Identification and characterization of pathogenic Yersinia enterocolitica isolates by PCR and pulsed-field gel electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n=48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

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.     

Inhibition of Yersinia enterocolitica serotype O3 by natural microflora of pork

Yersinia enterocolitica serotype O3 did not grow but did survive in inoculated raw ground pork kept at 6 and 25 degrees C. The antagonistic effect of microbial flora, especially Hafnia alvei and environmental Yersinia organisms, on the growth of Y. enterocolitica serotype O3 in raw ground pork was evident. These results were supported by evidence of the inhibition of growth of Y. enterocolitica serotype O3 by Enterobacteriaceae, especially H. alvei and environmental Yersinia organisms, in mixed cultures at 6 and 25 degrees C. We suggest that naturally contaminated pork is a source of human infection, since Y. enterocolitica serotype O3 was capable of surviving in the raw pork for a long time.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat.

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Inhibition of Yersinia enterocolitica serotype O3 by natural microflora of pork.

Yersinia enterocolitica serotype O3 did not grow but did survive in inoculated raw ground pork kept at 6 and 25 degrees C. The antagonistic effect of microbial flora, especially Hafnia alvei and environmental Yersinia organisms, on the growth of Y. enterocolitica serotype O3 in raw ground pork was evident. These results were supported by evidence of the inhibition of growth of Y. enterocolitica serotype O3 by Enterobacteriaceae, especially H. alvei and environmental Yersinia organisms, in mixed cultures at 6 and 25 degrees C. We suggest that naturally contaminated pork is a source of human infection, since Y. enterocolitica serotype O3 was capable of surviving in the raw pork for a long time.     

Use of a single procedure for selective enrichment, isolation, and identification of plasmid-bearing virulent Yersinia enterocolitica of various serotypes from pork samples.

Many selective enrichment methods for the isolation of Yersinia enterocolitica from foods have been described. However, no single isolation procedure has been described for the recovery and identification of various plasmid-bearing serotypes. A single improved procedure for selective enrichment, isolation, identification, and maintenance of plasmid-bearing virulent serotypes of Y. enterocolitica from pork samples was developed. Enrichment at 12 degrees C in Trypticase soy broth containing yeast extract, bile salts, and Irgasan was found to be an efficient medium for the recovery of plasmid-bearing virulent strains of Y. enterocolitica representing O:3; O:8; O:TACOMA; O:5, O:27; and O:13 serotypes. MacConkey agar proved to be a reliable medium for the isolation of presumptive colonies, which were subsequently confirmed as plasmid-bearing virulent strains by Congo red binding and low calcium response. Further confirmation by multiplex PCR employed primers directed at the chromosomal ail and plasmid-borne virF genes, which are present only in pathogenic strains. The method was applied to pig slaughterhouse samples and was effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Strains isolated from ground pork and tongue expressed plasmid-associated phenotypes and mouse pathogenicity.     

Identification and Characterization of Pathogenic Yersinia enterocolitica Isolates by PCR and Pulsed-Field Gel Electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n = 48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

New enrichment method for isolation of pathogenic Yersinia enterocolitica serogroup O:3 from pork

A new enrichment medium for the recovery of pathogenic Yersinia enterocolitica serogroup O:3 from naturally infected meat products based on three selective agents, Irgasan, ticarcillin, and potassium chlorate (ITC), was compared with several other one- or two-step enrichments. Y. enterocolitica serogroup O:3 was recovered from 96.5% of 29 pork tongues, 24% of 50 ground pork samples, 16% of 25 masseter muscle samples, and 61% of tonsils. ITC was by far the most sensitive method for the recovery of Y. enterocolitica O:3, especially from ground meat and masseter muscles, while cold and two-step enrichments yielded better results for nonpathogenic strains. Plating of ITC enrichments onto SS-deoxycholate-calcium agar gave overall better results than plating onto cefsulodin-Irgasan-novobiocin agar for serogroup O:3.     

New enrichment method for isolation of pathogenic Yersinia enterocolitica serogroup O:3 from pork.

A new enrichment medium for the recovery of pathogenic Yersinia enterocolitica serogroup O:3 from naturally infected meat products based on three selective agents, Irgasan, ticarcillin, and potassium chlorate (ITC), was compared with several other one- or two-step enrichments. Y. enterocolitica serogroup O:3 was recovered from 96.5% of 29 pork tongues, 24% of 50 ground pork samples, 16% of 25 masseter muscle samples, and 61% of tonsils. ITC was by far the most sensitive method for the recovery of Y. enterocolitica O:3, especially from ground meat and masseter muscles, while cold and two-step enrichments yielded better results for nonpathogenic strains. Plating of ITC enrichments onto SS-deoxycholate-calcium agar gave overall better results than plating onto cefsulodin-Irgasan-novobiocin agar for serogroup O:3.     

Rapid 5' nuclease (TaqMan) assay for detection of virulent strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5' nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be >/=10(2) CFU/ml in pure cultures and >/=10(3) CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5' nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

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.     

Note: Isolation, characterization and epidemiology of Yersinia enterocolitica from humans and animals

During an 11-year period (1983 to 1994), 51 strains of Yersinia enterocolitica were isolated from humans and animals. Specimens were collected from a total of 3601 sources consisting of 956 patients with enteritis, 300 patients with urinary tract infection, 1564 healthy humans, 510 swine, 38 guinea-pigs, 118 rats and 115 rabbits. Five strains of Y. enterocolitica , bio/serogroups 2/O:9 and 4/O:3, virulence positive, were recovered from patients. Forty-two variants of Y. enterocolitica belonging to pathogenic serogroup O:3, Voges-Proskauer-negative biogroup 3 were recovered from swine, rats and rabbits. The rate of isolation of Y. enterocolitica from diarrhoeal swine was apparently greater than those from healthy swine. The incidence of human infections due to Y. enterocolitica was very low and bioserogroups of isolates were different from the strains which were isolated from animals. There was no evidence to suggest that swine were the source of Y. enterocolitica in humans.     

Isolation of Yersinia enterocolitica and related species from foods in France

The occurrence of Yersinia enterocolitica and related species (Y. intermedia, Y. frederiksenii, Y. kristensenii) in foods from France was investigated by using different enrichment procedures. Initially, seven procedures were evaluated with pork products. These methods included a cold preenrichment in yeast extract-rose bengal broth or in phosphate-sorbitol-bile medium, followed by selective enrichment either in Pastone-sucrose-Tris-azide broth, in modified Rappaport broth, or in bile-oxalate-sorbose broth, and then isolation onto Hektoen or cefsulodin-irgasan-novobiocin agar with or without KOH pretreatment. The best enrichment procedure in terms of percentage of positive samples obtained within the shortest time was the combination of phosphate-sorbitol-bile and bile-oxalate-sorbose with alkali treatment before isolation onto cefsulodin-irgasan-novobiocin agar. This system was then used to analyze foods other than pork. An average contamination rate of 33.5% was observed for 666 samples analyzed; pork products were by far the most contaminated, especially the so-called tartinette (96.8% of positive samples) which contained up to five different strains of Yersinia spp. Environmental serogroups of Y. enterocolitica O:5, O:39,41, O:6, and O:7,8 were predominant, but no isolate of either human pathogenic type (O:3 or O:9) was obtained.     

Isolation of Yersinia enterocolitica and related species from foods in France.

The occurrence of Yersinia enterocolitica and related species (Y. intermedia, Y. frederiksenii, Y. kristensenii) in foods from France was investigated by using different enrichment procedures. Initially, seven procedures were evaluated with pork products. These methods included a cold preenrichment in yeast extract-rose bengal broth or in phosphate-sorbitol-bile medium, followed by selective enrichment either in Pastone-sucrose-Tris-azide broth, in modified Rappaport broth, or in bile-oxalate-sorbose broth, and then isolation onto Hektoen or cefsulodin-irgasan-novobiocin agar with or without KOH pretreatment. The best enrichment procedure in terms of percentage of positive samples obtained within the shortest time was the combination of phosphate-sorbitol-bile and bile-oxalate-sorbose with alkali treatment before isolation onto cefsulodin-irgasan-novobiocin agar. This system was then used to analyze foods other than pork. An average contamination rate of 33.5% was observed for 666 samples analyzed; pork products were by far the most contaminated, especially the so-called tartinette (96.8% of positive samples) which contained up to five different strains of Yersinia spp. Environmental serogroups of Y. enterocolitica O:5, O:39,41, O:6, and O:7,8 were predominant, but no isolate of either human pathogenic type (O:3 or O:9) was obtained.     

Development of a two-step enrichment procedure for recovery of Yersinia enterocolitica from food.

Two new enrichment media were formulated for the recovery of Yersinia enterocolitica from foods: (i) yeast extract-rose bengal broth for preenrichment at 4 or 10 degrees C; and (ii) bile-oxalate-sorbose broth, a selective enrichment incubated at 22 degrees C. Comparison of these media in a two-step enrichment procedure against cold enrichment and modified Rappaport broth showed improved and more rapid recovery of human strains of Y. enterocolitica from inoculated foods. The use of bile-oxalate-sorbose broth as a selective enrichment also improved the performance of cold enrichment with phosphate-buffered saline. Determination of the best enrichment system for recovery of Y. enterocolitica from samples of retail pork and fresh pork tongues depended on whether the criterion was the number of positive samples, the variety of different serotypes recovered, or the ability to recover the important human serotype O:3. A single enrichment system with the widest selectivity would include preenrichment at 4 degrees C with either phosphate-buffered saline for 14 days or yeast extract-rose bengal broth for 9 days followed by selective enrichment with bile-oxalate-sorbose broth at 22 degrees C for 5 days.     

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.     

Isolation of virulent Yersinia enterocolitica from porcine tongues.

Thirty-one tongues from apparently normal, freshly slaughtered pigs were assayed for the presence of Yersinia enterocolitica by different enrichment and postenrichment techniques. Sixteen different isolates were recovered, including six of serotype O:8, four of serotype O:6,30, two of serotype O:3 phage type IXb, and one each of serotypes O:13,7, O:18, and O:46. One isolate was not typable. Cold enrichment in phosphate-buffered saline followed by treatment with dilute KOH or subsequent enrichment in modified Rappaport broth recovered 12 and 7 isolates, respectively. Four of the same isolates were recovered from the same tongues by both procedures. Cold enrichment without a selective postenrichment treatment recovered two isolates. Direct enrichment in modified Rappaport broth or modified selenite broth was ineffective in recovering yersiniae, as no isolates were obtained by either method. All of the serotype O:8 isolates were virulent to mice, causing the death of adults after oral challenge. This is the first report that associates Y. enterocolitica serotype O:8 with a natural reservoir.     

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.