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A total of 300 food samples including 180 milk and 120 meat products have been examined for the presence of Yersinia spp. using the ISO 10273 and the cold enrichment method. The overall prevalence of Yersinia spp. was 84 (28%). Yersinia enterocolitica was isolated from 18 (6%) of the 300 samples. The other Yersinia species were detected in the samples Yersinia rohdei 15 (5%), Yersinia intermedia 14 (4.7%), Yersinia pseudotuberculosis 12 (4%), Yersinia ruckeri 12 (4%), Yersinia mollaretii 5 (1.7%), Yersinia bercovieri 4 (1.3%), and atypical Yersinia spp. 4 (1.3%). The conventionally identified Y. enterocolitica strains were also confirmed by the 16S rRNA gene sequencing. All Y. enterocolitica strains biotyped as 1A had negative results in the phenotypic virulence tests. The 84 Yersinia strains were also examined genotypically for the presence of virulence genes. None of the Y. enterocolitica and other Yersinia strains contained the ail, ystA, yadA, and virF except only 1 Y. intermedia and 2 Y. enterocolitica strains that were found to be positive for ystB. Antimicrobial resistance of 84 Yersinia to 16 antimicrobial agents was determined by the disk diffusion method. All strains were sensitive to tobramycine and imipenem while resistant to clindamycin. Although 84.5% of the strains were resistant to at least 3 or more antimicrobial agents, 64.3% of them were resistant to 4 or more antimicrobial agents., (© 2015 Institute of Food Technologists®)

Pasteurized bottled milk supplied by a single dairy was frequently found to be contaminated with Yersinia spp. Investigations were carried out at the dairy in an effort to pinpoint the source of these organisms. Viable counts obtained from milk bottle rinses indicated that bottle washing was often unsatisfactory, and on one occasion Y. frederiksenii was isolated from the pooled rinse water of six bottles. Samples of milk were taken on arrival at the dairy and at various stages following pasteurization. Heat resistance tests carried out on strains of yersinia isolated from pasteurized milk indicated that they would not survive the pasteurization process. However two strains of yersinia were isolated from a sample of milk taken immediately after pasteurization but before bottling. The thermograph indicated that the time/temperature conditions applied during pasteurization were adequate. The presence of yersinia strains in the milk at this stage therefore suggests that undetectable levels of raw milk were being allowed to contaminate the pasteurized milk. The absence of yersinia in cartoned samples produced on the same day as contaminated bottled samples indicated that environmental contamination of the bottle filler valve also may have occurred at times. Results of this investigation indicate that increased vigilance is required to ensure proper operation of pasteurizers and bottle washers.

We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.

At 5 degrees C four strains of fermentative, gram-negative bacteria (Serratia liquefaciens, Yersinia enterocolitica, Enterobacter cloacae, and Aeromonas hydrophila) grew aerobically and anaerobically on adipose tissue removed from beef muscle of low pH (5.4 to 5.6). All four strains also grew aerobically and anaerobically on muscle tissue of high pH (6.0 to 6.3). However, none of the four grew anaerobically on beef muscle of low pH, and the aeromonad strain also failed to grow aerobically on such muscle. Growth of S. liquefaciens and E.cloacae on vacuum-packaged beef muscle was dependent on the pH of the tissue and the oxygen transmission rate of the packaging film. Although the four strains grew in broth buffered at pH 5.55, L-lactate, at the concentration found in muscle of low pH (ca. 100 mM), prevented anaerobic growth of all four isolates and prevented the aerobic growth of th aeromonad. At pH 6.1 in buffered broth, the concentration of L-lactate occurring in muscle of high pH did not prevent aerobic or anaerobic growth of any of the strains.

Retail packs of mixed, prepared salad vegetables from two different manufacturers were stored at 7 degrees C until the end of storage-life (sell-by date plus 1 d), when the microbial flora was examined. The quality of the salads was acceptable at the end of storage life. The oxygen concentrations in packs were lower, and the carbon dioxide concentrations were higher, than those in air. High numbers of bacteria were present, with Pseudomonas spp. and Enterobacter agglomerans predominating in packs of both salads, together with lactic acid bacteria in one of the salads. Significant numbers of pectolytic bacteria including Pseudomonas spp. and Erwinia carotovora were detected. Despite the presence of high numbers of coliform bacteria, Escherichia coli was not detected in batches of one salad, and was detected in relatively low numbers in batches of the other. Yersinia spp., predominantly environmental strains of Yersinia enterocolitica, were isolated by enrichment from all samples tested; Staphylococcus aureus and enterococci were not detected.

A new culture method employing a potassium hydroxide treatment was compared with the conventional cold enrichment method for efficacy in recovering Yersinia sp. from naturally and artificially contaminated food. The new method increased the yield of Yersinia sp. fourfold and the sensitivity 100-fold, shortened the incubation period, and appreciably decreased the growth of non-Yersinia bacteria from a variety of meats, shellfish, and vegetables.

One hundred and twenty raw ground beef, pork and chicken samples from ten local grocery stores during a one year period were assayed for the presence of Yersinia spp., using direct and postenrichment KOH treatment. Seven thousand and fifty-five different isolates were recovered from 119 (99%) samples of beef and pork and 118 (98%) samples of chicken, including 27 Yersinia enterocolitica serotype 03 biotype 3B phage type 2 (4 pork samples), 4 Y. enterocolitica serotype 03 biotype 4 phage type 8 (3 pork samples), 4,066 Y. enterocolitica biotype 1, 2,194 Yersinia intermedia, 509 Yersinia frederiksenii, and 251 Yersinia kristensenii. Y. enterocolitica serotype 03 was isolated from 6 pork samples, including simultaneously isolation of both biotypes 3B and 4 from one sample. There was the negative correlation between the incidence of isolation of serotype 03 and the environmental temperature, but other environmental Yersinia spp. were frequently isolated through the year. Y. enterocolitica serotype 03 isolates were counted by alkali direct treatment at less than 10(3) cells per g of pork samples, and environmental Yersinia spp. at less than 10(5) cells per g of beef, pork and chicken samples. This may be the first evidence that some pork is contaminated with at least 10(3) Y. enterocolitica serotype 03 cells per g.

Tests have demonstrated that current enrichment methods may fail to detect low levels of some clinical strains of Yersinia enterocolitica inoculated into meats and oysters and need improvement. The results of cold enrichment with phosphate and tripolyphosphate buffers were very poor and the sensitive clinical strain failed to grow in Wauters' MgCl2 broth. Twenty typical clinical strains of Y. enterocolitica belonging to Nilehn's biotypes 2, 3, and 4 had the ability to invade HeLa cells tissue culture but none of the 52 atypical clinical and food strains belonging to Nilehn's biotype 1 or rhamnose-positive were invasive. Although temperature sensitive, this test is reproducible and useful for screening noninvasive isolates.

Seventeen Yersinia enterocolitica-like isolates from vacuum-packaged beef and lamb are described. A majority of the isolates were motile at 25 degrees C but not at 36 degrees C, utilized citrate at 25 degrees C (delayed or negative at 36 degrees C) and produced acid from rhamnose, raffinose and melibiose. Increases in count of Y. enterocolitica occurred on raw beef stored at 0--1 degrees C over a 10-day period. No viable Y. enterocolitica were detected in beef roasts which were inoculated with about 3 x 10(6) cells/g and subsequently heated to a final internal temperature of 60--62 degrees C. Y. enterocolitica counts of inoculated beef steaks stored at 1--5 degrees C for 21--35 days were consistently higher in the more oxygen-permeable films than in vacuum packages. Colonies of Y. enterocolitica and related bacteria from meats produced shiny, enamel-black colonies on bismuth sulfite agar with plate incubation at 25 degrees C for 3--5 days.

Yersinia enterocolitica is one of the few human pathogens that grows at the proper food refrigeration temperatures of 0 to 5 degrees C. Although the isolation of environmental biotypes of Y. enterocolitica from many types of food and water has been reported in the literature, the recovery of the sensitive strains inoculated into foods has been slow and uncertain. Rapid recovery of several clinical strains inoculated into meats was accomplished by using two modified selenite broths without added nutrients. It was critical to restrict the sample size of the blended meat suspension at the 0.2-g/100 ml level, thereby restricting the growth of the total bacterial population in the selenite enrichment media. Otherwise, the slower growing Y. enterocolitica would be overwhelmed by the faster growing normal bacterial flora from the meat. Both the resistant serotype O:3 and the sensitive O:8 clinical isolates of Y. enterocolitica were recovered from the modified selenite enrichment media after 2 and 3 days of incubation at 22 degrees C.

Isolation of Yersinia enterocolitica was performed on healthy humans, animals and food, and growth of Y. enterocolitica on sliced ham at various temperature ranges was also examined. It was noticed that the most prevalent 0 group (03) in human infection was isolated from swine, pork and chopping boards. The storage of food in the refrigerator (5 degrees C) possibly increases the risk of infection since Y. enterocolitica is multipliable at low temperatures more readily than many other pathogenic bacteria. Pork and meat products contaminated with Y. enterocolitica, either primarily or secondarily, are probably the most important source of human infection.