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.     

A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica

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.     

A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica.

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.     

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.     

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.     

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.     

Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization.

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.     

Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.     

Laboratory investigation of virulence among strains of Yersinia enterocolitica and related species isolated from pigs and pork products.

Eighty strains of Yersinia enterocolitica and related species isolated from slaughtered pigs and pork products were tested for possession of virulence-associated phenotypes by employing 12 in vivo and in vitro assays. The isolates could be broadly divided into two groups: (i) strains belonging to pathogenic bioserotypes of Y. enterocolitica that displayed virulence-associated characteristics in most or all assays and (ii) strains belonging to Y. enterocolitica biotype 1A and to related species that were largely negative in these assays. No individual test was found as a single reliable measure of virulence. All strains belonging to Y. enterocolitica serotype O:1,2,3 were pyrazinamidase positive (indicates avirulence) and autoagglutination negative but were positive in all other virulence assays. Salt aggregation was found to be a better indicator of virulence than latex particle agglutination, both of which measure surface hydrophobicity. Overall, tissue culture cell invasion provided the best selection of a subpopulation of yersiniae that are potentially virulent. However, crystal violet and Congo red binding assays among others provided good prediction of virulence at the time of testing. Our results provide further evidence that swine may constitute an important reservoir of human pathogenic strains.     

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.     

Detection of pathogenic Yersinia enterocolitica in foods and water by immunomagnetic separation, nested polymerase chain reactions, and colorimetric detection of amplified DNA.

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.     

Detection of pathogenic Yersinia enterocolitica using a digoxigenin labelled probe targeting the yst gene

A 145 base pair digoxigenin-d-UTP-labelled probe, specific for pathogenic Yersinia enterocolitica heat-stable enterotoxin yst gene, was prepared by PCR. The probe was used in DNA-DNA colony hybridization and dot-blot hybridization assays. The specificity of the probe was confirmed using 52 strains representing all Yersinia spp., except Y. pestis . Out of a total of 25 Y. enterocolitica strains screened, the probe correctly identified all 18 pathogenic strains. Among the other Yersinia spp. screened, only one strain of Y. kristensenii was positively detected by the yst probe but could be differentiated by its weak signal response as compared with that obtained by pathogenic strains of Y. enterocolitica .     

Direct detection and isolation of plasmid-bearing virulent serotypes of Yersinia enterocolitica from various foods.

A procedure was developed for direct detection, isolation, and maintenance of plasmid-bearing virulent serotypes of Yersinia enterocolitica from different food sources. Plasmid-bearing virulent strains of Y. enterocolitica representing five serotypes were simultaneously detected and isolated from enriched swab samples of artificially contaminated pork chops, ground pork, cheese, and zucchini, using Congo red binding and low-calcium-response tests. The method was also effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Virulence of the strains isolated from these foods was confirmed by PCR, the expression of plasmid-associated phenotypes, and mouse pathogenicity.     

Detection of Yersinia enterocolitica in food by PCR amplification

A polymerase chain reaction (PCR) assay was developed for detection of pathogenic, virulent strains of Yersinia enterocolitica . By using both virulence loci virF and ail as markers for pathogenicity, detection of species with a virulence factor present was possible. DNA preparation in the presence of hexadecyl trimethy ammonium bromide (CTAB) was followed by two 44 cycle amplification reactions, one for each of the markers. As few as 10² Y. enterocolitica cells were detected in ground pork in the presence of 10⁵–10⁶ bacteria of other species. The described PCR assay provides a sensitive robust assay for the detection of virulent Y. enterocolitica in food.     

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.     

A new chromogenic agar medium for detection of potentially virulent Yersinia enterocolitica

Several outbreaks of foodborne yersiniosis have been documented and this disease continues to be source of infections transmitted through foods. The selective agars most commonly used to isolate Yersinia enterocolitica in clinical, food and environmental samples, cefsulodin-irgasan-novobiocin (CIN) and MacConkey (MAC) agars, lack the ability to differentiate potentially virulent Y. enterocolitica from other Yersinia that may be present as well as some other bacterial spp. This study proposes the use of an agar medium, Y. enterocolitica chromogenic medium (YeCM), for isolation of potentially virulent Y. enterocolitica. This agar contains cellobiose as the fermentable sugar, a chromogenic substrate and selective inhibitors for suppression of colony formation by many competing bacteria. All strains of potentially virulent Yersinia of biotypes 1B, and biotypes 2-5 formed convex, red bulls-eye colonies on YeCM that were very similar to those described for CIN agar. However, Y. enterocolitica biotype 1A and other related Yersinia formed colonies that were purple/blue on YeCM while they formed typical red bulls-eye colonies on CIN agar. When a mixture of potentially virulent Y. enterocolitica biotype 1B, Y. enterocolitica biotype 1A and 5 other bacterial species was used to artificially contaminate tofu and then spread-plated on three selective agars, Y. enterocolitica biotype 1B colonies were easily distinguished from other strains on YeCM. However, Y. enterocolitica biotype 1B colonies were indistinguishable from many other colonies on CIN and only distinguishable from those of C. freundii on MAC. When colonies were picked and identified from these agars, typical colonies from YeCM were confirmed only as Y. enterocolitica biotype 1B. Typical colonies on CIN and MAC were found to belong to several competing species and biotypes.     

Digoxigenin-labelled inv- and ail-probes for the detection and identification of pathogenic Yersinia enterocolitica in clinical specimens and naturally contaminated pig samples

A non-radioactive colony hybridization method was developed for the rapid detection of Yersinia enterocolitica in primary isolates and for differentiation between pathogenic and non-pathogenic strains. The method is based on, respectively, the presence of the inv -locus in all Yersinia spp. and the presence of the ail -gene in pathogenic Y. enterocolitica only. Hybridization results with ail -probes of 132 strains of Y. enterocolitica were in good agreement with pathogenicity phenotypes as indicated by a tissue culture invasion (TCI) assay and by serotyping. All TCI⁺ strains and only two TCI^- strains were positive by hybridization with ail. Hybridization results with inv - or ail -probes of 150 primary isolates of human, animal or slaughterhouse origin were compared with those of conventional methods to detect and identify Y. enterocolitica. All samples that were positive for Yersinia spp. by cultivation (four of 66) or were positive for pathogenic Y. enterocolitica by cultivation and serotyping (six of 84) were also positive by hybridization with, respectively, the inv - or ail -probe. In three slaughterhouse swab samples, in which Yersinia spp. were not detected by cultivation (2%), strong positive hybridization signals were obtained with the inv - and/or ail -probe. Four other swab samples which were negative by cultivation produced weak positive signals by hybridization with inv - and/or ail - probes. These results indicate that the method can be used for (1) the identification of pathogenic Y. enterocolitica isolates and (2) the detection of Yersinia spp. in primary isolates of naturally contaminated samples.