Enterocolitica Isolates Research Articles

Production of Yersinia stable toxin (YST) and distribution of yst genes in biotype 1A strains of Yersinia enterocolitica

Two hundred and fifty nine isolates of Yersinia enterocolitica and related species were examined for the production of heat-stable enterotoxin (Yersinia stable toxin; YST) as well as for the prevalence of enterotoxin genes, viz. ystA, ystB and ystC. Under the conventional conditions used for the production of Y. enterocolitica enterotoxin, i.e. in tryptic soy broth (TSB) supplemented with yeast extract at 28 degrees C for 48 h, 77.7 % of clinical isolates and 62.3 % of swine isolates showed enterotoxigenicity in infant mice. All isolates that produced enterotoxin at 28 degrees C also showed enterotoxic activity at 37 degrees C after 48 h incubation under an alkaline pH of 7.5, the pH present in the ileum. All Yersinia intermedia and Yersinia frederiksenii isolates were negative for enterotoxin production. All clinical isolates and 96.3 % of Y. enterocolitica isolates from swine hybridized with a probe for ystB, which indicated that the ystB gene was most prevalent in Y. enterocolitica biotype 1A strains. None of the Y. enterocolitica isolates showed hybridization with oligonucleotide probes for ystA or ystC. The study indicated that YST-b was the major contributor to diarrhoea produced by biotype 1A strains of Y. enterocolitica.

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.

Typing of Yersinia Enterocolitica Isolates by ITS profiling, REP- and ERIC-PCR.

Thirty-five Yersinia enterocolitica strains isolated from humans, pigs and foxes were analysed by genotyping including intergenic transcribed sequence (ITS) profiling, REP- and ERIC-PCR. ERIC-PCR revealed the presence of seven different genotypes. Amplification of the 16S-23S rDNA spacer region by ITS profiling gave similar results with nine different genotypes. REP-PCR was found to be more discriminatory for typing of Y. enterocolitica than ERIC-PCR and ITS profiling. Fifteen different DNA patterns were obtained by this technique. Based on data obtained by three methods it was found that: (i) Y. enterocolitica strains belonging to the same serotype can represent different genotypes and vice versa; (ii) isolates recovered from humans, pigs and foxes exhibit limited heterogeneity and, independent of the origin, one or two prevailing genotypes were always observed; and (iii) many human Y. enterocolitica isolates shared common genotypes with porcine isolates.

Frequency of Yersinia species infection in paediatric acute diarrhoea in Tehran

This study determined the frequency of Yersinia enterocolitica infection in 300 children with acute diarrhoea aged 0-12 years who were attending a paediatric hospital in Tehran. Over the 5-month study (May-September 2002), Yersinia species and other organisms were cultured and serotyped from stool samples or swabs. Yersinia spp. were found in 8 cases (2.7%). Enteropathogenic Escherichia coli was isolated in 5.7% of cases, Shigella spp. in 3.0% and Salmonella spp. in 2.0%. None of the Y. enterocolitica belonged to the common serotypes of O:3 and O:9. Atypical Yersinia spp. (Y. intermedia and Y. frederiksenii) were isolated. All Y. enterocolitica isolates had a similar pattern of antimicrobial resistance. Yersinia spp. infections are not common in the summer months in Tehran.

Evaluation of a one-step biochemical screening test to determine pathogenic strains of Yersinia enterocolitica.

The use of D-arabitol can be considered a reliable, inexpensive, one-step procedure for screening pathogenicity of Y. enterocolitica isolates. We believe that diagnostic laboratories will find this test to be more convenient and easier to interpret than the existing pathogenicity screening methods currently available. All suspect Y. enterocolitica isolates, whether determined to be potentially pathogenic or non-pathogenic by screening methods, should be referred to a reference laboratory for further testing. Biochemical confirmation, biotyping, serotyping, pathogenicity testing and molecular characterization performed by reference laboratories can provide useful epidemiological information and allow for early recognition of outbreaks.

In vitro antibiotic susceptibilities of Yersinia enterocolitica biotype 1A

Antibiotic susceptibilities of 80 strains of Yersinia enterocolitica biotype 1A isolated from human, swine and various aquatic sources to 20 β-lactam and 26 non-β-lactam antibiotics were studied. Most isolates were resistant to penicillins, first-generation cephalosporins, macrolides and lincosamides, while sensitive to aminoglycosides and quinolones. In comparison to earlier studies, the majority of the strains were either intermediately sensitive or resistant to piperacillin. Relatively decreased susceptibility or resistance to third-generation cephalosporins was also observed in several Y. enterocolitica isolates.

DV005 Validation of a combination of pre-enrichment and yopT-based PCR assay to detect pathogenic Yersinia enterocolitica in organs and lymphoid tissue of slaughtered pigs

Slaughter pigs are the main reservoir for pathogenic Yersinia (Y.) enterocolitica isolates. Particularly meatproducts derived from swine asymptomatically infectedare a potential source of human Yersiniosis. Due to thisfact it is important to have simple, rapid and specificmethods available for the detection of pathogenic Y. enterocolitica in tissue samples from slaughtered pigs.The aim of this study was to validate a 24 h preenrichmentstep in Yersinia-PCR-compatible-enrichment (YPCE) broth followed by a yopT-based PCR-Method forthe rapid detection of pathogenic Y. enterocolitica inorgans and lymphoid tissue of slaughtered pigs. Untilnow, the bacteriological identification of pathogenic Yersinia, described in ISO 10273, is the goldstandard. To compare the PCR results with the goldstandard we choose for the cultural detection a 48hour pre-enrichment step at 25 C in ITC broth followedby plating on CIN agar plates and incubation for 24hours at 30°C. For the validation of the PCR method itwas important to have available samples fromasymptomatically infected pigs. To generate thosesamples Yersinia-free pigs (n = 9) were intragastricallyinfected with a plasmid bearing Y. enterocolitica isolate (DSM 13030). Two additional pigs served asuninoculated controls. Four weeks after infection pigswere slaughtered and 13 samples from each pig weretaken aseptically.In comparison to the conventional cultural examinationthe combination of pre-enrichment in YPCE brothfollowed by yopT-PCR saved about four days ofdiagnostic time. Particularly, in the examined samplesof the palatine tonsil, an organ with the highestcontamination rate in slaughter pigs, demonstrated thePCR method a significantly better sensitivity than thecultural method. In summary, the 24 h pre-enrichmentand subsequent yopT-PCR represents a rapid andsensitiv diagnostic tool for pathogenic Y. enterocolitica in asymptomatically infected pigs at the slaughterhouselevel.

05. Comparison of Y. enterocolitica isolates from humans and food-producing animals

05. Comparison of Y. enterocolitica isolates from humans and food-producing animals

Detection of Salmonella spp., Yersinia enterocolitica and verocytotoxin-producing Escherichia coli O157 in pigs at slaughter in Italy

From December 1999 to December 2000, 150 pigs were randomly selected in two large abattoirs of northern Italy. Caecal material and carcass swabs were collected and examined for Salmonella, Yersinia enterocolitica, and Escherichia coli O157. Tonsils were examined for Salmonella and Y. enterocolitica. Salmonella was isolated from the intestinal content of 55 (36.7%) specimens, from 8 (5.3%) tonsils, and from 9 (6.0%) carcasses. Ten different serotypes were detected; the more common were Salmonella derby (37.8%), Salmonella bredeney (21.6%), and Salmonella typhimurium (14.8%). S. typhimurium isolates that belonged to phage-types DT104 and DT208 were 45% and 27.3%, respectively; 18.2% belonged to U302 and 9.1% were non-typeable. Y. enterocolitica was detected in the intestinal matter of 6 (4.0%) slaughtered pigs and in 22 (14.7%) tonsils; however, this pathogen was not found on carcasses. The majority of Y. enterocolitica isolates (82.1%) belonged to serotype O:3 biotype 4, one (3.6%) belonged to serotype O:9, and 13% did not belong to any known biotype. Verocytotoxin-producing E. coli (VTEC) O157 was isolated from the intestinal content of one (0.7%) slaughtered pig and from one (0.7%) carcass; four (2.7%) faecal samples contained E. coli O157 strains negative for the presence of both eae and VT genes.

Detection of a novel repeated sequence useful for epidemiological typing of pathogenic Yersinia enterocolitica

AbstractStrains (n=203) of Yersinia species were used in genotyping and PCR experiments in order to evaluate the genotyping potential of the YeO:3RS probe. This probe comprises a 12.5 kb genomic fragment of the Y. enterocolitica O:3 lipopolysaccharide O-antigen gene cluster cloned into plasmid pBR322. The genotyping potential of YeO:3RS was shown to reside in the region upstream of the O-antigen gene cluster, i.e., in the first 1.65 kb of the cloned genomic fragment that contains a repeated sequence (RS) present in multiple copies in the genome. In genotyping, the YeO:3RS probe was hybridised to DNA of Yersinia enterocolitica isolates (n=112) from humans, animals and food, along with strains of other Yersinia species (n=5) and Salmonella enterica strains (n=3). The YeO:3RS probe efficiently detected and subtyped all European pathogenic Yersinia enterocolitica isolates of the serobiotypes O:3/4, O:9/2 and O:5,27/2 studied (n=87), whereas it hybridised only weakly or not at all with the other strains. Within Yersinia enterocolitica serobiotype O:3/4 strains, YeO:3RS genotyping was as discriminatory as genotyping by pulsed-field gel electrophoresis (PFGE) of XbaI-NotI digested genomic DNA. When these two methods were combined, YeO:3RS genotyping divided both of the two predominant PFGE types into six subtypes, thus increasing the discrimination. In PCR screening of additional 86 Yersinia strains, the 1.65 kb region was detected in European pathogenic serotypes O:1 and O:2 in addition to serotypes O:3, O:5,27 and O:9, indicating that it can be exploited in detecting and typing of European pathogenic serotypes in general.

Characterisation of sucrose-negative Yersinia enterocolitica 4/O:3 isolates recovered from pig tonsils

Sucrose-negative Yersinia enterocolitica isolates of bioserotype 4/O:3 have been recovered for the first time. They were found in 2% of the tonsils of clinically healthy fattening pigs. These sucrose-negative Y. enterocolitica isolates could not be differentiated from Y. kristensenii isolates using API 20E; thus, they were identified using PCR and sequencing. Using pulsed-field gel electrophoresis (PFGE), NotI profiles of sucrose-negative Y. enterocolitica 4/O:3 isolates showed a high similarity to sucrose-positive Y. enterocolitica 4/O:3 isolates. This study demonstrated that sucrose-negative Y. enterocolitica 4/O:3 isolates of porcine origin can harbour virulence genes; plasmid-encoded virulence markers were found in 8 out of 11 isolates and all isolates contained chromosomal-encoded virulence markers. Thus, the pathogenicity of sucrose-negative Yersinia isolates should always be assessed.

Loss of crystal violet binding activity in stationary phase Yersinia enterocolitica following gamma irradiation

Ionizing radiation can eliminate virulent Yersinia enterocolitica from meat. It is possible, however unlikely, that a small number of Y. enterocolitica could survive the pasteurization process. The virulence ofY. enterocolitica is dependent upon the presence of a 70 kb plasmid. The effect of low-dose ionizing radiation on the plasmid-associated virulence trait of crystal violet binding was investigated. Y. enterocolitica strains which carried the virulence plasmid were suspended in Butterfield's Phosphate Buffer or raw ground pork and irradiated to a dose of 1·0 or 1·25 kGy, respectively. Loss of crystal violet binding increased 10-fold following exposure to ionizing radiation, regardless of the suspending medium. Polymerase chain reaction analysis of Y. enterocolitica isolates that did not bind crystal violet following irradiation indicated that loss of the virulence plasmid, as opposed to mutation of a single plasmid-encoded gene, was the major mechanism for elimination of the crystal violet binding trait.

Arsenite-induced multiple antibiotic resistance phenotype in environmental isolates of Yersinia enterocolitica.

Minimal inhibitory concentrations (MICs) of five antibiotics namely amikacin, gentamicin, tetracycline, ciprofloxacin, and nitrofurantoin for pork isolates of Yersinia enterocolitica increased two- to eightfold after bacteria were grown in the presence of 5 mm arsenite. For Y. enterocolitica isolates obtained from wastewater (sewage effluents), an unequivocal increase in MICs was seen with amikacin and gentamicin. No change was discernible in the outer-membrane proteins after isolates were grown in the presence of arsenite.

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°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:52, 53, 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.

Serological and biochemical characteristics of virulence plasmid of Yersinia enterocolitica isolates from chicken in the Islamic Republic of Iran

Pathogenic Yersinia enterocolitica harbour plasmid that is essential for virulence. We studied the characteristics of virulence plasmid using serological, biochemical and bioassay tests in Y. enterocolitica isolates of chicken using plasmid curing. Plasmid-cured isogenic derivatives (2029c and 2150c) were obtained from two isolates of Y. enterocolitica (RTCC 2029 and RTCC 2150). The results demonstrated that plasmid-bearing isolates (2029 and 2150) were human-serum-resistant when grown at 37 degrees C, but were sensitive when grown at 25 degrees C, whereas plasmid-cured isolates (2029c and 2150c) were sensitive when grown at both temperatures. Also autoagglutination, calcium-dependency tests and experimental infection in mice demonstrated that these phenotypes were associated with the virulence plasmid.

Survey for psychrotrophic bacterial pathogens in minimally processed lettuce.

A total of 120 minimally processed, cut and packaged lettuce samples were purchased from retail supermarkets or provided by a salad production facility over an 8-month period. The samples were tested for total aerobic plate counts and for the presence of potentially pathogenic species belonging to the genera of Listeria, Aeromonas and Yersinia. The aerobic plate counts ranged from 103 to 109 colony forming units (cfu) g-1. Most samples (76%) contained between 105 and 107 cfu g-1 total aerobic bacteria. Listeria monocytogenes was isolated from three samples, Aeromonas hydrophila or Aeromonas caviae from 66 samples, and Yersinia enterocolitica from 71 samples. The pathogenic potential of Y. enterocolitica isolates was determined by screening for an array of biochemical, serological and genetic traits (heat-stable enterotoxin gene, the attachment and invasion gene locus, the invasin gene locus and the virulence plasmid). The Y. enterocolitica isolates lacked many of the phenotypic and genetic markers associated with virulence in primary pathogenic strains. As the roles of the reputed virulence factors of Aeromonas spp. in human infection are uncertain, the pathogenic potential of the Aeromonas isolates in lettuce remains unclear.

Identification of a streptogramin A acetyltransferase gene in the chromosome of Yersinia enterocolitica.

Streptogramins are polypeptide antibiotics inhibiting protein synthesis by the prokaryotic ribosome. Gram-positive organisms are susceptible to streptogramins, while most gram-negative bacteria are intrinsically resistant. We have found a genomic fragment from a Yersinia enterocolitica isolate with an open reading frame coding for a polypeptide similar to the virginiamycin acetyltransferases found in various plasmids from gram-positive bacteria. The susceptible Escherichia coli strain DB10 was transformed to resistance to the type A streptogramins and to mixed (A + B) streptogramins upon introduction of a plasmid containing that gene. In addition, we showed streptogramin acetylating activity in vitro dependent on the presence of the Y. enterocolitica sat gene. Southern blot hybridization experiments showed that the sat gene was present in all the Y. enterocolitica isolates examined. These data together show that the gene in the Y. enterocolitica chromosome encoded an active streptogramin acetyltransferase. The deduced sequence of the Y. enterocolitica Sat protein was close to those of sat gene products found in gram-positive bacteria and cyanobacteria, suggesting a common evolutionary origin.

In Vitro Susceptibility of Yersinia enterocolitica Isolated from the Oral Cavity of Swine

Antimicrobial susceptibility of 181 (107 ail-harboring isolates and 74 non-ail-harboring) Yersinia enterocolitica isolates obtained from the oral cavity of swine was determined against 24 antimicrobial agents. All Y. enterocolitica isolates were susceptible to sulfamethoxazole/trimethoprim, enrofloxacin, aminoglycosides, and nitrofurantoin. Susceptibility to tetracycline appeared to vary by lot of origin. Isolates were resistant to sulfonamides (other than sulfamethoxazole/trimethoprim), penicillin, ampicillin, ticarcillin, cephalothin, macrolides, and tiamulin.

Variations in the 165 rRNA gene sequence of Yersinia enterocolitica isolates influence the specificity of molecular identification systems

Four identification systems were used to type Yersinia enterocolitica strain Y11 and Yersinia enterocolitica sensu strictoT. Two systems based on biochemical reaction patterns identified both strains as Yersinia enterocolitica. Two molecular assays targeting the 16S rRNA gene failed to identify either strain Y11 or the type strain. Therefore, both strains were typed by the classical taxonomical approach requiring a determination of the overall base composition and the base sequence similarity using hybridization. Again both strains were classified as Yersinia enterocolitica isolates. Consequently, the sequences of the 16S rRNA gene of both strains were determined and compared. The strains differed in a region where nucleotide changes between species of the genus Yersinia had been described earlier. These differences may explain the failure of the molecular assays to identify the strains. They also demonstrate an independent evolution of the 16S rRNA genes in the species Yersinia enterocolitica sensu stricto suggesting an amendment to the nomenclature to be used in the future.

Antibiotic susceptibilities of Yersinia enterocolitica and Y. intermedia isolates from aquatic environments.

Of 37 Yersinia isolates from various aquatic environments, seven were Y. enterocolitica and 30 Y. intermedia. These isolates were biotyped, serotyped and tested for their susceptibility to 20 antibiotics. All Y. enterocolitica isolates were of biovar 1; those of Y. intermedia were distributed amongst four biovars (1, 2, 4 and 6). On the basis of combined biotyping and serotyping results, Y. enterocolitica isolates were distributed in five and Y. intermedia in 17 groups. With the exception of one Y. enterocolitica isolate which was resistant to tetracycline and streptomycin, the isolates were sensitive to the non-beta-lactam antibiotics. In contrast, various patterns of beta-lactam insensitivity were detected, including ampicillin and ticarcillin (35 isolates), cephalothin (33 isolates), carbenicillin (32 isolates), amoxycillin/clavulanate (23 isolates) and cefoxitin (22 isolates). No correlation between biotype or serotype and the susceptibility pattern of the isolates was apparent. Both inducible cephalosporinase activity against third-generation cephalosporins and inhibition of resistance to penicillins were detected in all Y. enterocolitica and Y. intermedia isolates by double-disk tests.