Enterocolitica Strains Research Articles

A PCR System for Yersinia pseudotuberculosis Identification

Pseudotuberculosis is an acute infectious disease caused by Yersinia pseudotuberculosis. In the 1950 and 1960s, outbreaks of a new, previously unknown disease were registered in the Far East of Russia, called the “Far East scarletlike fever” (FESLF). FESLF is pseudotuberculosis, which has a specific clinical form. The disease is endemic to the Far Eastern, Siberian, and Northwestern regions of Eurasia. At present, there is only one system designed to detect Y. enterocolitis and Y. pseudotuberculosis for diagnostics by polymerase chain reaction. The disadvantage of this system is the lack of the possibility for the specific detection of Y. pseudotuberculosis. In this work, 35 strains of Y. pseudotuberculosis were characterized by the detection of the plasmid gene encoding the adhesin (yadA), MLST typing, and detection of the chromosomal gene of the Y. pseudotuberculosis—the cytotoxic necrotizing factor (cnfY). Detection of the yadA gene correlated with presence of the plasmid (pYV). The strains belonged to five MLST sequence types (STs), one of which, ST240, was described for the first time. All the studied strains of Y. pseudotuberculosis showed the presence of the cnfY gene. Additionally, the results of 68 previously described Y. pseudotuberculosis strains were analyzed. A total of 103 strains showed two allelic variants. Allele 1 was found in all strains isolated from patients with FESLF. The cnfY gene was not specifically associated with the presence of plasmids. The cnfY gene was absent in the Y. enterocolitica strains. Thus, the cnfY gene is a stable and specific marker of Y. pseudotuberculosis. On the basis of the obtained data, a PCR diagnostic system was developed for the detection and intraspecific differentiation of Y. pseudotuberculosis by determining the cytotoxic necrotizing factor gene in the biomaterial.

Identification of Yersinia at the Species and Subspecies Levels Is Challenging

The genus Yersinia currently includes 18 species, of which Y. enterocolitica and Y. pseudotuberculosis are enteropathogenic. The identification of Y. enterocolitica in particular is very demanding because it consists of a group of very heterogeneous bacteria, including pathogenic and non-pathogenic strains. In this study, we provide recent information on the characteristics and identification of Yersinia spp. and the sources of enteropathogenic Yersinia spp. Identification of Yersinia spp. is still mainly based on biochemical tests and serotyping, but molecular methods have increasingly also been used. Pathogenic Y. enterocolitica strains of different bioserotypes have newly been identified from various animal sources. Moreover, the virulence gene ail has been detected in non-pathogenic Yersinia strains, especially from wild animals. Correct identification of pathogenic Yersinia strains is essential in assessing the health risk for humans and animals. Sequencing the whole genome enables more accurate identification of enteropathogenic Yersinia spp.

Sequence variants of Yersinia enterocolitica ystB gene detected in wild animals in Poland.

The purpose of the study was to analyze a part of the nucleotide sequences of ystB gene Y. enterocolitica strains isolated from wild animals. The material for the study consists of 30 Y. enterocolitica biotype 1A strains obtained from different wild animal species and belonging to different genotypes. Phylogenetic analysis of ystB nucleotide sequences belonging to four regular genotypes G1, G2, G3, G4 and to five groups of variations V1, V2, V3, V4, V5 revealed significant differences of Y. enterocolitica strains isolated from wild animals. The most phylogenetically distant were strains belonging to V5.

Characterisation of ail-positive Yersinia enterocolitica of different biotypes using HRMA

Yersiniosis is one of the four most frequent foodborne zoonotic diseases in Europe, and Yersinia enterocolitica is the primary agent in human infections. The ail gene is an important chromosomal virulence marker of Y. enterocolitica which encodes Ail, a 17-kDa outer membrane protein that promotes attachment and invasion. In the present study, ail-positive Y. enterocolitica strains of different biotypes were examined using high resolution melting analysis (HRMA) and DNA sequencing. Genotype data relating to Y. enterocolitica strains isolated from different sources and belonging to different biotypes were compared. Applied method allowed efficient distinguishing of three genotypes and phylogenetic groups: 1A – included non-pathogenic Y. enterocolitica strains; 1B – consisted of highly pathogenic Y. enterocolitica strains and 2/4 – involved weakly pathogenic Y. enterocolitica strains. Amplicon genotyping based on HRMA supports rapid identification of ail SNPs correlated with biotype of examined Y. enterocolitica strains.

Validation of EN ISO method 10273 - Detection of pathogenic Yersinia enterocolitica in foods

EN ISO 10273 method for the detection of pathogenic Yersinia enterocolitica in foods was validated in the project Mandate M/381 funded by European Commission. A total of 14 laboratories from five European countries participated in the interlaboratory study (ILS) organized during 2013 and 2014. Before the ILS, the method was revised by an international group of experts and the performance of the revised method was assessed in an ILS study. The results are published as a part of the standard EN ISO 10273 revision. The study included three rounds with different sample types; raw milk, iceberg lettuce and minced meat, inoculated with a low and high level of pathogenic Y. enterocolitica strains representing major pathogenic bioserotypes 4/O:3 and 2/O:9. The homogeneity and stability of the samples were verified before dispatching them to the laboratories. The results demonstrated the method sensitivity of 96% in raw milk, 97% in minced meat, and 98% in lettuce at high inoculation level of pathogenic Y. enterocolitica. The specificity was 100% in raw milk, 96% in minced meat, and 98% in lettuce. The level of detection, LOD50, varied between study rounds, being 9.4 CFU/25 ml in raw milk, 9.9 CFU/25 g in minced meat and 63 CFU/25 g in lettuce samples. During the study, confirmation by using real-time PCR method ISO/TS 18867 together with pyrazinamidase testing was also validated, as alternative to conventional biochemical confirmation. When comparing different isolation steps used in the revised method during the study rounds, PSB enrichment and plating on CIN after alkaline (KOH) treatment showed the highest sensitivity (52–92%) in raw milk and minced meat samples. In lettuce samples, however, ITC with KOH treatment before plating on CIN showed higher sensitivity (64% at low level; 82% at high level) than plating on CIN from PSB with KOH treatment (44% at low level; 74% at high level). Statistical analysis of different isolation steps supported the use of two enrichment media, PSB and ITC, in the revised method. Recovery of pathogenic Y. enterocolitica on CIN was most efficient after KOH treatment and, based on the analysis, plating on CIN agar without KOH treatment could be left as optional procedure in the method.

Revealing the inhibitory potential of Yersinia enterocolitica on cysteine proteases of the papain family

Cysteine proteases of the papain family, including mammalian cathepsins, play important physiological roles, however, their excessive activity may contribute to the development of various pathologies. Therefore, cysteine cathepsin inhibitors are being considered as promising drugs to treat cathepsin-driven diseases. Diverse saprophytic and parasitic microbes produce such inhibitors, which target the host's proteases playing pivotal roles in immune responses, thus leading to the survival of microbes within their host. Yersinia enterocolitica is a Gram-negative zoopathogenic coccobacillus, which has developed several mechanisms to evade the host's immune system. Nevertheless, the bacterium has not yet been shown to produce any cysteine protease inhibitors. Here we demonstrate that Y. enterocolitica strains of different bioserotypes and genotypes synthesize papain and human cathepsin L inhibitors, but not bovine cathepsin B inhibitors. By employing fluorimetry and zymography, the cell-surface inhibitors were shown to associate peripherally with the outer membrane, while the inhibitors present in cell-free extracts proved to: interact reversibly with their target enzymes, exhibit thermolability and stability in a range of pH values (5–9), and have high molecular weights. Batch affinity chromatography on papain–agarose resin was then undertaken to isolate putative inhibitors of cysteine proteases from the bacterial extract. The isolated 18 kDa protein was identified by LC–MS/MS as the periplasmic chaperone Skp. The Skp-containing eluate inhibited the activity of cysteine cathepsins produced by human dermal fibroblasts. The homologous Skp protein was also isolated from the extract of Escherichia coli. Our results point to a possible new biological role of the bacterial chaperone Skp.

Occurrence of Extended Spectrum β-Lactamases, KPC-Type, and MCR-1.2-Producing Enterobacteriaceae from Wells, River Water, and Wastewater Treatment Plants in Oltrepò Pavese Area, Northern Italy.

To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepò Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. Enterobacteriaceae were also characterized for the Extended-Spectrum-β-Lactamases (ESBLs), carbapenemases, and mcr-1 genes presence. From December 2014 to November 2015, 246 water samples were filtered, plated on Plate Count Agar, MacConkey Agar, and MacConkey Agar with cefotaxime. Isolates were species identified using AutoSCAN-4-System and ESBLs, carbapenemases, and colistin resistance determinants were characterized by PCR, sequencing, and microarray. Plasmid conjugative transfer experiments, PCR-based Replicon typing, Pulsed-Field Gel Electrophoresis, Multi-Locus-Sequence-Typing, and in-silico plasmid characterization were performed. A total of 132 enterobacteria isolates grew on MacConkey agar with cefotaxime: 82 (62.1%) were obtained from streams, 41 (31.1%) from treatment plants, and 9 (6.8%) from wells. Thirty out of 132 (22.7%) isolates, mainly belonging to Escherichia coli (n = 15) species, showed a synergic effect with piperacillin-tazobactam. A single ESBL gene of blaCTX−M-type was identified in 19/30 isolates. In further two E. coli strains, a blaCTX−M−1 gene co-existed with a blaSHV-type ESBL determinant. A blaSHV−12 gene was detected in two isolates of E. coli (n = 1) and Klebsiella oxytoca (n = 1), while any ESBL determinant was ascertained in seven Yersinia enterocolitica strains. A blaDHA-type gene was detected in a cefoxitin resistant Y. enterocolitica from a stream. Interestingly, two Klebsiella pneumoniae strains of ST307 and ST258, collected from a well and a wastewater treatment plant, resulted KPC-2, and KPC-3 producers, respectively. Moreover, we report the first detection of mcr-1.2 ST10 E. coli on a conjugative IncX4 plasmid (33.303 bp in size) from a stream of Oltrepò Pavese (Northern Italy). Both ESBLs E. coli and ESBLs/carbapenemase-producing K. pneumoniae strains showed clonal heterogeneity by Pulsed-Field Gel Electrophoresis and Multi-Locus-Sequence-Typing. During one-year study and taking in account the whole Gram-negative bacterial population, an average percentage of cefotaxime resistance of 69, 32, and 10.3% has been obtained for the wastewater treatment plants, streams, and wells, respectively. These results, of concern for public health, highlight the need to improve hygienic measures to reduce the load of discharged bacteria with emerging resistance mechanisms.

Determining the prevalence of inv-positive and ail-positive Yersinia enterocolitica in pig tonsils using PCR and culture methods.

Yersiniosis is believed to be the third most common intestinal zoonosis in the European Union, after campylobacteriosis and salmonellosis. Yersinia enterocolitica is the most common species responsible for human infections. Pigs are regarded as the biggest reservoir of pathogenic Y. enterocolitica strains, which are mainly isolated from pig tonsils. The aim of this paper is to examine the prevalence of inv-positive and ail-positive Y. enterocolitica in pigs which were slaughtered in a Polish abattoir. Real-time PCR and culture methods were used to assess the prevalence of patho- genic Y. enterocolitica strains in pig tonsils. Real-time PCR was applied to detect inv-positive and ail-positive Y. enterocolitica. Y. enterocolitica was also isolated by applying direct plating, unselective (tryptic soy broth) and selective (irgasan-ticarcillin-potassium chlorate bouillon) enrichment. A total of 180 pigs were studied, of which 85% and 32% respectively were found to be infected with inv-positive and ail-positive Y. enterocolitica. The 92 inv-positive and ail-positive isolates, from 57 culture- positive tonsils, underwent bio- and serotyping. The most common was bioserotype 4/O:3, which was found in 53 (93%) out of 57 culture-positive tonsils. Strains of bioserotypes 2/O:5, 2/O:9 and 2/O:5.27 occurred in significantly lower numbers. The prevalence of inv-positive and ail-positive Y. enterocolitica was found to be high in the ton- sils of slaughtered pigs, using real-time PCR. The real-time PCR method for the detection and identification of pathogenic Y. enterocolitica is sensitive and specific, which has been verified by specificity and sensitivity tests using the pure cultures. Serotypes were distinguished from each other using PCR serotyping. The PCR method was essential in forming our conclusions.

Detection of Yersinia Enterocolitica Species in Pig Tonsils and Raw Pork Meat by the Real-Time Pcr and Culture Methods.

The aim of the present study was to establish a rapid and accurate real-time PCR method to detect pathogenic Yersinia enterocolitica in pork. Yersinia enterocolitica is considered to be a crucial zoonosis, which can provoke diseases both in humans and animals. The classical culture methods designated to detect Y. enterocolitica species in food matrices are often very time-consuming. The chromosomal locus _tag CH49_3099 gene, that appears in pathogenic Y. enterocolitica strains, was applied as DNA target for the 5' nuclease PCR protocol. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). The real-time PCR cycling parameters included 41 cycles. A Ct value which reached a value higher than 40 constituted a negative result. The developed for the needs of this study qualitative real-time PCR method appeared to give very specific and reliable results. The detection rate of locus _tag CH49_3099 - positive Y. enterocolitica in 150 pig tonsils was 85 % and 32 % with PCR and culture methods, respectively. Both the Real-time PCR results and culture method results were obtained from material that was enriched during overnight incubation. The subject of the study were also raw pork meat samples. Among 80 samples examined, 7 ones were positive when real-time PCR was applied, and 6 ones were positive when classical culture method was applied. The application of molecular techniques based on the analysis of DNA sequences such as the Real-time PCR enables to detect this pathogenic bacteria very rapidly and with higher specificity, sensitivity and reliability in comparison to classical culture methods.

Prevalence of Yersinia enterocolitica Bioserotype 3/O:3 among Children with Diarrhea, China, 2010-2015.

Yersinia enterocolitica is thought to not significantly contribute to diarrheal disease in China, but evidence substantiating this claim is limited. We determined the prevalence of Y. enterocolitica infection and strain types present among children <5 years of age with diarrhea in China. The overall prevalence of pathogenic isolates was 0.59%. Prevalence of pathogenic bioserotype 3/O:3 varied geographically. In this population, the presence of fecal leukocytes was a characteristic of Y. enterocolitica infection and should be used as an indication for microbiological diagnostic testing, rather than for the diagnosis of bacillary dysentery. In contrast with Y. enterocolitica isolates from adults, which were primarily biotype 1A, isolates from children were primarily bioserotype 3/O:3. Most pathogenic isolates from children shared pulsed-field gel electrophoresis patterns with isolates from pigs and dogs, suggesting a possible link between isolates from animals and infections in children. Our findings underscore the need for improved diagnostics for this underestimated pathogen.

The Role of OmpR in the Expression of Genes of the KdgR Regulon Involved in the Uptake and Depolymerization of Oligogalacturonides in Yersinia enterocolitica.

Oligogalacturonide (OGA)-specific porins of the KdgM family have previously been identified and characterized in enterobacterial plant pathogens. We found that deletion of the gene encoding response regulator OmpR causes the porin KdgM2 to become one of the most abundant proteins in the outer membrane of the human enteropathogen Yersinia enterocolitica. Reporter gene fusion and real-time PCR analysis confirmed that the expression of kdgM2 is repressed by OmpR. We also found that kdgM2 expression is subject to negative regulation by KdgR, a specific repressor of genes involved in the uptake and metabolism of pectin derivatives in plant pathogens. The additive effect of kdgR and ompR mutations suggested that KdgR and OmpR regulate kdgM2 expression independently. We confirmed that kdgM2 occurs in an operon with the pelP gene, encoding the periplasmic pectate lyase PelP. A pectinolytic assay showed strong upregulation of PelP production/activity in a Y. enterocolitica strain lacking OmpR and KdgR, which corroborates the repression exerted by these regulators on kdgM2. In addition, our data showed that OmpR is responsible for up regulation of the kdgM1 gene encoding the second specific oligogalacturonide porin KdgM1. This indicates the involvement of OmpR in the reciprocal regulation of both KdgM1 and KdgM2. Moreover, we demonstrated the negative impact of OmpR on kdgR transcription, which might positively affect the expression of genes of the KdgR regulon. Binding of OmpR to the promoter regions of the kdgM2-pelP-sghX operon, and kdgM1 and kdgR genes was confirmed using the electrophoretic mobility shift assay, suggesting that OmpR can directly regulate their transcription. We also found that the overexpression of porin KdgM2 increases outer membrane permeability. Thus, OmpR-mediated regulation of the KdgM porins may contribute to the fitness of Y. enterocolitica in particular local environments.

Genetic diversity, virulotyping and antimicrobial resistance susceptibility of Yersinia enterocolitica isolated from pigs and porcine products in Malaysia.

The objectives of the present study were to determine the antimicrobial resistance, virulotypes and genetic diversity of Yersinia enterocolitica isolated from uncooked porcine food and live pigs in Malaysia. Thirty-two non-repeat Y. enterocolitica strains of three bioserotypes (3 variant/O:3, n = 27; 1B/O:8, n = 3; 1A/O:5, n = 2) were analysed. Approximately 90% of strains were multidrug-resistant with a multiple antibiotic resistance index < 0.2 and the majority of the strains were resistant to nalidixic acid, clindamycin, ampicillin, ticarcillin, tetracycline and amoxicillin. Yersinia enterocolitica could be distinguished distinctly into three clusters by pulsed-field gel electrophoresis, with each belonging to a particular bioserotype. Strains of 3 variant/O:3 were more heterogeneous than others. Eleven of the 15 virulence genes tested (hreP, virF, rfbC, myfA, sat, inv, ail, ymoA, ystA, tccC, yadA) and pYV virulence plasmid were present in all the bioserotpe 3 variant/03 strains. The occurrence of virulent strains of Y. enterocolitica in pigs and porcine products reiterated that pigs are important reservoirs for Y. enterocolitica. The increasing trend of multidrug resistant strains is a public health concern. This is the first report on the occurrence of potential pathogenic and resistant strains of Y. enterocolitica in pigs in Malaysia. © 2017 Society of Chemical Industry.

Antimicrobial resistance and plasmid replicons in Yersinia enterocolitica strains isolated in Brazil in 30 years

Some studies evaluated the resistance profile of the Y. enterocolitica strains isolated in diverse countries. However, in Brazil the isolation and the study of Y. enterocolitica are not common and therefore information about the antimicrobial resistance profile of this species in this country is scarce. Therefore, the aim of this study was to evaluate the antimicrobial resistance of Y. enterocolitica of biotypes 1A, 2 and 4 isolated from clinical and non-clinical sources between 1979 and 2012, in Brazil. This study showed that some Yersinia enterocolitica of different biotypes remain susceptible to antimicrobials used for gastroenteritis treatment. Moreover, neither acquired resistance genes nor diversity of plasmids replicons were found; however, variation in the in vitro intrinsic resistant pattern was detected, except the non-resistance to cefoxitin in all strains. Notwithstanding, due to epidemiological link between Y. enterocolitica and the pork production chain, monitoring plasmid acquired resistance in Y. enterocolitica could also be considered for antimicrobial resistance control purposes and food safety measures.

Draft Genome Sequences of Four Yersinia enterocolitica Strains, Isolated from Wild Ungulate Carcasses.

ABSTRACTThis study describes the draft genome sequences of four Yersinia enterocolitica strains, originally isolated from ungulate carcasses. These isolates were typed biochemically and two were determined to be highly virulent (biotype 1B). The draft genome sequences had a mean size of 4.77 Mb and a mean G+C content of 47.1%.

Evaluation of virulence genes in Yersinia enterocolitica strains using SYBR Green real-time PCR

Yersinia enterocolitica comprises six biotypes 1A, 1B, 2, 3, 4, and 5. The virulence of the strains belonging to biotypes 1B and 2–5 depends on the presence of both chromosomal and plasmid-borne genes. Strains belonging to biotype 1A do not carry the virulence plasmid pYV. However, they carry other virulence genes, such as ystB and hreP. The aim of this study was to investigate the distribution of yadA, virF, inv, ystA, ystB, myfA, hreP and ymoA virulence genes in Y. enterocolitica strains in order to select the target genes that could be used for the development of a probe-specific real-time PCR to determine the presence of Y. enterocolitica in food samples. A total of 161 Y. enterocolitica strains isolated in eight countries and grouped into biotypes 1A, 2 (serotypes O3, O5 and O9), 3 (serotypes O3 and O9) and 4 (serotype O3) were examined for virulence genes. The most common virulence-associated gene in pathogenic Y. enterocolitica proved to be ystA, which can therefore be considered the best target gene to be amplified in order to evaluate the presence of pathogenic biotypes. By contrast, to identify Y. enterocolitica 1A strains, ystB, which codes for the enterotoxin YstB, can be proposed. This has been found in all non-pathogenic biotypes studied, but never in pathogenic biotypes.

A study of single nucleotide polymorphism in the ystB gene of Yersinia enterocolitica strains isolated from various wild animal species.

Y. enterocolitica is the causative agent of yersiniosis. The objective of the article was a study of single nucleotide polymorphism in the ystB gene of Y. enterocolitica strains isolated from various wild animal species. High-resolution melting (HRM) analysis was applied to identify single nucleotide polymorphism (SNP) of ystB gene fragments of 88 Y. enterocolitica biotype 1A strains isolated from wild boar, roe deer, red deer and wild ducks. HRM analysis revealed 14 different melting profiles - 4 of them were defined as regular genotypes (G1, G2, G3, G4), whereas 10 as variations. 24 of the examined Y. enterocolitica strains were classified as G1, 18 strains as a G2, 21 strains as a G3, and 15 strains as a G4. Nucleotide sequences classified as G1 revealed 100% similarity with the Y. enterocolitica D88145.1 sequence (NCBI). Analysis of G2 revealed one point mutation - transition T111A. One mutation was also found in G3, but SNP was placed in a different gene region - transition G193A. Two SNPs - transitions G92C and T111A - were identified in G4. Direct sequencing of 10 variations revealed 5 new variants of the ystB nucleotide sequence: V1 - transition G129A (3 strains); V2 - transitions T111A and G193A (2 strains); V3 - transitions C118T and G193A (1 strain); V4 - transitions C141A and G193A (2 strains); and V5 characterized by 19 SNPs: G83A, T93A, A109G, G114T, C116T, A123G, T134C, T142G, T144C, A150C, G162A, T165G, T170G, T174A, T177G, G178A, A179G, A184G and G193A (2 strains). The predominant genotype in isolates from wild ducks was G1; in red deer G2; in wild boar G3; in roe deer G1 and G4. The proposed HRM method could be used to analyze Y. enterocolitica biotype 1A strains isolated from different sources, including humans.

Yersinia enterocolitica, a Neglected Cause of Human Enteric Infections in Côte d’Ivoire

BackgroundEnteropathogenic Yersinia circulate in the pig reservoir and are the third bacterial cause of human gastrointestinal infections in Europe. In West Africa, reports of human yersiniosis are rare. This study was conducted to determine whether pathogenic Yersinia are circulating in pig farms and are responsible for human infections in the Abidjan District.Methodology/Principal findingsFrom June 2012 to December 2013, pig feces were collected monthly in 41 swine farms of the Abidjan district. Of the 781 samples collected, 19 Yersinia strains were isolated in 3 farms: 7 non-pathogenic Yersinia intermedia and 12 pathogenic Yersinia enterocolitica bioserotype 4/O:3. Farm animals other than pigs and wild animals were not found infected. Furthermore, 2 Y. enterocolitica 4/O:3 strains were isolated from 426 fecal samples of patients with digestive disorders. All 14 Y. enterocolitica strains shared the same PFGE and MLVA profile, indicating their close genetic relationship. However, while 6 of them displayed the usual phage type VIII, the other 8 had the highly infrequent phage type XI. Whole genome sequencing and SNP analysis of individual colonies revealed that phage type XI strains had unusually high rates of mutations. These strains displayed a hypermutator phenotype that was attributable to a large deletion in the mutS gene involved in DNA mismatch repair.Conclusions/SignificanceThis study demonstrates that pathogenic Y. enterocolitica circulate in the pig reservoir in Côte d'Ivoire and cause human infections with a prevalence comparable to that of many developed countries. The paucity of reports of yersiniosis in West Africa is most likely attributable to a lack of active detection rather than to an absence of the microorganism. The identification of hypermutator strains in pigs and humans is of concern as these strains can rapidly acquire selective advantages that may increase their fitness, pathogenicity or resistance to commonly used treatments.

Prevalence, biofilm formation and virulence markers of Salmonella sp. and Yersinia enterocolitica in food of animal origin in Poland

Examination of 330 food samples (meat, white raw sausage, smoked meat and cheeses) was conducted and the Salmonella spp. and Y. enterocolitica prevalence were determined. For isolated strains of Salmonella spp., it was determined whether they were of S. Typhi, S. Typhimurium or S. Enteritidis serotypes. Isolated Y. enterocolitica strains were biotyped. Moreover, the strains' ability to form a biofilm and presence of virulence factors was determined. Salmonella spp. were found in 16 (5.5%) samples, whereas Yersinia enterocolitica in 7 (2.1%) samples. Serotyping resulted in classifying 4 strains as S. Typhimurium, 2 as S. Enteritidis and none as S. Typhi. Ten strains were not classified as any of the determined serotypes. The gene invA was found in all Salmonella strains and spvC was found in 3 (18.7%) strains. All of the Y. enterocolitica were classified as biotype 1A, none of the strains had the genes ail or ystA, and the ystB gene was found in five strains. None of the 16 strains of Salmonella spp. and the 7 strains of Y. enterocolitica showed any strong ability to form a biofilm, while 7 strains of Salmonella spp. and 3 strains of Y. enterocolitica showed a moderate ability to form a biofilm.

Characterization of Yersinia enterocolitica strains potentially virulent for humans and animals in river water.

The aim of this study was to isolate and identify potentially pathogenic strains of Yersinia enterocolitica in water samples collected from the upstream section of the Drwęca River in Poland. Thirty-nine water samples were collected. Strains were isolated, identified with the use of the API(®) 20E test kit (Biomerieux, Marcy l'Etoile, France) at 37°C, serotyped and subjected to a molecular analysis. Multiplex PCR was carried out to amplify three virulence genes: ail, ystA and ystB. Fragments of ail and ystA genes were not identified in the genetic material of the analysed strains. The ystB gene was identified in four strains. Yersinia enterocolitica strains of biotype 1A, which contain the ystB gene, may cause gastrointestinal problems. In our study, Y.enterocolitica strains of biotype 1A/ystB with serotypes 0:3, 0:5 and 0:8 were identified in samples collected from the Drwęca River which flows through the areas protected by Natura 2000, one of the largest networks of nature conservation areas in the European Union. The presence of Y.enterocolitica in the Drwęca River indicates that the analysed bacteria colonize natural water bodies. Most research focuses on food or sewage as a source of Y.enterocolitica infections. Little is known about the occurrence of this pathogen in natural waters. Our results show that natural waters are also a potential threat to human and animal health.

МОЛЕКУЛЯРНО-БИОЛОГИЧЕСКАЯ ХАРАКТЕРИСТИКА YERSINIA PSEUDOTUBERCULOSIS И YERSINIA ENTEROCOLITICA, ВЫДЕЛЕННЫХ В СИБИРИ И НА ДАЛЬНЕМ ВОСТОКЕ

A special feature of Y. pseudotuberculosis strains is its biochemical uniformity irrespective of the time and location of the causative agent isolation and the existence of 21 serological variants. Y. enterocolitica is a quite a heterogeneous species and is classified into 6 biochemical types associated with 29 serological variants. 221 Y. pseudotuberculosis and 447 Y. enterocolitica strains in total isolated in Siberia and in the Far East were characterized. Y. pseudotuberculosis genotype dominating in the Siberian and Far Eastern regions is presented by О:1b serotype of the first genogroup (pYV+, ympA+, HPI-) in two- (47:82 МDа) or single-plasmid (47 МDа) variants. Ribotyping and fingerprinting revealed 8 and 10 Y. pseudotuberculosis genotypes, respectively, that indicated relative heterogeneity of the circulated strains. Regional difference of ribotypes and fingerprints was noted. 401 of 447 Y. enterocolitica strains were classified as biotype A1 including 11 serotypes (O:4,32; O:4,44; O:5; O:6,30; O:6,31; O;7,8; O:12,25; O:13,7; O:19,8; O:41,43) and 46 strains belonged to biotypes 2–4 of O:3 and O:9 serotypes. Y. enterocolitica strains of biotypes 1A were isolated both from the environments, animals and patient samples as like the representatives of biotypes 2–4. The differentiating tests of fucose and sorbose made it possible to identify two species new for the Russian Federation – Y. mollaretii and Y. bercovierii. Y. enterocolitica biotypes 2–4 carried pYV plasmid and chromosomal ail, ystA virulence genes. These strains were referred to phagotypes Х3 (2/O:9) and VIII (4/O:3) and also to phagotype Xz (3/O:3), unique for Russia. Y. enterocolitica biotype 1A containing ystB thermostable toxin gene was confirmed to be an infectious etiological agent.