Drug resistance and lecithinase activity of Yersinia enterocolitica isolated from buffalo milk

Two-hundred-and-seven samples of raw buffalo milk and 60 samples of pasteurized buffalo milk were screened for presence of Yersinia enterocolitica. The prevalence of Y. enterocolitica was found to be 24.1% in raw milk, however, no isolation could be made from the pasteurized milk samples. Cold enrichment in trypticase soy broth and alkali treatment methods were followed in this study. The majority of the isolates (62%) were found sensitive to all the antibiotics used and only a few (16%) were resistant to two or more than two antibiotics. The incidence of Y. enterocolitica showed seasonal variations. Incidence was much higher (25-50%) during the winter season as compared to the summer (0-17%). The incidence of lecithinase production was high (40-50%) in Yersinia isolates resistant to one or two antibiotics.     

Occurrence of Yersinia enterocolitica in milk and dairy products in Morocco.

A total of 227 samples of milk and dairy products were examined for the presence of Yersinia enterocolitica. Yersinia spp. were recovered from 11 of 30 raw milks (36.6%), one of 20 pasteurized milks (5%), 15 of 63 traditional fermented milks (23.8%), seven of 94 cheeses and one of 20 cream samples (5%). The overall incidence of Y. enterocolitica in milk and dairy products was 6.6%. The other Yersinia species were Y. intermedia, Y. kristensenii, Y. frederiksenii and Y. pseudotuberculosis. Y. enterocolitica was detected only in raw milk (30% of the samples), in traditional fermented milks (6.3%) and in raw milk-made cheese (4%). The majority of the Y. enterocolitica isolates were of biotype 1 (environmental strains). The Celfulodin-Irgasan-Novobiocin (CIN) Agar was found to be more efficient than the Mac Conkey Agar in the isolation of Yersinia organisms.     

Isolation of Yersinia enterocolitica from foods.

Many selective enrichment and plating media for the isolation of Yersinia enterocolitica from foods are described. However, at present no single isolation procedure is available for the recovery of all pathogenic strains of Yersinia enterocolitica. Cold enrichment in phosphate-buffered saline plus 1% sorbitol and 0.15% bile salts (PBSSB) and two-step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth are very efficient methods for the recovery of a wide spectrum of serotypes of Y. enterocolitica. Enrichment in irgasan ticarcillin chlorate (ITC) broth was found to be the most efficient method for the recovery of strains of serotype 0:3, which is the most common clinical serotype of Y. enterocolitica in Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsulodin irgasan novobiocin (CIN) agar and Salmonella-Shigella deoxycholate calcium chloride (SSDC) agar are the most commonly used plating media. For the recovery of serotype 0:8 strains, the common clinical isolates in North America, enrichment in BOS and plating on CIN seems the most efficient procedure. Selection of the proper enrichment procedure will depend on the bio/serotypes of Yersinia spp. sought and on the type of food to be examined. The use of more than one medium for both enrichment and plating will result in higher recovery rates of Yersinia spp. from foods. Parallel use of the following two isolation procedures is recommended. (1) Enrichment in ITC for 2 days at 24 degrees C; plating on SSDC agar (2 days at 30 degrees C). (2) Pre-enrichment in TSB for 1 day at 24 degrees C; enrichment in BOS for 5 days at 24 degrees C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaCl for 5 s); plating on CIN agar (2 days at 24 degrees C).     

Isolation of Salmonella typhimurium from outbreak-associated cake mix

During May and June of 2005, 26 persons in several states were infected by a single strain (isolates indistinguishable by pulsed-field gel electrophoresis) of Salmonella enterica serotype Typhimurium after eating cake batter ice cream. The cake mix used to prepare the cake batter in the ice cream was implicated by epidemiologic investigation as the source of Salmonella contamination. Initial tests did not detect Salmonella in cake mix collected during the outbreak investigation. The objective of this study was to evaluate different procedures to isolate Salmonella from the implicated cake mix, cake, and ice cream. All outbreak-associated food samples (14 samples) were collected during the outbreak investigation by health departments of several of the states involved. Different combinations of Salmonella isolation procedures, including sample size, preenrichment broth, enrichment broth, enrichment temperature, and isolation medium, were used. Salmonella Typhimurium was isolated from two cake mix samples; the food isolates were indistinguishable from the outbreak pattern by pulsed-field gel electrophoresis subtyping. Universal preenrichment broth was substantially better than was lactose broth for preenrichment, and tetrathionate broth was better than was Rappaport-Vassiliadis broth for isolating Salmonella from the two positive cake mix samples. Although more typical Salmonella colonies were observed on plates from enrichment cultures grown at 35 degrees C, more confirmed Salmonella isolates were obtained from plates of enrichment cultures grown at 42 degrees C. Brilliant green agar, xylose lysine tergitol 4 agar, xylose lysine desoxycholate agar, Hektoen enteric agar, and bismuth sulfite agar plates were equally effective in isolating Salmonella from cake mix. The best combination of preenrichment-enrichment conditions for isolating the outbreak strain of Salmonella was preenrichment of cake mix samples in universal preenrichment broth at 35 degrees C for 24 h, followed by enrichment in tetrathionate broth at 42 degrees C for 24 h.     

Yersinia enterocolitica in milk and dairy products

Yersinia enterocolitica was first recognized during the 1960's as an important human enteropathogen. The species as later redefined includes both pathogenic and nonpathogenic forms. Pathogenic strains that retain the virulence plasmid can be identified in several animal models and four indirect tests (calcium dependency, autoagglutination, Congo red uptake, serological detection of outer membrane antigen) and by tissue culture assay, serotype, and biotype. Y. enterocolitica and related bacteria have frequently been isolated from raw milk, but none of the isolates, with the possible exception of serotype 05,27, are recognizable as pathogens. Under normal circumstances Y. enterocolitica does not survive pasteurization. If introduced into pasteurized milk, it can grow well at refrigeration temperatures. Two outbreaks of yersiniosis have occurred that involved pasteurized milk. Pigs, which frequently carry pathogenic Y. enterocolitica in their throat, were the probable source in one of these outbreaks. The most rapid enrichment procedure available for isolation of Y. enterocolitica requires 6 d. No isolation method is available for selective isolation of pathogenic Y. enterocolitica in the presence of related bacteria common in milk and other foods.     

Prevalence of virulent Yersinia enterocolitica in bulk raw milk and retail cheese in northern-west of Iran

To determine the prevalence of virulent Yersinia enterocolitica, 554 samples consisting of 354 bulk raw milks and 200 traditional cheeses were collected from different parts of Eastern-Azerbaijan province, during a 23-month period from 2008 to 2010. The occurrence of virulent strains of Y. enterocolitica in samples enriched in peptone sorbitol bile broth (PSBB) was evaluated via the detection of attachment invasion locus (ail) gene by PCR. The viability of virulent Y. enterocolitica in the PCR-positive samples was tested using conventional culture method and the isolates were confirmed by the second-phase ail-PCR. According to the results, 8.66% of total samples including 7.62% of bulk raw milks and 10.5% of raw milk cheeses were found ail-positive by PCR method; subsequently Y. enterocolitica was isolated by the culture method and confirmed by the second phase ail-PCR in 2.88% of total samples including 2.26% of raw milks and 4% of cheese samples. It was concluded that, a sample enrichment followed by ail-PCR was more sensitive and robust to detect and distinguish the virulent strains of Y. enterocolitica compared to the conventional culture method.     

Yersinia enterocolitica biogroup 1A, serotype O:5 in chicken carcasses

To evaluate the presence of Yersinia enterocolitica in raw chicken, 70 samples of chicken carcasses were obtained from a Buenos Aires, Argentina, processing plant. The detection of this psychotropic microorganism was carried out using the whole carcass rinse method and enrichment in phosphate-buffered saline, 0.067 M, pH 7.6, at 4 degrees C, followed by alkaline treatment and isolation on cefsulodin-irgasan-novobiocin nutrient agar. Y. enterocolitica or related species were detected in 7 of 70 samples. From them, 4.3% were identified as Y. enterocolitica, 1.4% as Yersinia intermedia, and 4.3% as Yersinia frederiksenii. The serotype and phagotype of Y. intermedia isolates were O:5(2), 5(3), and Xz and those of Y. frederiksenii were O:4.16 and Xo. All Y. enterocolitica isolates belong to the biogroup 1A, serotype O:5, phagotype Xz, which presents uncertain pathogenic potential. These findings reinforce the worldwide concern on the microbiological quality of food products. Quality assurance programs on the whole poultry process are increasingly being adopted in Argentina.     

Evaluation of methods for recovery of Salmonella from dairy cattle, poultry, and swine farms

Current official methods for detection and isolation of Salmonella are mostly designed for foods. The objective of this study was to determine optimal methods for detection and isolation of Salmonella from animal and environmental samples of dairy, poultry, and swine farms. Preenrichment in lactose broth versus direct enrichment (no preenrichment) prior to selective enrichment in Rappaport-Vassiliadis, selenite cystine, and tetrathionate incubated at 35 and 42 degrees C and in four differential/selective plating media (brilliant green, bismuth sulfite, Hektoen enteric, and xylose-lysine-tergitol 4 agar base) were evaluated for their ability to recover Salmonella from artificially contaminated samples. The effects of pH adjustments to samples on Salmonella recovery were determined. A pH adjustment of the enrichment broth to 6.8 +/- 0.2 after addition of samples significantly improved recovery of Salmonella. The most effective medium combinations for isolation of Salmonella from farm samples depended on the type of samples. Generalizations of protocols for recovery of Salmonella from farm samples might result in poor recovery, increased recovery time, and increased sample processing costs.     

Enrichment procedures and plating media for isolation of Yersinia enterocolitica

A shortened enrichment procedure (25 degrees C for 24 h) was compared with cold enrichment procedures (4 degrees C for 1 to 3 weeks) and direct plating for isolation of Yersinia enterocolitica from commercial ground meat samples. The combined data of all recovery procedures showed that this organism was isolated from 34% of the ground beef samples. The highest isolation rate was 32% for the 4 degrees C/3-week enrichment, followed by 28% for the 4 degrees C/2-week enrichment, 26% for the 25 degrees C/24-h enrichment, 22% for the 4 degrees C/1-week enrichment, and 10% for direct plating. No significant differences (P > 0.05) in isolation rate occurred between the 4 degrees C/3-week, 4 degrees C/2-week, 25 degrees C/24-h, and 4 degrees C/1-week enrichments. The combined data of all recovery procedures showed that Y. enterocolitica was isolated from 64% of ground pork samples. The highest isolation rate was 48% for the 4 degrees C/3-week enrichment, followed by 40% for the 25 degrees C/24-h enrichment, 34% for the 4 degrees C/2-week enrichment, 24% for the 4 degrees C/1-week enrichment, and 24% for direct plating. No significant differences (P > 0.05) in isolation rate occurred between the 4 degrees C/3-week, 25 degrees C/24-h, and 4 degrees C/2-week enrichments. During the plating phase of the experiment, the efficiency of a dye-containing, Yersinia-selective medium (KV202) was compared with that of a commercially available cefsulodin-irgasan-novobiocin medium. Recovery rates were similar for both media. However, KV202 agar differentiated Y. enterocolitica from such contaminating bacteria as Enterobacter, Serratia, and Salmonella by colony morphologic characteristics and color.     

Recovery of Salmonella from refrigerated preenrichment and refrigerated enrichment media

The productivity of the standard cultural procedure for the isolation of Salmonella using preenrichment in buffered peptone water (BPW) followed by inoculation into Rappaport-Vassiliadis (RV), tetrathionate (TBG) (Difco) and selenite-cystine (SC) (Difco) enrichment broths, was compared with that using preenrichment and enrichment cultures which had been held under refrigeration (72 h at 5–10°C). Seventy-seven of 251 samples of food products were found to contain Salmonella. Refrigerated preenrichment cultures inoculated into RV medium (43°C), TBG broth (43°C) and SC broth (37°C) yielded salmonellae from 93.5, 85.7 and 54.5% contaminated samples, respectively. Refrigerated cultures in RV, TBG and SC broths, inoculated onto three selective plating media, identified 100, 87.0 and 41.6% of the contaminated samples, respectively.The selective plating medium brilliant green deoxycholate agar was at least as productive as brilliant green sulpha agar and bismuth sulphite agar, when streaked from RV and TBG broths, but was less effective when streaked from SC broth.     

Genotypic and phenotypic characteristics of Yersinia enterocolitica isolated from the surface of chicken eggshells obtained in Argentina

The prevalence of Yersinia enterocolitica on chicken eggshell surfaces in San Luis, Argentina, was investigated. The pathogenic potential of recovered isolates was assessed by means of phenotypic virulence tests and the presence of the 72-kb pYV plasmid. Antimicrobial susceptibility was determined by the agar diffusion method. DNA digested with XbaI was analyzed by pulsed-field gel electrophoresis (PFGE), and relationships between genomic DNA profiles were established. Eight Y. enterocolitica B2 O:9 strains were recovered after enrichment, for a prevalence of 2.27%. All strains harbored the virulence pYV plasmid, bound Congo red, grew in a low-calcium medium, and autoagglutinated at 37 degrees C. They lacked pyrazinamidase activity and did not hydrolyze esculin. These Y. enterocolitica strains were susceptible to amikacin, ciprofloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole and were resistant to rifampin. According to the genomic DNA patterns obtained by PFGE, the isolates clustered into two groups, I and II. The highest similarity coefficient observed between Y. enterocolitica strains was 0.947. Microbiological controls on production stages of eggs and good culinary practices are necessary to reduce the risk of Y. enterocolitica infection for consumers.     

Expression of major cold shock proteins and genes by Yersinia enterocolitica in synthetic medium and foods

Yersinia enterocolitica is a psychrotrophic foodborne pathogen that has been implicated in outbreaks of foodborne illness involving cold-stored foods, especially milk and pork. A major mechanism bacteria use to adapt to cold is expression of cold shock proteins. The objective of this research was to study the expression of major cold shock proteins of Y. enterocolitica in Luria-Bertani (LB) broth, milk, and pork following a temperature downshift from 30 to 4 degrees C. Y. enterocolitica was inoculated into 10 ml of LB broth, sterile skim milk, or pork, and the samples were stored at 4 degrees C (cold shock) or 30 degrees C (control) for 0, 4, 8, 12, and 24 h. At each sampling time, total protein and total RNA were extracted from Y. enterocolitica harvested from LB broth, milk, and pork and subjected to two-dimensional gel electrophoresis and dot blot analysis. Two major cold shock proteins (CspA1 and CspA2) of approximately 7 kDa and their genes were expressed by Y. enterocolitica following cold shock. However, the CspA1 and CspA2 proteins were not expressed by Y. enterocolitica at 30 degrees C. Y. enterocolitica CspA1 and CspA2 were observed as early as 2 h of cold shock in cultures from LB broth and milk and at 8 h of cold shock in cultures from pork.     

Slaughter pigs and pork as a source of human pathogenic Yersinia enterocolitica.

Pathogenic Yersinia enterocolitica strains (serogroups 0:3;0:9 and 0:5.27) were isolated from 36 (42%) of 86 porcine tonsils, 8 (20%) of 40 tongues, 17 (17%) of 100 rectal swabs and from 4 (1%) of 400 pork samples. Pathogenic Yersinia strains were not isolated from samples of 210 pig carcasses and from 20 samples of porcine head meat. These results confirm that pigs are an important reservoir of pathogenic Y. enterocolitica. However, contamination of carcasses during the slaughtering process with Yersinia from either faecal material or from the tonsillary region does not seem to occur frequently and this may also explain the low contamination rate of pathogenic Y. enterocolitica found for pork. For the isolation of pathogenic Y. enterocolitica strains from foods, enrichment in irgasan-ticarcillin-chlorate broth (ITC) and isolation on SS-deoxycholate-calcium agar (SSDC) is recommended.     

Occurrence of Yersinia spp. in foods in Brazil.

Over the past 9 years, 468 bacterial strains isolated from raw and pasteurized milk, beef and pork, bovine and chicken liver, chicken heart, gizzards and lung sausage, hamburger, cheese and lettuce in different regions of the State of S?o Paulo and in the city of Rio de Janeiro were received by the Reference Laboratory for Yersinia in Brazil. All were confirmed to be Yersinia spp. The 468 Yersinia isolates were grouped as 184 strains because some of the bacteria isolated from the same food sample belonged to the same species, and were considered to be a single strain. The Yersinia food strains were classified as Y. enterocolitica (46), Y. intermedia (67), Y. frederiksenii (20), Y. kristensenii (8) and 43 of them were biochemically atypical. Pathogenic types were not detected.     

Evaluation of a 5'-nuclease (TaqMan) assay with the thin agar layer oxyrase method for the detection of Yersinia enterocolitica in ground pork samples

A 5'-nuclease (TaqMan) assay was evaluated for its capability to recover and detect stressed Yersinia enterocolitica. Sensitivity studies of a 5'-nuclease assay for detecting Y. enterocolitica 0:8 in a pure culture system and spiked ground pork samples demonstrated that the assay has reliable sensitivity with a detection limit of 3 to 4 log CFU/ml or CFU/g. The PCR 5'-nuclease (TaqMan) assay was evaluated with the Thin Agar Layer Oxyrase method (TALO, overlaying 14 ml of Trypticase soy agar with a 1:30 dilution of "Oxyrase for Agar" onto a prepoured pathogen-specific, selective medium), and it was compared against the selective medium cefsulodin-irgasan-novobiocin (CIN) for recovering and detecting Y. enterocolitica from inoculated nonfrozen and frozen (-15 degrees C, 2 days) ground pork samples. The TALO method showed more sensitivity (detection limit, 2 log CFU/ml), and it has greater recovery capability (0.5 to 1 log CFU/ml) than CIN (P < 0.05). The 5'-nuclease assay provided rapid detection processing (5 versus 24 h after an 18-h enrichment). The sensitivity per PCR was calculated to as low as 0 to 1 log CFU per PCR reaction; however, in the method's current developmental stage, target pathogens should be enriched to 3 to 4 log CFU/ml or CFU/g to show consistent results. In a survey of 100 ground pork samples using TALO, CIN, and PCR methods, no Y. enterocolitica was recovered. A combined cultivation and an automated PCR TaqMan could be used as a presumptive screening test for detecting Y. enterocolitica in food samples.     

Detection, isolation and enumeration of Yersinia enterocolitica from raw pork

The methods available for the isolation of Yersinia enterocolitica from foods are generally considered to be less than optimal, and methods for estimation of numbers are lacking. Such methods are needed to understand better the significance of foodborne yersiniosis and to provide data for exposure assessment. We describe a method for the detection and enumeration of Y. enterocolitica containing the pYV virulence plasmid (YeP+) in samples from pork surfaces. The method uses a multiplex PCR targeting the ail and virF genes to detect Y. enterocolitica after incubation of surface swabs in Yersinia enrichment broth according to Ossmer. Enumeration was achieved by adapting the enrichment to a most probable number (MPN) method format. A presumptive result was available within 24 h of sample receipt, and YeP+ isolates were confirmed within four days. The presence/absence and MPN methods were evaluated in a pilot survey of 34 packs of raw pork meat purchased from retail outlets in Christchurch, New Zealand. YeP+ was detected by PCR on meat from 32% of the packs, and YeP+ isolates were obtained from 18% of the samples. YeP+ were present at numbers ranging from 0.30 to 5.42 MPN/cm(2). This improved method for the detection and enumeration of YeP+ from meat samples can be used for microbiological surveys to obtain data for assessments of consumer exposure to virulent Y. enterocolitica, and in outbreak investigations.     

Evaluation of immunomagnetic separation and plating media for recovery of Salmonella from meat

Immunomagnetic separation (IMS) was compared with selective enrichment in selenite cystine (SC) broth for isolation of Salmonella from 86 artificially contaminated ground beef samples. Both Rambach agar (RA) and Hektoen enteric (HE) agar were used as selective plating media. The highest count of Salmonella colonies per plate was obtained after enrichment in SC broth and plating on RA (mean value: 111.1+/-58.1 CFU per plate); the lowest count was obtained after IMS and plating on HE agar (mean value: 65.4+/-36.6 CFU per plate). Salmonella in preenrichment was concentrated 1.7-fold by IMS and represented 31% of the microbial population captured by the beads, but nonspecific binding was high. As a result of the large numbers of competing bacteria, isolations on both RA and HE agar following IMS were quite difficult (mean value for Salmonella colonies: 79.9+/-42.7 CFU per plate; mean value for non-Salmonella colonies: 144.1+/-75.7 CFU per plate; ratio of Salmonella to non-Salmonella colonies: 0.8). This study indicates that SC broth is superior to IMS in the isolation of Salmonella from raw ground meat.     

Prevalence, antibiotic susceptibility, and virulence factors of Yersinia enterocolitica and related species from ready-to-eat vegetables available in Korea

A total of 673 ready-to-eat vegetable samples were collected in Korea from 2001 to 2002 and analyzed for the presence of Yersinia spp. We analyzed biotypes, serotypes, and susceptibility to 12 antibiotics and tested for virulence genes of pathogenic Yersinia enterocolitica isolates by PCR assay. Among the samples, 27 (4.0%) were found to be contaminated with Yersinia spp. Among the 27 strains of Yersinia spp. isolates, 18 strains (66.7%) of Y. enterocolitica, 5 strains (18.5%) of Y. frederiksenii, 3 strains (11.1%) of Y. intermedia, and 1 strain (3.7%) of Y. kristensenii were identified. According to the serotypes of Y. enterocolitica isolates, O:3 (11.1%) and O:5 (11.1%) were the most predominant, followed by O:8 (5.6%) and others (72.2%). For biotypes of Y. enterocolitica isolates, 1A (77.8%) was the most predominant, followed by 3B (11.1%), 3 (5.6%), and 5A (5.6%). Also, an antibiotic susceptibility test showed that Y. enterocolitica isolates were very susceptible to the antibiotics tested but highly resistant to ampicillin (94%), cephalothin (100%), and carbenicillin (83%). PCR assays with specific primers derived from yst and ail genes of Y. enterocolitica were applied to confirm the presence of pathogenic Y. enterocolitica. Among the 18 strains of Y. enterocolitica isolates, only 3 strains (O:3/1A, UT/3B, and UT/1A isolated from Chinese cabbage, onion, and spinach, respectively) were shown to have a virulence gene.     

Isolation of Yersinia enterocolitica from ready-to-eat foods and pork by a simple two step procedure

Out of 119 ready-to-eat food samples and pork processed for isolation of Yersinia enterocolitica, 15 samples (12·6%) were positive for the presence of Y. enterocolitica by a two step procedure in a modified trypticase soy broth containing 0·25% yeast extract, 0·2% bile salts and 4 μg ml^-1 Irgasan at pH 7·6 and upon incubating at 10°C and 22°C for 6 days. Only five samples (4·2%) were positive by cold enrichment in trypticase soy broth containing 0·2% yeast extract, incubated at 4°C for 14 days. Four strains of Y. enterocolitica and one strain of Y. intermedia were isolated from pork samples processed by cold enrichment. Out of 15 strains of Y. enterocolitica isolated from pork (four strains) and ready-to-eat food samples (10 strains) by two step procedure only three strains belonged to pathogenic serotype O:3 and which were isolated only from pork samples. Overall recovery of Y. enterocolitica was much better by the two step procedure as compared to cold enrichment (P < 0·05).     

Isolation of Yersinia from raw meat (pork and chicken) and precooked meat (porcine tongues and sausages) collected from commercial establishments in Mexico City

A total of 160 meat product samples were collected from commercial outlets in Mexico City to investigate the presence of different species of Yersinia by the 4 degrees C enrichment method after 1, 3, 5, and 7 days of incubation using alkaline treatment and isolating in cefsulodin-Irgasan-novobiocin and MacConkey agars with Tween 80. Overall, Yersinia spp. were isolated from 27% of the samples analyzed, whereas 40% of the raw and only 13% of the precooked samples were contaminated. Although 2,970 colonies showed Yersinia characteristics, only 706 (24%) actually corresponded to this genus: 49% were Yersinia enterocolitica, 25% Yersinia kristensenii, 15% Yersinia intermedia, 9% Yersinia frederiksenii, and 2% Yersinia aldovae; 10% corresponded to biotype 2, 2% to biotype 3, and 4% to biotype 4. The presence of Yersinia in raw and cooked meat products represents a health risk for consumers in Mexico, where further clinical studies are needed to assess the epidemiological importance of this pathogen.