Enterocolitica Biotype Research Articles

Development and implementation of a core genome multilocus sequence typing scheme for Yersinia enterocolitica: a tool for surveillance and outbreak detection.

Yersinia enterocolitica (Y. enterocolitica) is the most frequent etiological agent of yersiniosis and has been responsible for several national outbreaks in Norway and elsewhere. A standardized high-resolution method, such as core genome Multilocus Sequence Typing (cgMLST), is needed for pathogen traceability at the national and international levels. In this study, we developed and implemented a cgMLST scheme for Y. enterocolitica. We designed a cgMLST scheme in SeqSphere + using high-quality genomes from different Y. enterocolitica biotype sublineages. The scheme was validated if more than 95% of targets were found across all tested Y. enterocolitica: 563 Norwegian genomes collected between 2012 and 2022 and 327 genomes from public data sets. We applied the scheme to known outbreaks to establish a threshold for identifying major complex types (CTs) based on the number of allelic differences. The final cgMLST scheme included 2,582 genes with a median of 97.9% (interquartile range 97.6%-98.8%) targets found across all tested genomes. Analysis of outbreaks identified all outbreak strains using single linkage clustering at four allelic differences. This threshold identified 311 unique CTs in Norway, of which CT18, CT12, and CT5 were identified as the most frequently associated with outbreaks. The cgMLST scheme showed a very good performance in typing Y. enterocolitica using diverse data sources and was able to identify outbreak clusters. We recommend the implementation of this scheme nationally and internationally to facilitate Y. enterocolitica surveillance and improve outbreak response in national and cross-border outbreaks.

Detection and Characterization of Zoonotic Pathogens in Game Meat Hunted in Northwestern Italy.

Wildlife can represent a reservoir of zoonotic pathogens and a public health problem. In the present study, we investigated the spread of zoonotic pathogens (Salmonella spp., Yersinia enterocolitica, Listeria monocytogenes, Shiga-toxin-producing Escherichia coli (STEC), and hepatitis E virus (HEV)) considering the presence of virulence and antibiotic resistance genes in game meat from animals hunted in northwest Italy. During two hunting seasons (2020 to 2022), samples of liver and/or muscle tissue were collected from chamois (n = 48), roe deer (n = 26), deer (n = 39), and wild boar (n = 35). Conventional microbiology and biomolecular methods were used for the detection, isolation, and characterization of the investigated pathogens. Two L. monocytogenes serotype IIa strains were isolated from wild boar liver; both presented fosfomycin resistance gene and a total of 22 virulence genes were detected and specified in the text. Eight Y. enterocolitica biotype 1A strains were isolated from chamois (2), wild boar (5), and deer (1) liver samples; all showed streptogramin and beta-lactam resistance genes; the virulence genes found were myfA (8/8 strains), ymoA (8/8), invA (8/8), ystB (8/8), and ail (4/8). Our data underscore the potential role of wildlife as a carrier of zoonotic and antibiotic-resistant pathogens in northwest Italy and a food safety risk for game meat consumers.

Isolation and characterization of Yersinia enterocolitica from foods in Apulia and Basilicata regions (Italy) by conventional and modern methods.

Yersiniosis is the third most reported food-borne zoonosis in Europe. The aim of the present study was to perform the search for Yersinia enterocolitica in food samples collected from Apulia and Basilicata regions (Southern Italy) and to characterize any isolates by classical and modern analytical methods. A total of 130 samples were analyzed between July 2018 and July 2019: most of them were raw milk and dairy products made from it. Furthermore, 8 out of 130 samples were individual milk samples collected from bovines reared in a Brucella-free farm which showed false positive serological reaction for brucellosis due to the presence of pathogenic Y. enterocolitica O:9 biotype 2 in faeces. The Real Time PCR targeting the ail gene and the culture method were performed to detect pathogenic Y. enterocolitica. Isolates were subjected to API 20E (Biomerieux) and MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization Time-of-Flight) for species identification. All samples were negative for the ail gene. The culture method allowed to isolate suspicious colonies from 28 samples. The API 20E system and the MALDI-TOF MS technique identified 20 Y. enterocolitica and 1 Y. intermedia in a concordant way. The remaining 7 strains were all identified as Y. enterocolitica by the API 20E system, while the MALDI-TOF MS recognized 4 Y. intermedia, 1 Y. bercovieri and 2 Y. massiliensis. Genotypic characterization of the discordant strains was performed by rMLST and it confirmed the MALDI-TOF MS’ results. Only non-pathogenic Y. enterocolitica biotype 1A strains were found, although with a non-negligible prevalence (P = 0.15 with CI 95% = ± 0.06). This study indicates a poor circulation of pathogenic Y. enterocolitica in food products made and marketed in the investigated areas. However, the small number of samples, insufficient for some food categories such as meat and vegetable, does not allow to exclude the presence of pathogenic strains at all.

Prevalence and Persistence of Multidrug-Resistant Yersinia enterocolitica 4/O:3 in Tonsils of Slaughter Pigs from Different Housing Systems in Croatia.

Yersinia enterocolitica is one of the priority biological hazards in pork inspection. Persistence of the pathogen, including strains resistant to antimicrobials, should be evaluated in pigs from different housing systems for risk ranking of farms. In this 2019 study, tonsils were collected from 234 pigs, of which 69 (29.5%) were fattened on 3 big integrated farms, 130 (55.5%) on 10 medium-sized farms, and 35 (15%) on 13 small family farms. In addition, 92 pork cuts and minced meat samples from the same farms were tested for the presence of Y. enterocolitica using the culture method. Phenotypic and genetic characteristics of the isolates were compared with previously collected isolates from 2014. The overall prevalence of Y. enterocolitica in pig tonsils was 43% [95% CI 36.7–49.7]. In pigs from big integrated, medium-sized, and small family farms, the prevalence was 29%, 52%, and 40%, respectively. All retail samples of portioned and minced pork tested negative for pathogenic Y. enterocolitica, likely due to high hygienic standards in slaughterhouses/cutting meat or low sensitivity of culture methods in these matrices. The highest recovery rate of the pathogen from tonsils was found when alkali-treated PSB and CIN agar were combined. The biosecurity category of integrated and medium farms did not affect the differences in prevalence of Y. enterocolitica (p > 0.05), in contrast to family farms. Pathogenic ail-positive Y. enterocolitica biotype 4 serotype O:3 persisted in the tonsils of pigs regardless of the type of farm, slaughterhouse, and year of isolation 2014 and 2019. PFGE typing revealed the high genetic concordance (80.6 to 100%) of all the Y. enterocolitica 4/O:3 isolates. A statistically significant higher prevalence of multidrug-resistant Y. enterocolitica 4/O:3 isolates was detected in the tonsils of pigs from big integrated farms compared to the other farm types (p < 0.05), with predominant and increasing resistance to nalidixic acid, chloramphenicol, and streptomycin. This study demonstrated multidrug resistance of the pathogen in pigs likely due to more antimicrobial pressure on big farms, with intriguing resistance to some clinically relevant antimicrobials used in the treatment of yersiniosis in humans.

Toxigenic Properties of Yersinia enterocolitica Biotype 1A

Yersinia (Y.) enterocolitica, an etiological agent of yersiniosis, is a bacterium whose pathogenicity is determined, among other things, by its ability to produce toxins. The aim of this article was to present the most important toxins that are produced by biotype 1A strains of Y. enterocolitica, and to discuss their role in the pathogenesis of yersiniosis. Y. enterocolitica biotype 1A strains are able to synthesize variants of thermostable YST enterotoxin and play a key role in the pathogenesis of yersiniosis. Biotype 1A strains of Y. enterocolitica also produce Y. enterocolitica pore-forming toxins, YaxA and YaxB. These toxins form pores in the cell membrane of host target cells and cause osmotic lysis, which is of particular importance in systemic infections. Insecticidal toxin complex genes have been detected in some clinical biotype 1A strains of Y. enterocolitica. However, their role has not yet been fully elucidated. Strains belonging to biotype 1A have long been considered non-pathogenic. This view is beginning to change due to the emerging knowledge about the toxigenic potential of these bacteria and their ability to overcome the defense barriers of the host organism.

Verification of analytical bacterial spectrum of QIAstat-Dx® GI V2 and Novodiag® Bacterial GE+ V2-0 diagnostic panels.

BackgroundImplementing multiplex PCR or syndromic panel-based testing platforms to detect microbial species that cause acute diarrhoea may guide patient management more effectively and efficiently.ObjectivesTo assess and compare the performance of two syndromic panel-based testing systems, QIAstat-Dx® Gastrointestinal Panel V2 (QGI) and the Novodiag® Bacterial GE+ V2-0 (NGE).MethodsThe QGI and NGE panels include 16 and 14 bacterial gastrointestinal pathogens, respectively. The performance of the panels was tested retrospectively using 141 positive clinical stool specimens, External Quality Assessment (EQA) panels and spiked faecal specimens.ResultsFor Campylobacter jejuni and coli (n = 20), Salmonella (n = 24), Shigella (n = 13), Yersinia enterocolitica (non-1A biotypes) (n = 8), Clostridioides difficile (n = 24) and Vibrio parahaemolyticus (n = 2), QGI correctly verified 19/20, 20/24, 13/13, 8/8, 23/24 and 2/2, whereas NGE correctly verified 20/20, 17/24, 13/13, 8/8, 14/24 and 1/2. Among diarrhoeagenic Escherichia coli (n = 29), QGI reported one Shiga toxin-producing E. coli (STEC) stx1a O26:H11 as STEC serotype O157:H7 and NGE failed on one enteropathogenic E. coli, one enteroaggregative E. coli and one STEC (stx2e). Y. enterocolitica biotype 1A (non-pathogenic) (n = 6) were all positive in QGI, but negative in NGE.ConclusionsBoth QGI and NGE testing panels can improve laboratory workflow and patient management by providing user-friendly platforms that can rapidly detect a number of targets with one specimen. QGI was significantly more sensitive in identifying C. difficile. Both methods had suboptimal detection of Salmonella and this needs to be examined further. The short hands-on time and turnaround time are of value for on-demand testing and use in a high-throughput setting.

Two copies of the ail gene found in Yersinia enterocolitica and Yersinia kristensenii

Yersinia enterocolitica is the most common Yersinia species causing foodborne infections in humans. Pathogenic strains carry the chromosomal ail gene, which is essential for bacterial attachment to and invasion into host cells and for serum resistance. This gene is commonly amplified in several PCR assays detecting pathogenic Y. enterocolitica in food samples and discriminating pathogenic isolates from non-pathogenic ones. We have isolated several non-pathogenic ail-positive Yersinia strains from various sources in Finland. For this study, we selected 16 ail-positive Yersinia strains, which were phenotypically and genotypically characterised. Eleven strains were confirmed to belong to Y. enterocolitica and five strains to Yersinia kristensenii using whole-genome alignment, Parsnp and the SNP phylogenetic tree. All Y. enterocolitica strains belonged to non-pathogenic biotype 1A. We found two copies of the ail gene (ail1 and ail2) in all five Y. kristensenii strains and in one Y. enterocolitica biotype 1A strain. All 16 Yersinia strains carried the ail1 gene consisting of three different sequence patterns (A6-A8), which were highly similar with the ail gene found in high-pathogenic Y. enterocolitica biotype 1B strains (A2). The Ail protein encoded by the ail1 gene was highly conserved compared to the Ail protein encoded by the ail2 gene. Multiple sequence alignment of the ail gene and Ail protein were conducted with MAFF. In total, 10 ail sequence variations have been identified, of which 8 conserved ones belonged to the ail1 gene. According to our results, the detection of ail alone is not sufficient to predict the pathogenicity of Yersinia isolates.

Yersinia enterocolitica biotype 1B case report: an unusual pathogen in an osteoarticular infection on device

BackgroundYersinia enterocolitica is an aero-anaerobic Gram-negative coccobacilli of the Enterobacteriaceae family, rarely reported in osteoarticular infection.Case presentationThis report case described a rare septic osteoarticular infection on device due to Yersinia enterocolitica biotype 1B. A purulent fistula appeared after osteosynthesis with plate performed abroad 27 days prior to the presentation for a distal femoral fracture. The treatment consisted of surgical irrigation and washing of the femoral plate and a bitherapy by levoflaxacine and ceftriaxone during 3 months.ConclusionY. enterocolitica biotype 1B is extremely rare in France. Moreover, the strain implicated in this european case is extremely close from the USA reference strain (with only 2 SNP difference) described in a septicemia in Ohio. The extreme proximity of the strains underlines the need for a sustained surveillance of the spread of this pathogen in France.

Exploring the genetic determinants underlying the differential production of an inducible chromosomal cephalosporinase - BlaB in Yersinia enterocolitica biotypes 1A, 1B, 2 and 4

Yersinia enterocolitica is an enteric bacterium which can cause severe gastroenteritis. Beta-lactams are the most widely used antibiotics against Y. enterocolitica. Y. enterocolitica produces two chromosomal β-lactamases, BlaA and BlaB. BlaB is an Ambler Class C inducible broad spectrum cephlaosporinase which showed differential enzyme activity in different biotypes of Y. enterocolitica. The expression of blaB is mainly regulated by ampR- the transcriptional regulator and, ampD - which helps in peptidoglycan recycling. The aim of this study was to identify and characterize genetic determinants underlying differential enzyme activity of BlaB in Y. enterocolitica biotypes 1 A, IB, 2 and 4. Thus, ampR, blaB and ampD were PCR-amplified and modeled in silico. The intercistronic region containing promoters of ampR and blaB was also investigated. Our results indicated that blaB was more inducible in biotypes 2 and 4, than in biotypes 1 A and 1B. Superimposition of in silico modeled proteins suggested that variations in amino acid sequences of AmpR, BlaB and AmpD were not responsible for hyper-production of BlaB in biotypes 2 and 4. Analysis of promoter regions of ampR and blaB revealed variations at −30, −37 and −58 positions from blaB transcription start site. Studies on relative expression levels of blaB in different biotypes by qRT-PCR indicated that nucleotide variations at these positions might contribute to a higher enzyme activity of BlaB in biotypes 2 and 4. However, this is a preliminary study and further studies including more strains of each biotype are required to strengthen our findings. Nevertheless, to the best of our knowledge, this is the first study which has investigated the genetic determinants underlying differential inducible production of BlaB in different biotypes of Y. enterocolitica.

PREVALENCE OF YERSINIA AMONG WILD SIKA DEER (CERVUS NIPPON) AND BOARS (SUS SCROFA) IN JAPAN

We examined the prevalence of Yersinia, including pathogenic species such as Yersinia enterocolitica and Yersinia pseudotuberculosis, among wild sika deer (Cervus nippon) and boars (Sus scrofa) captured in Japan. The prevalence of Yersinia in the wild deer was 75% (207/277) and in the boars was 74% (40/54). A total of 417 isolates of nine Yersinia species were isolated from the animals examined: the largest number of isolates (48%, 200/417) were Y. enterocolitica biotype 1A. Pathogenic Y. enterocolitica 1B/O:8 were also isolated from two deer, and Y. pseudotuberculosis serogroups 3 and 4 were isolated from two boars and a deer, respectively. The pathogenic Y. enterocolitica 1B/O:8 isolates carried four virulence genes (ail, ystA, yadA, and virF), and Y. pseudotuberculosis serogroups 3 and 4 isolates carried three virulence genes (inv, yadA, and lcrF). Although the Y. enterocolitica 1B/O:8 and Y. pseudotuberculosis isolates were sensitive to almost all the antimicrobials tested, the two Y. enterocolitica 1B/O:8 isolates were resistant to azithromycin and ampicillin, and the three Y. pseudotuberculosis isolates were resistant only to azithromycin. These findings suggested that wild deer and boars might be important reservoirs for the agent causing human yersiniosis.

The design, validation and clinical verification of an in-house qualitative PCR to detect Yersinia enterocolitica and Yersinia pseudotuberculosis in faeces

Yersiniosis is a zoonotic foodborne infection of public health significance. The aim of this study was to design and validate a simple, accurate and cost-effective polymerase chain reaction (PCR) to detect pathogenic Yersinia spp. in faecal samples. An intercalating dye (EvaGreen)-based real-time multiplex PCR assay was designed to detect yadA, ystB and inv by melt curve analysis, allowing undifferentiated detection of all Yersinia enterocolitica biotypes, including biotype 1A, and Yersinia pseudotuberculosis. The assay was validated using cultured bacteria and clinical samples. A total of 107 positive and 51 negative samples were tested. The sensitivity and specificity was 98% and 100%. The limit of detection was 104-105 CFU/g faeces. A total of 605 samples (9 positive) were tested in the clinical verification with an accuracy and negative predictive value of 99% [95% confidence interval (CI) 97.9-99.6%] and 99.8% (95% CI 97.9-99.6%), respectively. This is an accurate, simple and cost-effective assay for the detection of pathogenic Yersinia spp.

Antimicrobial resistance and its relationship with biofilm production and virulence-related factors in Yersinia enterocolitica biotype 1A

The aim of the present study was to determine antimicrobial susceptibilities, biofilm production and, to discern a relationship between antimicrobial resistance, biofilm potential and virulence-related genes in strains of Yersinia entercocolitica biotype 1A. Thirty strains of Y. enterocolitica biotype 1A including clinical and non-clinical strains were investigated. Antimicrobial susceptibility for 15 antibiotics (representing different classes) was determined by disk-diffusion assay. Biofilm potential was determined on two different culture media using crystal violet assay. Also, a co-relation was studied between antimicrobial susceptibilities, biofilm production and virulence-related genes. All strains of biotype 1A produced biofilms and exhibited varied level of susceptibilities for different antibiotics. More than 60% of the strains were strong to moderate biofilm producers and, were exclusively associated with REP/ERIC clonal group B. Moderate and strong biofilm producers exhibited both sensitive and resistant phenotypes towards different antibiotics. Interestingly, weak biofilm producers were resistant to amoxicillin, amoxicillin-clavulanate and cefazolin. Analysis of antimicrobial susceptibilities, biofilm potential and virulence-related genes did not reveal any unequivocal relationships. The differential biofilm potential of Indian strains of Y. enterocolitica biotype 1A, suggests that biotype 1A strains are heterogeneous in nature.

Molecular analysis of ampR and ampD to understand variability in inducible expression of “BlaB-like” cephalosporinase in Yersinia enterocolitica biotype 1A

Yersinia enterocolitica strains produce two chromosomal β‑lactamases, BlaA - a constitutively produced penicillinase, and BlaB - an inducible “AmpC-type” cephalosporinase. As in other members of Enterobacteriaceae, expression of ampC in Y. enterocolitica is regulated by the genes - ampR and ampD. The ampR encodes a transcriptional regulator which represses the expression of ampC and, ampD encodes a cytoplasmic N‑acetyl‑anhydromuramyl‑l‑alanine amidase which participates in recycling of peptidoglycan. Exposure of bacteria to antibiotics like imipenem and cefoxitin results in generation and accumulation of large quantities of muropeptides in cytoplasm which is beyond the recycling capability of AmpD. These muropeptides bind to AmpR, converting it into an activator of ampC expression (ampC de-repression). Earlier studies from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype 1A strains produced a “BlaB-like” enzyme which was non-heterogeneous and showed a differential expression when induced with imipenem. The detection of “BlaB-like” cephalosporinase which was also induced differentially in Y. enterocolitica biotype 1A strains presented an opportunity to discern newer mechanisms, if any, which may underlie inducible expression of “AmpC-type” cephalosporinases. Thus, the objective of the present study was to understand the role of ampR and ampD in regulating differential expression of “BlaB-like” cephalosporinases in biotype 1A strains. Analysis of promoters and amino acid sequences of AmpR revealed that these were conserved in all strains of biotype 1A. Analysis of AmpD amino acid sequences revealed that five variants of AmpD were present which did not contribute to hyper-inducible production of “BlaB-like” enzyme. In-silico prediction of the mRNA secondary structures of ampD revealed significant differences, which might have affected the rate of translation of ampD and accumulation of un-recycled muropeptides inside the cell leading to hyper production of “BlaB-like” cephalosporinases in some Y. enterocolitica biotype 1A strains. The findings provide newer insights to our understanding of the mechanisms underlying regulation of expression of “AmpC-type” β‑lactamases.

Molecular Characteristics of "BlaB-Like" Chromosomal Inducible Cephalosporinase of Yersinia enterocolitica Biotype 1A Strains.

Yersinia enterocolitica biotype 1A strains are emerging pathogens, frequently isolated from clinical samples across the globe. Y. enterocolitica strains produce two chromosomal β-lactamases, BlaA and BlaB. BlaA is a constitutively expressed, Ambler class A, penicillinase, whereas BlaB is Ambler class C-type, inducible cephalosporinase. An earlier study from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype 1A produced a "BlaB-like" enzyme. The objective of this work was to study the molecular characteristics of "Bla-B like" β-lactamases produced by biotype 1A strains to discern their similarity with AmpC-type β-lactamases and the basis of varied levels of minimum inhibitory concentration (MIC) to cefotaxime. Thus, the promoters and blaB genes were investigated in four strains of biotype 1A. Three-dimensional structures of the "BlaB-like" enzymes were modeled, and docked in silico with cefotaxime to understand how specific substitutions in gene sequences affect antibiotic susceptibilities. Our results indicated that all the reported key catalytic residues were present in variants of "Bla-B-like" enzymes, discerned in biotype 1A strains, but at different positions. Molecular docking of enzyme variants with cefotaxime revealed that lesser was the number of the H-binding residues with cefotaxime in a strain, lower was the MIC of cefotaxime in that strain. To the best of our knowledge, this is the first study in which the molecular characteristics and enzymatic interactions of "BlaB-like" cephalosporinases of Y. enterocolitica biotype 1A strains have been reported.

Microbial contamination of moose (Alces alces) and white-tailed deer (Odocoileus virginianus) carcasses harvested by hunters

Hunting is currently a very popular activity, and interest in game meat is increasing. However, only limited research is available on the bacterial quality and safety of moose (Alces alces) and white-tailed deer (Odocoileus virginianus) harvested by hunters. Poor hunting hygiene can spread bacteria onto the carcasses, and inadequate chilling of the carcasses may increase the bacterial load on the carcass surface. We studied the bacterial contamination level on carcasses of 100 moose and 100 white-tailed deer shot in southern Finland. Hunters eviscerated carcasses in the field and skinned them in small slaughter facilities. During the sampling, same person visited 25 facilities located in 12 municipalities of four provinces. Moose carcasses had mean mesophilic aerobic bacteria (MAB), Enterobacteriaceae (EB) and Escherichia coli (EC) values of 4.2, 2.6 and 1.2 log10 cfu/cm2, respectively, while deer carcass values were 4.5, 1.5 and 0.7 log10 cfu/cm2, respectively. Moose carcasses were significantly more contaminated with EB and EC than deer carcasses. High bacterial counts (MAB>5.0 log10 cfu/cm2 and EB > 2.5 log10 cfu/cm2) on the carcasses were associated with the smallest facilities having only one room. The outdoor temperature and days between hunting and sampling affected the bacterial counts. High EB counts on the carcasses indicated a gut hit. Male gender was significantly more contaminated by EC and meat-borne pathogenic bacteria: Campylobacter spp., Salmonella spp., enteropathogenic Yersinia spp., stx-harbouring EC (STEC) and Listeria monocytogenes. STEC (28/200) and L. monocytogenes (20/200) were the most commonly detected bacteria by PCR. L. monocytogenes isolates of different sequence types (ST7, 18, 29, 37, 249, 412, 451 and 611) belonged to serotypes 1/2a (seven isolates) and 4b (three isolates). The virulence gene ail was detected in four Yersinia enterocolitica biotype 1A isolates and one Yersinia kristensenii isolate. The bacterial counts on the moose and deer carcasses varied highly, and more attention should be paid to hunting hygiene and training of hunters. Game meat may be a source of meat-borne pathogens, and close attention should therefore be paid when handling and preparing game.

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.

Yersinia enterocolitica biotype 1A: a possible new trigger of reactive arthritis.

Yersinia enterocolitica (YE) biotype 1A is generally considered non-pathogenic, and the role of it in causing reactive musculoskeletal complications is unclear. We evaluated the capability of YE biotype 1A to induce reactive arthritis (ReA) and other reactive musculoskeletal symptoms. Analysis of self-reported musculoskeletal symptoms was supplemented with a telephone interview (with a permission to acquire copies of patient files from a local physician or hospital) and/or clinical examination of subjects with recent musculoskeletal symptoms after a positive stool culture for YE. The diagnoses of ReA and reactive tendinitis and enthesitis (ReTe) were defined as "definite" when based on clinical examination and/or on interview by phone and "probable" when based solely on the questionnaire. Of 120 subjects, who reported musculoskeletal symptoms, 100 were included in the final analysis. Among these 100 patients, 68% had YE biotype 1A, 16% YE bio/serotype 4, and 1% biotype 2 infection; the remaining 15% had different YE-like strains or a non-biotypable strain. Of the 21 patients with ReA and of the 14 patients with ReTe, the diagnosis was definite in 9 and 7 patients and probable in 12 and 7 patients, respectively. The clinical picture of ReA caused by YE biotype 1A was similar with other bio/serotypes of YE. The definite ReA due to YE biotype 1A occurred in middle-aged adults (5 men, 4 women) with the most frequently affected joints being the knees and ankles. We suggest that YE biotype 1A should be taken into account as a new trigger of ReA.

Development of a novel multiplexed qPCR and Pyrosequencing method for the detection of human pathogenic yersiniae

The purpose of this study was to develop a novel and robust molecular assay for the detection of human pathogenic yersiniae (i.e. Yersinia enterocolitica, Y. pseudotuberculosis and Y. pestis) in complex food samples. The assay combines multiplexed real-time PCR (qPCR) and Pyrosequencing for detecting and differentiating human pathogenic yersiniae with high confidence through sequence based confirmation. The assay demonstrated 100% specificity and inclusivity when tested against a panel of 14 Y. enterocolitica, 22 Y. pestis, 24 Y. pseudotuberculosis and a diverse selection of 17 other non-Yersinia bacteria. Pyrosequencing reads ranged from 28 to 40bp in length and had 94–100% sequence identity to the correct species in the GenBank nr database. Microbial enrichments of 48 ready-to-eat foods collected in the Greater Toronto Area from March 2014 to May 2014, including 46 fresh sprout and 2 salad products, were then tested using the assay. All samples were negative for Y. pestis and Y. pseudotuberculosis. Both salads (n=2) and 35% of sprout products (n=46) including 7.1% of alfalfa sprouts (n=14), 81% of bean sprouts (n=16), 12% of mixed sprouts (n=8) tested positive for Y. enterocolitica which was not detected in broccoli sprouts (n=5), onion sprouts (n=1), and pea sprouts (n=2). Cycle thresholds (Ct) of positive samples for Y. enterocolitica were between 23.0 and 37.9 suggesting post enrichment concentrations of approximately 1×102 to 1×106Y. enterocolitica per 1mL of enriched broth. An internal amplification control which was coamplified with targets revealed PCR inhibition in five samples which was resolved following a one in ten dilution. Pyrosequencing of qPCR amplicons suggests monoclonality and revealed a single nucleotide polymorphism that is present in Y. enterocolitica biotype 1A suggesting low pathogenicity of the detected strains. This study is the first to combine Pyrosequencing and qPCR for the detection of human pathogenic yersiniae and is applicable to a broad range of complex samples including ready-to-eat food samples.

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.

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.