrees C). Mol. Microbiol. 28, 555 – 569. Grant, T., Bennett-Wood, V., Robins-Browne, R.M., 1998. Identification of virulence-associated characteristics in clinical isolates of Yersinia enterocolitica lacking classical virulence markers. Infect. Immun. 66, 1113 – 1120. Gripenberg-Lerche, C., Zhang, L., Ahtonen, P., Toivanen, P., Skurnik, M., 2000. Construction of urease-negative mutants of Yersinia enterocolitica serotypes O:3 and O:8: role of urease in virulence and arthritogenicity. Infect. Immun. 68, 942 – 947. Hanna, M.O., Stewart, J.C., Zinc, D.L., Carpenter, Z.L., Vanderzant, C., 1977. Development of Yersinia enterocolitica in raw and cooked beef and pork at different temperatures. J. Food Sci. 42, 1180 – 1184. ISO, 1994. Microbiology, General Guidance for the Detection of Presumptive Pathogenic Yersinia enterocolitica International Organization for Standardization, Genf Standard 10273. Jiang, G.C., Kang, D.H., Fung, D.Y., 2000. Enrichment procedures and plating media for isolation of Yersinia enterocolitica. J. Food Prot. 63, 1483 – 1486. Johannessen, G.S., Kapperud, G., Kruse, H., 2000. Occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products determined by a PCR method and a traditional culturing method. Int. J. Food Microbiol. 54, 75 – 80. Jourdan, A.D., Johnson, S.C., Wesley, I.V., 2000. Development of a fluorogenic 5Vnuclease PCR assay for detection of the ail gene of pathogenic Yersinia enterocolitica. Appl. Environ. Microbiol. 66, 3750 – 3755. Nesbakken, T., Kapperud, G., Dommarsnes, K., Skurnik, M., Hornes, E., 1991. Comparative study of a DNA hybridization method and two isolation procedures for detection of Yersinia enterocolitica 0:3 in naturally contaminated pork products. Appl. Environ. Microbiol. 57, 389 – 394.
F. Hilbert et al. / International Journal of Food Microbiology 84 (2003) 111–115 Robins-Browne, R.M., Miliotis, M.D., Cianciosi, S., Miller, V.L., Falkow, S., Morris Jr., J.G., 1989. Evaluation of DNA colony hybridization and other techniques for detection of virulence in Yersinia species. J. Clin. Microbiol. 27, 644 – 650. Tauxe, R.V., Vandepitte, J., Wauters, G., Martin, S.M., Goossens, V., De Mol, P., Van Noyen, R., Thiers, G., 1987. Yersinia enterocolitica infections and pork: the missing link. Lancet 1, 1129 – 1132. Varnam, A.H., Evans, M.G., 1996. Yersinia. In: Varnam, A.H., Evans, M.G. (Eds.), Foodborne Pathogens. Manson Publishing, London, pp. 129 – 155.
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Wauters, G., Goossens, V., Janssens, M., Vandepitte, J., 1988. New enrichment method for isolation of pathogenic Yersinia enterocolitica serogroup 0:3 from pork. Appl. Environ. Microbiol. 54, 851 – 854. Weenk, G.H., van den Brink, J.A., Struijk, C.B., Mossel, D.A.A., 1995. Modified methods for the enumeration of spores of mesophilic Clostridium species in dried foods. Int. J. Food Microbiol. 27, 185 – 200.
Short communication
Rapid urease screening of Yersinia on CIN agar plates Friederike Hilbert *, Sigrid Mayrhofer, Frans J.M. Smulders Institute of Meat Hygiene, Meat Technology and Food Science, University of Veterinary Medicine Vienna, Veterinaerplatz 1, A-1210 Vienna, Austria Received 1 May 2002; accepted 18 July 2002
Abstract Yersinia enterocolitica is an important foodborne pathogen, but isolation of virulent Yersinia from food sources is still time consuming and requires skills. In this article, we describe a rapid urease screening on cefsulodin – irgasan – novobiocin (CIN) agar plates with an agar overlay assay. This test is simple to perform, all colonies on a plate can be checked simultaneously, it only takes minutes for detection of urease-positive colonies and the colonies survive for transfer, further characterisation, and storage. Additionally, this method is useful to isolate virulent (urease-positive and pYV harbouring) Y. enterocolitica from foodstuffs. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Yersinia; Food; Urease; Virulence
1. Introduction Infections with Yersinia enterocolitica are acquired by ingestion of contaminated food or water. Alongside to foodborne infections caused by Salmonella and Campylobacter, Yersinia is the third frequent cause of foodborne diseases in Austria (Anonymous, 2001). As an invasive enteric pathogen, Y. enterocolitica causes a variety of symptoms in humans such as gastroenteritis, terminal ileitis, mesenterical lymphadenitis (pseudoappendicitis), septicemia and reactive arthritis (Bottone, 1997). Especially in children and individuals with underlying disease, symptoms may
* Corresponding author. Tel.: +43-1-25077-3316; fax: +43-125077-3390. E-mail address: [email protected] (F. Hilbert).
be severe (Cornelis et al., 1987; Cover and Aber, 1989; Attwood et al., 1989). As swine are the only known reservoir for human pathogenic Y. enterocolitica, pork and contaminated sources represent special risk factors (Tauxe et al., 1987). Epidemiological studies in food microbiology revealed that refrigerated foods stored over a long period pose an additional risk, because Yersinia, as a psychrotrophic microbe, is able to grow at temperatures as low as 0 jC (Hanna et al., 1977; Bercovier and Mollaret, 1984; Goverde et al., 1998). To cause disease, Y. enterocolitica has to harbour certain virulence factors encoded on the chromosome as well as on the virulence plasmid, pYV (Cornelis and Wolf-Watz, 1997; Cornelis et al., 1998). Nevertheless, clinical isolates without the virulence plasmid have also been identified (Robins-Browne et al., 1989; Grant et al., 1998). However, all pathogenic strains isolated
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1605(02)00397-5
112
F. Hilbert et al. / International Journal of Food Microbiology 84 (2003) 111–115
so far have the capacity to produce urease and the role of the urease enzyme in acid survival and the induction of arthritis has been demonstrated and discussed (Gripenberg-Lerche et al., 2000). The urease in Yersinia is most active at acidic pH and helps Yersinia to overcome the gastric barrier by developing extensive acid resistance (De Koning-Ward and Robins-Browne, 1995, 1996). By hydrolysing urea into carbonic acid and ammonia, the net pH of the medium is increasing. Different procedures exist for isolating Y. enterocolitica from food especially from fresh meat, meat products and fish (Wauters et al., 1988; Nesbakken et al., 1991; ISO, 1994; Jiang et al., 2000; Jourdan et al., 2000; Johannessen et al., 2000). Nevertheless, competing microflora in raw meat and fish makes direct isolation, as is relatively easy to achieve for clinical samples, extremely skilful. Moreover, cold enrichment enhances growth of various other Yersinia species. The most widely used method for culturing Yersinia from food includes enrichment in liquid media, alkali treatment, selective plating on cefsulodin – irgasan –novobiocin (CIN) agar plates followed by differentiation through morphology (bulls-eye colonies) and identification by biochemical testing, biotyping and occasionally virulence testing (Varnam and Evans, 1996). However, CIN agar is not totally selective and morphological differentiation of colonies requires experience. Generally only a number of presumptive colonies are selected for further discrimination, with the associated risk of isolating false-negatives as well as a large number of false-positives that need to be further tested biochemically. We developed a direct method for urease testing of colonies on CIN agar plates. This test is simple to perform, all colonies on a plate can be checked simultaneously, it only takes minutes for detection of ureasepositive colonies and the colonies survive for transfer, further characterisation, and storage. In addition, this method is useful for isolating virulent (urease-positive and pYV harbouring) Y. enterocolitica from foodstuffs.
1975), Y. enterocolitica DSM 11502, 11503, 11504 as reference strains. We isolated and defined 90 biochemical different Y. enterocolitica food isolates, food isolates of Citrobacter freundii, Serratia marcescens,
2. Materials and methods 2.1. Bacterial strains Strains tested in our experiment included Y. enterocolitica W22703 plasmid cured (Cornelis and Colson,
Fig. 1. (A) CIN agar plate after plating 0.1 ml of alkali-treated enrichment broth (25 g minced meat in 250 ml PSS broth) and an incubation time of 24 h at 28 jC. (B) Same plate as in (A) after 8min incubation with brom-cresol-red soft agar. Urease-positive colonies develop a bright violet-blue colour and urease-negative are yellow to reddish.
F. Hilbert et al. / International Journal of Food Microbiology 84 (2003) 111–115
Serratia fonticola, Serratia liquefaciens, and Morganella morganii (by API 20E BioMe´rieux) (this work) out of 120 different food samples (45 raw pork, 30 chicken, 30 beef, and 15 fish samples). 2.2. Food samples and isolation of Yersinia Food samples included raw pork, chicken, beef, and fish. These samples were analysed according to ISO-10273 with minor modification: in brief, 25 g of the food sample was inoculated in peptone, sorbitol and sodium pyrovate broth (PSS, Biermayer, 1994) for 24 h at 28 jC. The enrichment culture was diluted 1:10 in 0.5% and 0.25% KOH for 30 s and 2 min (alkali treatment; Aulisio et al., 1980) and 0.1 ml of the mixture was immediately plated on CIN agar plates (Oxoid CM 653 and SR109E). After an incubation period of 24 h at 28 jC, colonies were analysed for colony morphology and further discriminated biochemically (API 20E, BioMe´rieux). 2.3. Urease detection 2.3.1. Rapid method CIN agar plates were overlayed with 0.25% agarose at 42 jC in 20 mM sodium acetate, 1 mM EDTA, containing 1% w/v urea and 0.25% w/v brom-cresolred. After an incubation period of 5 – 10 min at room temperature, urease-positive colonies displayed a brightly violet blue colour, whereas, urease-negative colonies were yellow to reddish (Fig. 1). Overlay assays have already been described as being useful for diagnostic food microbiology (Weenk et al., 1995). 2.3.2. Conventional urease testing Based on colony morphology on CIN agar, suspective colonies were isolated and incubated in urea broth (Oxoid CM 71 and SR 20) for 24 – 48 h at 28 jC. Urease-positive colonies were picked and transferred for further identification from both methods. 2.4. Biochemical differentiation All urease-positive isolates were biochemically differentiated using the API 20E system (BioMe´rieux) according to the standard protocol.
113
3. Results and discussion 3.1. Isolation of Y. enterocolitica from food The isolation of potential pathogenic Y. enterocolitica from different foods especially raw meat and meat products, fish, seafoods may cause difficulties, especially because of competing microflora. In contrast to clinical microbiological practice, direct isolation of Yersinia from unprocessed meat and fish requires skill. During the first enrichment step in peptone/sorbitol/bile salt (PSB) liquid media (according to ISO 10273—for the isolation of presumptively pathogenic Y. enterocolitica), the growth of Y. enterocolitica is enhanced provided sodium pyruvate is added as growth enhancer and bile salts are eliminated as enrichment component. From this enrichment media, direct plating on selective agar following enrichment is not advantageous because competing microflora will cover the plate and plating dilutions may result in a total disappearance of Y. enterocolitica colonies. Hence, alkali treatment prior to selective plating is required to avoid false-negative results. The cefsulodin – irgasan – novobiocin (CIN) agar is not totally selective and susceptive single colonies need to be further identified by biochemical differentiation. In our study C. freundii, S. marcescens, S. fonticola, S. liquefaciens, and M. morganii have been identified as the most important contaminants on CIN agar plates when the above procedure is followed. Some colonies of these species have a similar ‘bulls-eye’ appearance as Y. enterocolitica and hence further biochemical testing is essential. 3.2. Identification of presumptive Yersinia colonies Further biochemical testing is necessary to distinguish Yersinia from other species. Testing urease activity is widely used as initial screening reaction for colonies showing the typical morphology on CIN agar plates. Urease activity discriminates Yersinia from most other species and additionally, is required for assessing virulence. Urease-negative Y. enterocolitica have not been identified as pathogenic so far. Nevertheless, conventional methods for urease testing take 24 – 48 h. Single colonies have to be selected from CIN agar plates and must be grown in urea broth or agar. This is disadvantageous, not only the cultur-
114
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ing for reasons of time efficiency but also because preselection of colonies requires considerable experience. Consequently, less experienced persons will have to screen a large number of colonies with the risk of obtaining false-negative results. The rapid and direct urease detection we developed is shown in Fig. 1. We first tested Y. enterocolitica reference strains W22703 plasmid cured (Cornelis and Colson, 1975), DSM 11502, 11503 and 11504 by streaking them on CIN agar plates. After an incubation period of 24 h at 28 jC, the overlay assay was performed. All colonies tested positive for urease. Colonies positive for urease production are identified within 1– 5 min and our method allows simultaneous monitoring of all colonies on a plate. Furthermore, the bacterial colonies survive for transfer and subsequent characterisation. Then 45 raw pork, 30 chicken, 30 beef and 15 fish samples were examined. In total, 90 urease-positive Y. enterocolitica were isolated and further biochemically differentiated. All of them tested positive for urease in the rapid overlay assay as well as with the conventional method. A small number of competitive species growing on CIN agar plates—such as M. morganii and C. freundii—produce similar urease activity but these microorganisms are easily distinguished by their larger colony diameters. Acknowledgements This work was partly supported by the Austrian National Bank Jubilaeumsfonds Project No. 8559. We thank Erich Schopf for technical assistance. References Anonymous, 2001. Mitt. Sanitaetsverwalt. 102 (2), 47. Attwood, S.E., Cafferkey, M.T., Keane, F.B., 1989. Yersinia infections in surgical practice. Br. J. Surg. 76, 499 – 504. Aulisio, C.C.G., Mehlman, I.J., Sanders, A.C., 1980. Alkali method for rapid recovery of Yersinia enterocolitica and Yersinia pseudotuberculosis from foods. Appl. Environ. Microbiol. 39, 135 – 140. Bercovier, H., Mollaret, H.H., 1984. Yersinia. In: Krieg, N.R., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 1. Williams and Wilkins, Baltimore, pp. 498 – 506. Biermayer, J., 1994. Incidence and detection of Yersinia in Austrian slaughter pigs. Thesis, Veterinary Medical University of Vienna. Bottone, E.J., 1997. Yersinia enterocolitica: the charisma continues. Clin. Microbiol. Rev. 10, 257 – 276.
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