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.     

Evaluation of different antimicrobial agents used as selective supplements for isolation and recovery of Yersinia enterocolitica

Seventeen antimicrobial agents were evaluated separately or in combination for their efficiency as selective supplements in a broth medium against six different serotypes of Yersinia enterocolitica and 20 selected strains of different Gram-negative bacteria. Irgasan (DP300, 5-chloro-2-(2,4-dichlorophenoxy) phenol) at a concentration of 4 μg ml^-1 inhibited the growth of most Gram-negative bacteria with the exceptions of Aeromonas hydrophila, Morganella morganii, Pseudomonas aeruginosa and Serratia liquefaciens. Other antimicrobial agents incorporated in the growth medium, separately or in combination with Irgasan, either inhibited some strains of Y. enterocolitica or did not inhibit the growth of Irgasan-resistant Gram-negative bacteria.     

Modified enrichment broth for isolation of Yersinia enterocolitica from nonfood sources.

Sorbitol-bile salts broth was modified by the addition of 0.5% peptone for the enrichment of Yersinia enterocolitica in nonfood samples. A quantitative comparison was made of the growth of Y. enterocolitica in sorbitol-bile salts broth and peptone-sorbitol-bile salts broth at 22 degrees C. Incubation for 48 h resulted in an average 7.79 log10 increase in a viable cell number for six serotypes with peptone-sorbitol-bile salts broth compared with an average 1.85 log10 increase for sorbitol-bile salts broth. Recovery from seeded seawater showed a greater than 100-fold increase in sensitivity with peptone-sorbitol-bile salts broth enrichment. Isolation of indigenous Y. enterocolitica from fresh and marine waters was also significantly enhanced.     

Synthesis of a selective agar medium for Yersinia enterocolitica

A new agar medium for isolation of Yersinia enterocolitica was formulated based on growth studies which defined an optimum basal, and the evaluation of selective chemical agents, dyes, and antibiotics. The final formulation, designated cefsulodin-irgasan-novobiocin(CIN) agar, provided quantitative recovery of 40 different strains of Y. enterocolitica in 24 h using incubation at 32 degrees C or with 48 h of incubation at 22 degrees C. The medium was highly selective, especially against Pseudomonas aeruginosa. Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. Colony morphology coupled with a differential reaction resulting from mannitol fermentation permitted discrimination of Y. enterocolitica from most of those Gram-negative bacteria that were able to grow on the medium. Recovery and selective characteristics of CIN agar were stable during storage at room temperature for 9 days. CIN agar gave a higher recovery of Y. enterocolitica from feces both direct and with cold enrichment (0.4/1.5%) than Salmonella-Shigella (0.0/0.7%) and MacConkey (0.0/0.9%) agars while significantly reducing the level of background organisms.     

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.     

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.     

Modified enrichment broth for isolation of Yersinia enterocolitica from nonfood sources

Sorbitol-bile salts broth was modified by the addition of 0.5% peptone for the enrichment of Yersinia enterocolitica in nonfood samples. A quantitative comparison was made of the growth of Y. enterocolitica in sorbitol-bile salts broth and peptone-sorbitol-bile salts broth at 22 degrees C. Incubation for 48 h resulted in an average 7.79 log10 increase in a viable cell number for six serotypes with peptone-sorbitol-bile salts broth compared with an average 1.85 log10 increase for sorbitol-bile salts broth. Recovery from seeded seawater showed a greater than 100-fold increase in sensitivity with peptone-sorbitol-bile salts broth enrichment. Isolation of indigenous Y. enterocolitica from fresh and marine waters was also significantly enhanced.     

Beta-lactamases from Yersinia enterocolitica.

Two beta-lactamases, A and B, have been shown to be present in a strain of Yersinia enterocolitica (w222). Beta-Lactamase A hydrolyses a variety of penicillins and cephalosporins. This enzyme is sensitive to thiol reagents, is only partially inhibited by 0-1 mM-cloxacillin and has a molecular weight of approximatley 20,000.beta-Lactamase B shows strong cephalosporinase activity but does not hydrolyse some of the penicillins. It is more resistant than beta-lactamase A to thiol reagents, is completely inhibited by 0-1 mM-cloxacillin and has a molecular weight of about 34,000. With cephaloridine as a substrate, which is readily hydrolysed by both enzymes, about 85% of the total activity of a cell extract is due to beta-lactamase A and 15% to B. Addition of 6-aminopenicillanic acid to the culture during growth results in a 2-to4-fold selective increase in the amount of beta-lactamase B. Two beta-lactamases similar to enzymes A and B have been found in five other strains of Y. enterocolitica. In contrast, only one beta-lactamase, similar to enzyme B, has been detected in a different strain of Y. enterocolitica (H66), which is abnormal in that it is sensitive to ampicillin. Addition of 6-aminopenicillanic acid to cultures of this strain results in an 8-to 10-fold increase in beta-lactamase production.     

Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content

Aims: The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intestinal content. Methods and Results: The four methods comprised of 15 isolation steps using selective enrichments (irgasan–ticarcillin–potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25°C. Salmonella–Shigella‐desoxycholate–calcium chloride agar, cefsulodin–irgasan–novobiocin agar were used as plating media. The most sensitive method detected 78% (53/68) of the positive samples. Individual isolation steps using cold enrichment as the only enrichment or as a pre‐enrichment step with further selective enrichment showed the highest sensitivities (55–66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. Conclusions: Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. Significance and Impact of the Study: The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. enterocolitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica.     

Yersinia enterocolitica as a causative agent of septicaemia.

The authors describe the first case of isolation of Yersinia enterocolitica from blood in a septic infection in Czechoslovakia. They also isolated a strain of Y. enterocolitica from an inguinal tumour in the same patient. Both strains belonged to biotype 4, serotype 0:3 and phagotype 8 and were non-pathogenic for guinea pigs and hares.     

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.     

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.     

Highly selective medium for isolation of Listeria monocytogenes from food.

A new selective medium (Al-Zoreky-Sandine listeria medium [ASLM]) was formulated to recover Listeria monocytogenes from food specimens; the medium completely inhibited common food microflora. Recognition of Listeria colonies is evident by black discoloration of the medium due to esculin hydrolysis without need for special illuminating equipment. The medium contains acriflavin, ceftazidime, and moxalactam as selective agents. Compared with Listeria Selective Agar, ASLM was equally effective in recovering L. monocytogenes. However, ASLM inhibited micrococci, enterococci, and gram-negative bacteria, especially a strain that mimicked L. monocytogenes on Listeria Selective Agar. The new medium was able to recover heat injured cells with only 15% less count than the nonselective medium.     

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.     

Real-time PCR method for detection of pathogenic Yersinia enterocolitica in food

The current methods for the detection of pathogenic Yersinia enterocolitica bacteria in food are time consuming and inefficient. Therefore, we have developed and evaluated in-house a TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. Good precision, robustness, and efficiency of the PCR amplification were also established. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail gene. Since no conventional culture method could be used as a reference method, the PCR products amplified from these samples were positively verified by using conventional PCR and sequencing of the amplicons. A rapid and specific real-time PCR method for the detection of pathogenic Y. enterocolitica bacteria in food, as presented here, provides a superior alternative to the currently available detection methods and makes it possible to identify the foods at risk for Y. enterocolitica contamination.     

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.