Application of a simplified method for recovery of Yersinia enterocolitica from surface waters

Yersinia enterocolitica was recovered from surface water by a simplified method: samples were taken by means of the Moore tampon and, after a short, warm preincubation in peptone-sorbitol-bile salts broth, were quickly passed to an alkali solution. Immediately after this, samples were directly plated on MacConkey agar. Y. enterocolitica was isolated from 60% of the water samples, and the identification confirmation of the strains chosen was 100%.     

Association of Yersinia enterocolitica with the manufacture of cheese and occurrence in pasteurized milk.

Raw milk in southern Ontario frequently contains Yersinia enterocolitica. The potential for transmission of this organism by cheese manufactured from unpasteurized milk was evaluated by examination of milk and cheese curd samples from cheese manufacturing plants and finished cheddar and Italian cheeses. The incidence of Y. enterocolitica was lower in cheese curd samples (9.2%) than in raw milk (18.2%). Most of the curd samples showed a positive phosphatase test, indicating production from raw milk. One curd sample yielded Y. enterocolitica after 4 weeks of storage at 4 degrees C but was negative after 8 weeks. All samples of cheddar and Italian cheeses, most of which showed a positive phosphatase test, were negative for Y. enterocolitica. One out of 265 samples (0.4%) of pasteurized fluid dairy products contained Y. enterocolitica.     

Detection of pathogenic Yersinia enterocolitica in environmental waters by PCR

The isolation of pathogenic strains of Yersinia enterocolitica from food and water samples by culture is time-consuming and unreliable. A two-step PCR procedure has been developed which, after a period of bacterial enrichment, can detect and confirm the presence of pathogenic Y. enterocolitica within a single day. This PCR method works effectively for a range of environmental water types, including reticulated waters, reservoirs and creeks. A survey of environmental waters in Victoria, Australia, showed that the PCR method detected pathogenic Y. enterocolitica in water sampled from four separate sites (two creeks and two reservoirs). Repeat samplings of the two reservoirs yielded PCR-positive results on all but one occasion. Culture analysis of the same samples detected pathogenic Y. entrocolitica in only one sample, indicating that the PCR can detect pathogenic Y. enterocolitica which are undetectable by culture. Results from this study confirm that potentially pathogenic strains of Y. enterocolitica can exist in environmental waters.     

Detection of Yersinia enterocolitica O:3 in faecal samples and tonsil swabs from pigs using IMS and PCR

Dilutions of faecal samples spiked with Yersinia enterocolitica O:3 were analysed using immunomagnetic separation (IMS) followed by PCR. In 10% faecal dilutions with added Y. enterocolitica cells; the limit of detection was 200 cells g^-1 faeces. Faecal samples from 38 pigs were analysed by IMS-PCR in parallel with detection and quantification of Y. enterocolitica O:3 using cold pre-enrichment culturing. Of the 15 culture-positive samples, only two were detected with IMS-PCR. These two samples contained 40–400 Y. enterocolitica O:3 cells g^-1 faeces; the highest level found in the investigation. This indicated that the low sensitivity of IMS-PCR was due to low amounts of cells in the faecal samples. Swab samples from 195 pig tonsils, taken on a slaughterline were examined using IMS-PCR and culture detection. Of 164 culture-positive samples, 60 were positive with IMS-PCR. In addition, IMS-PCR was positive for three culture-negative samples. Forty-five of the samples were further examined by IMS-PCR after 7–10 d of cold pre-enrichment. All 31 culture-positive samples as well as five culture-negative samples were detected by IMS-PCR. From these data it can be concluded that IMS-PCR can be used to detect Y. enterocolitica O:3 cells after pre-enrichment, but direct detection needs further optimization of the sample preparation procedures.     

Detection of low numbers of pathogenic Yersinia enterocolitica in environmental water and sewage samples by nested polymerase chain reaction

Isolation of pathogenic Yersinia enterocolitica from water and sewage by traditional culture techniques is time-consuming and subsequent differentiation between pathogenic and non-pathogenic strains can be difficult and unreliable. A nested polymerase chain reaction (PCR) procedure was used for the detection of low numbers of Y. enterocolitica in spiked samples from natural surface sources with variable background flora ranging from oligotrophic water to sewage. Water and sewage samples were filtered and filters enriched overnight in a non-selective medium. Nested PCR conducted on enriched broth, prepared by use of a rapid and simple preparation step consisting of centrifugation, proteinase K treatment and boiling, enabled the detection of 8-17 cfu 100 ml-1 water with background levels of up to 8700 heterotrophic organisms ml-1 and 10,000 cfu coliform organisms 100 ml-1 water. The analysis can be completed within 2-3 d and should be a significant tool in monitoring environmental waters and drinking water sources for the presence of pathogenic Y. enterocolitica.     

Yersinia in the water of wells near an area of irrigation with the effluents from a swine-breeding farm complex

Water taken from wells situated in the zone of irrigation with sewage water from a swine-breeding complex was studied. In 25.4% of samples Y. enterocolitica and Y. intermedia were detected. In winter and spring months twice as many strains were isolated in comparison with the number of strains isolated in summer and autumn. No Yersinia were isolated from control water samples taken from wells remote from the irrigation zone. The epidemiological relationship between a case of Yersinia infection and the use of infected well water was established.     

High prevalence of Yersinia enterocolitica 4:O3 on pig offal in southern Germany: a slaughtering technique problem

Prevalence and contamination routes of pathogenic Yersinia enterocolitica were studied in Southern Germany. Tonsil and faeces samples of 50 fattening pigs, 140 offal samples and 120 minced meat samples were examined. Pig and offal samples were collected from a slaughterhouse approved by the European Union, and minced meat samples from two large meat factories. Yersinia enterocolitica was isolated using direct plating, overnight enrichment and selective enrichment in MRB and ITC broth. The isolates were bio- and serotyped, and pathogenicity was studied using two plasmid-encoded virulence markers: calcium dependence and Congo red absorption. The genotypes were studied with pulsed-field gel electrophoresis using NotI enzyme. Prevalence of pathogenic Y. enterocolitica 4:O3 was 60% and 10% in tonsils and faeces of fattening pigs, respectively. Besides tonsils, prevalence of pathogenic Y. enterocolitica 4:O3 was also high in other pluck set samples, including tongues, lungs, hearts, diaphragms and livers. However, the highest isolation rate was obtained from the tonsils. Kidneys, which were not attached to the pluck set and did not hang together with tonsils on the rack, had the lowest isolation rate. Yersinia enterocolitica 4:O3 was isolated from 12% of minced meat samples. A total of 25 NotI profiles were obtained from porcine samples. The most common genotype, NBI, found in tonsils was also the most common type recovered from offal and minced meat samples. The high contamination rate of tonsils, and the indistinguishable NotI profiles obtained from tonsils and offal indicate that the tonsils contaminate offal when they are removed and hung on the rack together. When the head, with the tonsils and tongue, is not removed prior to evisceration and is not handled and inspected separately, it is difficult to control the spread of Y. enterocolitica 4:O3 from tonsils to the carcass, and subsequently, to meat.     

Prevalence of pathogenic Yersinia enterocolitica strains in pigs in the United States

Yersinia enterocolitica is considered an important food-borne pathogen impacting the pork production and processing industry in the United States. Since this bacterium is a commensal of swine, the primary goal of this study was to determine the prevalence of pathogenic Y. enterocolitica in pigs in the United States using feces as the sample source. A total of 2,793 fecal samples were tested for its presence in swine. Fecal samples were collected from late finisher pigs from 77 production sites in the 15 eastern and midwestern pork-producing states over a period of 27 weeks (6 September 2000 to 20 March 2001). The prevalence of ail-positive Y. enterocolitica was determined in samples using both a fluorogenic 5' nuclease PCR assay and a culture method. The mean prevalence was 13.10% (366 of 2,793 fecal samples tested) when both PCR- and culture-positive results were combined. Forty-one of 77 premises (53.25%) contained at least one fecal sample positive for the ail sequence. The PCR assay indicated a contamination rate of 12.35% (345/2,793) compared to 4.08% (114/2,793) by the culture method. Of the 345 PCR-positive samples, 252 were culture negative, while of the 114 culture-positive samples, 21 were PCR negative. Among 77 premises, the PCR assay revealed a significantly (P < 0.05) higher percentage (46.75%, n = 36 sites) of samples positive for the pathogen (ail sequence) than the culture method (22.08%, n = 17 sites). Thus, higher sensitivity, with respect to number of samples and sites identified as positive for the PCR method compared with the culture method for detecting pathogenic Y. enterocolitica, was demonstrated in this study. The results support the hypothesis that swine are a reservoir for Y. enterocolitica strains potentially pathogenic for humans.     

Membrane filter technique for the isolation of Yersinia enterocolitica.

A membrane filter procedure was developed for the isolation of Yersinia enterocolitica from aquatic environments. Primary differentiation was based on the fermentation of sorbitol, the absence of lysine decarboxylase and arginine decarboxylase-dihydrolase activities, and the production of urease. Sodium deoxycholate was incorporated as an inhibitor of background organisms. The presumptive identification of Y. enterocolitica was accomplished in 50 h, and the rate of identity confirmation of typical colonies was 88%. The mean recovery rate of 15 strains from phosphate buffer suspensions was 91%, and quantitative recovery was demonstrated for low populations of the organism in both laboratory-prepared and naturally occurring mixed cultures. The technique was used to isolate 33 strains of Y. enterocolitica from 15 of 27 river water samples and from prechlorinated sewage effluent. Nine (27%) of the isolates were rhamnose positive, and only five (15%) were serotypable. Two isolates were identified as serotype O:4 (or O:4,32), two were O:17, and the fifth was O:40.     

Enteropathogenic bacteria in frozen chicken.

Eighty-two samples of frozen chicken from retail stores were examined for the presence of Campylobacter, Yersinia enterocolitica, and salmonellae. Aerobic plate counts and numbers of coliform bacteria at 37 degrees C were determined. Campylobacter fetus subsp. jejuni was found in 22% of the samples, Y. enterocolitica was found in 24.5% and Salmonella typhimurium was found in one sample (1.2%). The isolated strains of Y. enterocolitica belonged to serotypes 4, 5b, 6, and 8. Aerobic plate counts and numbers of coliform bacteria at 37 degrees C were not found to be noticeably higher in samples containing pathogens than in pathogen-free samples. This investigation showed that chicken does contain other pathogenic bacteria than salmonellae. Campylobacter and Y. enterocolitica were isolated in much higher frequencies than Salmonella.     

Isolation of Yersinia enterocolitica and related species from the faeces of cows

A total of 203 samples of faeces from 124 cows was examined for the presence of Yersinia enterocolitica and related species by a variety of isolation procedures. Cold enrichment at 5°C for three weeks, followed by plating on cefsulodin-irgasannovobiocin agar yielded Yersinia species most frequently. Yersinia enterocolitica or related species were isolated from 50% of the cows.     

Yersinia enterocolitica in Danish pigs

Yersinia enterocolitica serotype 0:3, the predominating pathogenic serotype in Danish pigs, was isolated consistently from the tonsils of pigs in six farms but not from those in another four farms during a one-year survey, indicating a herd-wise distribution. Only one positive culture was obtained from four specific-pathogen-free herds. The organisms were not recovered from samples of fodder, water and faeces from any of the infected farms. Strains of Y. enterocolitica were tested for sensitivity to antimicrobial agents.     

Prevalence of Yersinia enterocolitica in waters of the lower Chippewa River Basin, Wisconsin.

Water samples collected over a 14-month period from the lower Chippewa River drainage basin in Wisconsin were examined for Yersinia enterocolitica with either MacConkey-Tween 80 agar or Y-M agar (T. N. Saari and G. P. Jansen, Contrib. Microbiol. Immunol. 5:185-196) as the selective medium. A new method of isolation was developed. Of 303 water samples, 8.25% were positive. A seasonal variation was noticed, with winter isolations being most frequent.     

Isolation of Yersinia enterocolitica from well water and growth in distilled water.

Yersinia enterocolitica was recovered from well water during a large water-borne outbreak of gastrointestinal illness. Isolates were predominantly Nilehn biotype 1, of which 57% were serologically nontypable. Isolation and enumeration of these Y. enterocolitica strains were made on M-Endo broth. Laboratory studies were conducted on selected isolates to establish the growth of Y. enterocolitica in distilled water and the competitive growth of this organism in various enteric media. Growth was obtained in sterile distilled water without added nutrients at 4, 25, and 37 degrees C. M-Endo medium gave equal or better recovery of Y. enterocolitica in competitive growth studies than did other commonly used enteric media using the membrane filter technique and incubating at 35 degrees C. All well water isolates were confirmed biochemically at 25 and 35 degrees C and serotyped, and antibiotic susceptibility tests were performed.     

Isolation of Yersinia enterocolitica from well water and growth in distilled water.

Yersinia enterocolitica was recovered from well water during a large water-borne outbreak of gastrointestinal illness. Isolates were predominantly Nilehn biotype 1, of which 57% were serologically nontypable. Isolation and enumeration of these Y. enterocolitica strains were made on M-Endo broth. Laboratory studies were conducted on selected isolates to establish the growth of Y. enterocolitica in distilled water and the competitive growth of this organism in various enteric media. Growth was obtained in sterile distilled water without added nutrients at 4, 25, and 37 degrees C. M-Endo medium gave equal or better recovery of Y. enterocolitica in competitive growth studies than did other commonly used enteric media using the membrane filter technique and incubating at 35 degrees C. All well water isolates were confirmed biochemically at 25 and 35 degrees C and serotyped, and antibiotic susceptibility tests were performed.     

Prevalence of Yersinia enterocolitica in pigs slaughtered in Chinese abattoirs

The distribution of Yersinia enterocolitica in slaughtered pigs in China was studied. A total of 8,773 samples were collected and examined from different pig abattoirs in 11 provinces from 2009 to 2011. Of these, 4,495 were oral-pharyngeal swab (tonsils) samples from pigs, 1,239 were from intestinal contents, and 3,039 were feces samples from abattoirs or local pigpens. The data showed that 1,132 strains were obtained, from which the isolation rate for Yersinia enterocolitica was 19.53% (878/4,495) from the tonsil samples, 7.51% (93/1,239) from intestinal contents, and 5.30% (161/3,039) from feces. Of the 850 pathogenic Yersinia strains, except for three of bioserotype 2/O:9 and three of bioserotype 4/O:3, most (844/850) were of bioserotype 3/O:3. Interestingly, pathogenic Y. enterocolitica accounted for the majority of the isolated strains from most provinces (85.17% to 100%), whereas from Heilongjiang, 96.52% (111/115) were classified as nonpathogenic biotype 1A with various serotypes, and only 3.48% of the strains (4/115) were pathogenic 3/O:3. All of the pathogenic strains were analyzed using pulsed-field gel electrophoresis (PFGE), and 49 patterns were obtained for the O:3 pathogenic strains; most of them were K6GN11C30021 (53.13%: 450/847) and K6GN11C30012 (21.37%: 181/847). Several strains from diarrhea patient samples revealed PFGE patterns identical to that from samples of local pigs, suggesting a possible link between porcine isolates and human infection. The results above suggested that Yersinia enterocolitica in slaughtered pigs from Chinese abattoirs was characterized by region-specific PFGE patterns and confirmed that strains isolated from pigs are closely related to those from human infections.     

The incidence of Yersinia enterocolitica and Yersinia enterocolitica-like organisms in raw and pasteurized milk in Northern Ireland

Yersinia enterocolitica and Y. enterocolitica -like bacteria were frequently isolated from samples of both raw bulked milk (34/150) and farm bottled (raw) milk (5/20). These bacteria were also found to contaminate creamery pasteurized milk (6/100 samples) and farm pasteurized milk (4/50 samples). Although Y. enterocolitica was the most commonly isolated species, Y. intermedia and Y. frederiksenii were also frequently obtained (52, 31 and 15% of isolates, respectively). Also, one atypical strain was identified as Y. aldovae . The Y. enterocolitica strains were largely biotype 1 (20/27) including five strains which could ferment lactose. One third of the Y. enterocolitica strains were not typable, but of those which were, the serotypes were 0:34 (18.5%), 0:5,27 (18.5%), 0:6,30 (15%), 0:4 (11%) and 0:7 (4%). Pre-enrichment in trypticase-soy broth (TSB) (at 22°C for 24 h) followed by selective enrichment in bile-oxalate-sorbose broth (at 22°C for 6 d) allowed the recovery of 92.3% of all isolates, as compared with 15.4% using cold enrichment in TSB at 4°C for 21 d.     

Comparative study of a DNA hybridization method and two isolation procedures for detection of Yersinia enterocolitica O:3 in naturally contaminated pork products

We compared a DNA-DNA hybridization assay, using a synthetically produced oligonucleotide probe, and two conventional isolation procedures (methods A and B) with regard to their relative efficiency in detecting Yersinia enterocolitica O:3 in naturally contaminated pork products. Method A was as described by Wauters et al. (Appl. Environ. Microbiol. 54:851-854, 1988). Method B has been recommended by the Nordic Committee on Food Analysis (method no. 117, 1987). The genetic probe was used in a colony hybridization assay to detect virulent yersiniae at each of the isolation steps with composed methods A and B. A total of 50 samples of raw pork products obtained from 13 meat-processing factories in Norway were examined. Y. enterocolitica serogroup O:3, biovar 4, was isolated from altogether 9 (18.0%) of the samples by using the two isolation procedures. In contrast, colony hybridization using the genetic probe indicated that 30 (60.0%) of the samples contained virulent yersiniae. All samples which were positive on cultivation were also positive by hybridization. The results indicate that the occurrence of pathogenic Y. enterocolitica in Norwegian pork products is substantially higher than previously demonstrated and, therefore, reinforce our suggestion that pork products represent an important potential source of human infection in Norway. The results also indicate that the use of conventional isolation procedures may lead to considerable underestimation of pathogenic Y. enterocolitica in pork products.     

Comparative study of a DNA hybridization method and two isolation procedures for detection of Yersinia enterocolitica O:3 in naturally contaminated pork products.

We compared a DNA-DNA hybridization assay, using a synthetically produced oligonucleotide probe, and two conventional isolation procedures (methods A and B) with regard to their relative efficiency in detecting Yersinia enterocolitica O:3 in naturally contaminated pork products. Method A was as described by Wauters et al. (Appl. Environ. Microbiol. 54:851-854, 1988). Method B has been recommended by the Nordic Committee on Food Analysis (method no. 117, 1987). The genetic probe was used in a colony hybridization assay to detect virulent yersiniae at each of the isolation steps with composed methods A and B. A total of 50 samples of raw pork products obtained from 13 meat-processing factories in Norway were examined. Y. enterocolitica serogroup O:3, biovar 4, was isolated from altogether 9 (18.0%) of the samples by using the two isolation procedures. In contrast, colony hybridization using the genetic probe indicated that 30 (60.0%) of the samples contained virulent yersiniae. All samples which were positive on cultivation were also positive by hybridization. The results indicate that the occurrence of pathogenic Y. enterocolitica in Norwegian pork products is substantially higher than previously demonstrated and, therefore, reinforce our suggestion that pork products represent an important potential source of human infection in Norway. The results also indicate that the use of conventional isolation procedures may lead to considerable underestimation of pathogenic Y. enterocolitica in pork products.     

The incidence of Yersinia enterocolitica and Yersinia enterocolitica-like organisms in raw and pasteurized milk in Northern Ireland

Yersinia enterocolitica and Y. enterocolitica-like bacteria were frequently isolated from samples of both raw bulked milk (34/150) and farm bottled (raw) milk (5/20). These bacteria were also found to contaminate creamery pasteurized milk (6/100 samples) and farm pasteurized milk (4/50 samples). Although Y. enterocolitica was the most commonly isolated species, Y. intermedia and Y. frederiksenii were also frequently obtained (52, 31 and 15% of isolates, respectively). Also, one atypical strain was identified as Y. aldovae. The Y. enterocolitica strains were largely biotype 1 (20/27) including five strains which could ferment lactose. One third of the Y. enterocolitica strains were not typable, but of those which were, the serotypes were 0:34 (18.5%), 0:5.27 (18.5%), 0:6.3 (15%), 0:4 (11%) and 0:7 (4%). Pre-enrichment in trypticase-soy broth (TSB) (at 22 degrees C for 24 h) followed by selective enrichment in bile-oxalate-sorbose broth (at 22 degrees C for 6 d) allowed the recovery of 92.3% of all isolates, as compared with 15.4% using cold enrichment in TSB at 4 degrees C for 21 d.