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.     

Improved procedure for recovery of Yersinia enterocolitica from meats.

A 1- to 3-day enrichment-KOH postenrichment procedure was evaluated and found to be as effective in recovering Yersinia enterocolitica from meats as a 14- to 21-day cold enrichment procedure, with or without KOH postenrichment. The shortened procedure consists of enriching 1.0- and 25-g samples of meat in phosphate-buffered saline (pH 7.2) at 25 degrees C. After incubation (48 and 72 h for 1.0-g samples and 24 and 48 h for 25-g samples); 0.5-ml portions of enrichment culture were treated with 4.5 ml of 0.25% KOH-0.5% NaCl for 2 min and 0.5% KOH-0.5% NaCl for 15 s, and 0.1-ml portions of treated culture were plated onto MacConkey or CIN agars or both. The procedure effectively recovered 2 to 12 cells of a number of both mouse-virulent and avirulent strains per g of ground beef with aerobic plate counts of approximately 10(6) to 10(7) CFU/g. Similarly, the procedure isolated both likely virulent and avirulent strains from porcine tongues (aerobic plate counts of 10(5) to 10(7) CFU/g) naturally contaminated with Y. enterocolitica. The organism was isolated from the tongues at similar rates by both shortened enrichment and cold enrichment procedures. Eight tongues were positive for serotype O:5,27 strains that agglutinate with WA-specific absorbed antiserum, an antiserum specific for mouse-virulent Y. enterocolitica (Doyle et al., Infect. Immun. 37:1234-1240, 1982), indicating that the oral cavity of swine is a reservoir of likely virulent serotype O:5,27 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.     

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.     

Isolation of Yersinia enterocolitica from raw milk

Four enrichment procedures were used for examining 131 raw milk samples for the presence of Yersinia enterocolitica. Forty-two isolations were obtained from 19 pooled- (31.1 percent positive) and 10 individual-producer samples (14.3 percent positive). Enrichment by Butterfields phosphate buffer incubated at 4 degrees C for 14 days and then inoculation of modified Rappaport broth incubated at 23 degrees C for 5 days produced the greatest number of isolations. The majority of isolates were biotype 1, and many were atypical from clinical isolates in being rhamnose positive (47.6 percent), citrate positive (16.7 percent), and lactose positive (26.2 percent). Thirteen isolates were serotypable, belonging to seven different O serotypes, with O:5 occurring most frequently.     

Isolation of Yersinia enterocolitica from raw milk.

Four enrichment procedures were used for examining 131 raw milk samples for the presence of Yersinia enterocolitica. Forty-two isolations were obtained from 19 pooled- (31.1 percent positive) and 10 individual-producer samples (14.3 percent positive). Enrichment by Butterfields phosphate buffer incubated at 4 degrees C for 14 days and then inoculation of modified Rappaport broth incubated at 23 degrees C for 5 days produced the greatest number of isolations. The majority of isolates were biotype 1, and many were atypical from clinical isolates in being rhamnose positive (47.6 percent), citrate positive (16.7 percent), and lactose positive (26.2 percent). Thirteen isolates were serotypable, belonging to seven different O serotypes, with O:5 occurring most frequently.     

Two modified selenite media for the recovery of Yersinia enterocolitica from meats.

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.     

Incidence of Yersinia enterocolitica in raw milk in eastern France.

A total of 75 raw milk samples collected from a central dairy or from retailers in Alsace, France, were analyzed for the presence of Yersinia enterocolitica. Three procedures were used: enrichment at 4 degrees C for 1 month; enrichment in modified Rappaport medium at room temperature for 72 h after a preenrichment at 4 degrees C for 1 month; and enrichment in a new medium containing sucrose, tris(hydroxymethyl)aminomethane, sodium azide, and ampicillin (PSTA) at 28 degrees C for 48 h after a preenrichment at 4 degrees C for 1 month. Isolation of Y. enterocolitica was made on Hektoen medium plus ampicillin. Sixty-one samples were positive (81.4%), but the PSTA medium produced the greatest number of isolates. Biochemical, serological, and phage typing of 40 isolates showed that chemotype 1 and serogroup O:5 were predominant. In seven cases, two different strains were obtained from the same samples. Most of the 66 isolates tested for their antimicrobial susceptibility were resistant to ampicillin and carbenicillin, and all were sensitive to tetracycline, chloramphenicol, streptomycin, sulfonamides, and mercuric ions.     

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.     

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.     

Isolation of Yersinia enterocolitica and related species from porcine samples obtained from an abattoir

H armon , M.C., S waminathan , B. & F orrest , J.C. 1984. Isolation of Yersinia enterocolitica and related species from porcine samples obtained from an abattoir. Journal of Applied Bacteriology 56 , 421–427.
Swabs of swine carcasses and samples of porcine tongue and trim obtained from an abattoir were examined for the presence of Yersinia enterocolitica and related species ( Y. intermedia, Y. kristensenii and Y. frederiksenii) . Three enrichment media (phosphate buffered saline, sorbitol-bile salts-phosphate buffered saline, and a modified Rappaport's broth) were compared at 4C for their efficiency of recovery of Y. enterocolitica and related species. Two secondary enrichment procedures (post-enrichment in modified Rappaport's broth for 2 d at 25C and treatment with 0.5% KOH in 0.5% NaCl) also were evaluated. The porcine isolates were characterised by biochemical and serological examination, speciation, and biotyping. Eight of 43 samples were positive for Y. enterocolitica and related species. The combination of incubation in sorbitol-bile salts-phosphate buffered saline for 21 d at 4C followed by post-enrichment in modified Rappaport's broth yielded maximum number of isolates. All isolates, except one, were avirulent as determined by auto-agglutination, calcium dependence at 37C, and HeLa cell invasiveness tests.     

The use of a surface adhesion immunofluorescent (SAIF) method for the rapid detection of Yersinia enterocolitica serotype O:3 in meat

This study examined the attachment kinetics of Yersinia enterocolitica serotype O:3 to determine the optimum conditions for its isolation from meat enrichment systems using a novel surface adhesion technique. Minced beef was inoculated with Y. enterocolitica at an initial level of 10 cfu g⁻¹ and incubated at 25 °C in an enrichment broth. Yersinia was recovered from enriched samples on polycarbonate membranes by surface adhesion and enumerated using immunofluorescence microscopy. The surface adhesion immunofluorescence technique (SAIF) had a minimum detection limit of approximately 4·0–4·5 log₁₀ cfu ml⁻¹ and provided good correlation between the estimation of the numbers of Yersinia in the enrichment broth derived from plate counts on Yersinia Selective agar (CIN) and those determined by SAIF ( r ² ₌ 0·94; rsd ₌ ± 0·21). A derived regression equation of the SAIF count vs plate counts was used to predict Y. enterocolitica numbers in spiked meat samples stored at 0 °C for up to 20 d. The numbers as predicted by the SAIF method showed good correlation with counts derived by plating techniques ( r ² ₌ 0·78; rsd ₌ ± 0·42). The application of the SAIF technique for the rapid detection of Y. enterocolitica serotype O:3 from meat is discussed.     

Efficient subtyping of Yersinia enterocolitica bioserotype 4/O:3 with pulsed-field gel electrophoresis

The purpose of this study was to evaluate the efficiency of pulsed-field gel electrophoresis (PFGE) for differentiation of isolates of Yersinia enterocolitica bioserotype 4/O:3 recovered from pig tongues at retail level in the Helsinki area during October and November of 1996. NotI generated 15 different PFGE patterns for 128 isolates. The discriminatory index did not exceed 74% due to the presence of two predominant PFGE-types, NA1 (58/128) and NB1 (25/128). After preliminary investigations with 35 enzymes, ApaI, XbaI, XhoI and SpeI were chosen for further characterization of the isolates. The discriminatory index increased from 74% to 93% and the number of different genotypes from 15 to 30 when isolates with the same PFGE pattern with NotI were further characterized with ApaI and XhoI, indicating that PFGE can be an efficient technique for characterization of bioserotype 4/O:3.     

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 10⁸ CFU/ml in UP medium in 18 h from an initial level ofca. 10² CFU/ml. Addition of Oxyrase_TM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24-h cultures were tested, but not when 18-h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with Oxyrase^TM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.     

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.     

Construction of a mobilizable Yersinia enterocolitica virulence plasmid

Virulence of Yersinia enterocolitica O:8 is associated with pO:8, a 42-megadalton plasmid. We constructed a mobilizable pO:8 derivative by successive in vitro and in vivo genetic manipulations. The in vitro constructed hybrid molecule pRK290B8-5 consisting of the mobilizable vector pRK290B and a 2.9-megadalton BamHI fragment of pO:8 was conjugally transferred to a Y. enterocolitica strain of serotype O:8 which harbored the virulence plasmid pO:8. From Yersinia transconjugants, a cointegrate was isolated which apparently formed by homologous recombination between the two component plasmids. The cointegrate was mobilized into plasmidless Y. enterocolitica strains of different serotypes. The transconjugants of serotype O:8 were found to express all four plasmid-associated phenotypes: (i) mouse lethality (Ml), (ii) conjunctivitis provocation in the guinea pig eye (Con), (iii) calcium requirement for growth at 37 degrees C (Mox), and (iv) agglutinogens (Ag8). The transconjugants of serotype O:3 expressed the phenotypes Con, Mox, and Ag8 but were nonlethal for mice (Ml-). The transconjugants of serotype O:5 remained avirulent for mice (Ml-) and for the guinea pig eye (Con-) but expressed the phenotypes Mox and Ag8. These data show that the virulence plasmid is probably not functionally interchangeable within different serotypes of Y. enterocolitica.     

Monoclonal antibodies directed against plasmid-encoded released proteins of enteropathogenic Yersinia

Abstract Yersinia enterocolitica of serotypes O:3, O:8, O:9 and O:5,27 and Yersinia pseudotuberculosis of serotypes I and III release plasmid-encoded proteins into calcium-deficient medium. Mouse monoclonal antibodies were elicited against plasmid-encoded released proteins of Y. enterocolitica of serotype O:9. As shown by immunoblot analysis the monoclonal antibody Mab9–200 recognized the 46-kDa protein of Y. enterocolitica of serotypes O:3, O:9 and O:5,27, the 58-kDa protein of Y. enterocolitica of serotype O:8 and the 67-kDa protein of Y. pseudotuberculosis of serotypes I and III. Mab9–15 reacted with the 36-kDa protein of Y. enterocolitica of serotypes O:9, O:3 and O:8, and the 34-kd protein of Y. enterocolitica of serotype O:5,27 and Y. pseudotuberculosis of serotypes I and III. The 25-kDa proteins of Y. enterocolitica of serotypes O:3, O:9, O:8 and O:5,27, but not those of Y. pseudotuberculosis were recognized by the monoclonal antibody Mab-128. This species-specific recognition of epitopes could not be achieved by mouse polyclonal antibodies.