A two-step polymerase chain reaction (PCR) procedure with two nested pairs of primers specific for the yadA gene of Yersinia enterocolitica was developed. The PCR assay identified all common pathogenic serogroups (O:3, O:5,27, O:8, O:9, O:13, and O:21) from three continents and differentiated pathogenic Y. enterocolitica from Y. pseudotuberculosis and from a variety of nonpathogenic yersiniae representing 25 serogroups and four species. The performance of the method was evaluated with seeded food and water samples. We compared two procedures for sample preparation prior to PCR: one was based on immunomagnetic separation of the target bacteria from the sample, using magnetic particles coated with immunoglobulin antibodies to Y. enterocolitica serogroup O:3, and the other method consisted of a series of centrifugation steps combined with proteinase treatment. Regardless of the method used, the PCR assay was capable of detecting 10 to 30 CFU/g of meat in 10(6)-fold excess of indigenous bacteria. When the samples were enriched overnight in a nonselective medium, the sensitivity was increased to approximately 2 CFU/g, except for samples with an extremely high background flora (> 10(7) CFU/g). We compared gel electrophoretic detection of PCR products with a colorimetric detection method designated DIANA (detection of immobilized amplified nucleic acids), which enabled easy visualization of amplified fragments in a microtiter plate format with an optical density reader. DIANA and gel electrophoresis showed complete concordance in their discrimination between positive and negative samples. The combination of immunomagnetic separation, nested PCR, and DIANA makes possible the development of a fully automated analytic process which requires a minimum of laboratory manipulations.
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 enterocolitica 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-degrees-C; plating on SSDC agar (2 days at 30-degrees-C). (2) Pre-enrichment in TSB for 1 day at 24-degrees-C; enrichment in BOS for 5 days at 24-degrees-C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaCl for 5 s); plating on CIN agar (2 days at 24-degrees-C).
In November 1992 and June and August 1993 rectal contents from 204 small mammals living in the wild were collected and examined for the presence of Yersinia enterocolitica serovar O:8 to clarify the source of human infections caused by this microbe in the Tsugaru Region of Aomori Prefecture, Japan. Serovar O:8 was isolated from 10 (5.2%) of 193 wild rodents trapped in June 1993 (9 of 107) and August 1993 (1 of 23) but not from animals trapped in November 1992 (0 of 63). This serovar was not isolated from 11 moles. From May to September 1993, 12 human patients were found to have become ill and to be infected with Y. enterocolitica O:8. The patients lived in the same districts where the wild rodents harboring serovar O:8 were trapped. Two different patterns by restriction enzyme analysis of the virulence plasmid were observed. One pattern obtained by restriction enzyme analysis of the virulence plasmid was observed in 20 isolates from 11 human patients and 9 wild rodents, and the other was observed in 2 isolates from 1 human patient and 1 wild rodent. These findings indicate that wild rodents seem to play an important role as a source of human Y. enterocolitica O:8 infection.
