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The objective of this study was to determine risk factors and sources attributed to yersiniosis in Aotearoa New Zealand (NZ). A risk factor questionnaire was administered to 247 notified yersiniosis cases and 258 control participants from the Canterbury and/or Wellington regions of NZ. Yersinia sp. isolates from clinical cases and a range of food sources were whole-genome sequenced and genetically compared. Yersinia enterocolitica (YE) bioserotype 2/3, O:9 [McNally multi-locus sequence type (ST) 12] and YE Biotype (BT) 1A (46 different STs) predominated within the consented cases (45 and 27%, respectively). Exposure to pork was identified as a significant risk factor for cases associated with YE ST12. The presence of YE ST12 was confirmed in retail raw meat, primarily raw pork. Single-nucleotide polymorphism (SNP) analysis identified multiple genomically very closely related clusters (0-5 SNPs) of YE ST12, predominately from raw pork with clinical cases from one or both regions. Risk factors associated with YE BT 1A included the consumption of cooked seafood, sushi, tofu, and some vegetable types. Analysis of specific risk factors and SNP analysis, combined, indicate that raw pork is a significant risk factor for exposure and infection to pathogenic YE cases, but not BT 1A cases., Competing Interests: The authors declare no conflict of interest.

Background: Yersiniosis is one of the most significant intestinal disorders caused by Yersinia enterocolitica and affects both humans and animals. This study aimed to investigate the prevalence of Y. enterocolitica in New Valley Governorate, Egypt in animals, humans, fresh milk and dried milk. Additionally, this study analyzed the presence of virulence genes, including ail and Yst in tested isolates and conducted a phylogenetic analysis to determine the genetic similarity between human, and animal Y. enterocolitica isolates. Finally, the antimicrobial resistance patterns of the isolates were examined., Results: Among the 982 samples examined, the prevalence of Y. enterocolitica based on ISO10273-2017 was 11.7% in animal samples including 12.8% of animal faeces, and 10.4% in milk samples. Moreover, the prevalence of Y. enterocolitica was 13.2% in human stool, and 9.5% in dried milk samples. The molecular characterization of the six randomly selected isolates showed that the 16S rRNA, ail and Yst genes were found in 50, 33.3 and 100% of the examined Y. enterocolitica isolates, respectively. Phylogenetic analysis of animal and human isolates based on the 16S rRNA gene revealed a high degree of similarity between the isolates. All the tested animal and human Y. enterocolitica isolates (100%) were resistant to ampicillin and cefotaxime, but highly sensitive to norfloxacin., Conclusions: The high prevalence of Y. enterocolitica in animal and human samples with high degrees of genetic similarity poses a threat to public and animal health. Animal faeces, milk and milk powder represent the main sources of Y. enterocolitica infection in humans. Additionally, high levels of antibiotic resistance of Y. enterocolitica can cause public health hazards by leading to the failure of disease prevention and treatment programs in humans and animals., (© 2024. The Author(s).)

Purpose: We aimed to characterise Yersinia enterocolitica from human clinical specimens in Switzerland using epidemiological, microbiological and whole-genome sequencing (WGS) data., Methods: Isolates (n = 149) were collected between January 2019 and December 2023. Epidemiological data was noted and strains were characterized by biochemical and serological typing, antimicrobial susceptibility testing (AST), and WGS-based analysis., Results: Most of the isolates (86%) were from stool specimens and 52% were from male patients. The patients' median age was 28 years (range < 1-94 years). Typing assigned the isolates to bioserotype 4/O:3 (44%), biotype 1A (34%), bioserotype 2/O:9 (21%), and bioserotype 3/O:3 (1%). WGS identified Y. enterocolitica (n = 147), Y. alsatica (n = 1) and Y. proxima (n = 1). Seven isolates were multidrug resistant (MDR) and harboured plasmid pAB829 carrying aph(3″)-Ib, aph(6)-Id, and tet(Y) (n = 1), pAC120 carrying aph(6)-Id and tet(A) (n = 2), or a 12.6 kb Tn2670-like transposon containing catA1, aadA12, sul1, and qacEΔ1 (n = 4). Virulence factors (VFs) included ail (n = 99), invB, (n = 145), ystA (n = 99), ystB (n = 48) and pYV-associated VFs (n = 93). MLST and cgMLST analysis showed that BT 1A strains consisted of several STs and were highly diverse, whereas BT 2/O:9 strains were all ST12 and clustered closely, and BT 4/O:3 strains mostly belonged to ST18 but were more diverse. SNP analysis revealed two highly clonal BT 4/O:3 subpopulations with wide spatio-temporal distribution., Conclusions: Y. enterocolitica BT 1A, BT 2/O:9 and BT 4/O:3 are frequently associated with human yersiniosis in Switzerland. WGS-based subtyping of Y. enterocolitica is a powerful tool to explore the genetic diversity and the pathogenic potential of human isolates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Yersinia enterocolitica (Ye) is a foodborne pathogen isolated from humans, food, animals, and the environment. Yersiniosis is the third most frequently reported foodborne zoonosis in the European Union. Ye species are divided into six biotypes 1A, 1B, 2, 3, 4, and 5, based on biochemical reactions and about 70 serotypes. Biotype 1A is non-pathogenic, 1B is highly pathogenic, and biotypes 2-5 have moderate or low pathogenicity. The reference analysis method for detecting pathogenic Ye species underestimates the presence of the pathogen due to similarities between Yersinia enterocolitica-like species and other Yersiniaceae and/or Enterobacteriaceae, low concentrations of distribution pathogenic strains and the heterogeneity of Yersinia enterocolitica species. In this study, the real-time PCR method ISO/TS 18867 to identify pathogenic biovars of Ye in bivalve molluscs was validated. The sensitivity, specificity and accuracy of the molecular method were evaluated using molluscs experimentally contaminated. The results fully agree with those obtained with the ISO 10273 method. Finally, we evaluated the presence of Ye in seventy commercial samples of bivalve molluscs collected in the Gulf of Naples using ISO/TS 18867. Only one sample tested resulted positive for the ail gene, which is considered the target gene for detection of pathogenic Ye according to ISO/TS 18867. Additionally, the presence of the ystB gene, used as target for Ye biotype 1A, was assessed in all samples using a real-time PCR SYBR Green platform. The results showed amplification ystB gene aim two samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Yersinia enterocolitica is a globally distributed food-borne gastrointestinal pathogen. The O-antigen variation-determined serotype is an important characteristic of Y. enterocolitica , allowing intraspecies classification for diagnosis and epidemiology purposes. Among the 11 serotypes associated with human yersiniosis, O:3, O:5,27, O:8, and O:9 are the most prevalent, and their O-antigen gene clusters have been well defined. In addition to the O-antigen, several virulence factors are involved in infection and pathogenesis of Y. enterocolitica strains, and these are closely related to their biotypes, reflecting pathogenic properties. In this study, we identified the O-AGC of a Y. enterocolitica strain WL-21 of serotype O:10, and confirmed its functionality in O-antigen synthesis. Furthermore, we analyzed in silico the putative O-AGCs of uncommon serotypes, and found that the O-AGCs of Y. enterocolitica were divided into two genetic patterns: (1) O-AGC within the hemH-gsk locus, possibly synthesizing the O-antigen via the Wzx/Wzy dependent pathway, and (2) O-AGC within the dcuC-galU-galF locus, very likely assembling the O-antigen via the ABC transporter dependent pathway. By screening the virulence genes against genomes from GenBank, we discovered that strains representing different serotypes were grouped according to different virulence gene profiles, indicating strong links between serotypes and virulence markers and implying an interaction between them and the synergistic effect in pathogenicity. Our study provides a framework for further research on the origin and evolution of O-AGCs from Y. enterocolitica , as well as on differences in virulent mechanisms among distinct serotypes.

Background: Commonly used approaches for genomic investigation of bacterial outbreaks, including SNP and gene-by-gene approaches, are limited by the requirement for background genomes and curated allele schemes, respectively. As a result, they only work on a select subset of known organisms, and fail on novel or less studied pathogens. We introduce refMLST, a gene-by-gene approach using the reference genome of a bacterium to form a scalable, reproducible and robust method to perform outbreak investigation., Results: When applied to multiple outbreak causing bacteria including 1263 Salmonella enterica, 331 Yersinia enterocolitica and 6526 Campylobacter jejuni genomes, refMLST enabled consistent clustering, improved resolution, and faster processing in comparison to commonly used tools like chewieSnake., Conclusions: refMLST is a novel multilocus sequence typing approach that is applicable to any bacterial species with a public reference genome, does not require a curated scheme, and automatically accounts for genetic recombination., Availability and Implementation: refMLST is freely available for academic use at https://bugseq.com/academic ., (© 2024. The Author(s).)

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., Competing Interests: The authors declare no conflict of interest.

Enteric yersiniosis, the third most common food-borne zoonosis in Europe, is mainly caused by the pathogen Yersinia enterocolitica . In France , the yersiniosis microbiological surveillance is conducted at the Yersinia National Reference Laboratory (YNRL). Since 2017, isolates have been characterized by whole genome sequencing (WGS) followed by a 500-gene Yersinia -cgMLST. We report here the data of the WGS-based surveillance on Y. enterocolitica isolates for the 2017-2021 period. The YNRL characterized 7,642 Y. enterocolitica strains distributed in 2,497 non-pathogenic isolates from lineages 1Aa and 1Ab, and 5,145 specimens belonging to 8 pathogenic lineages. Among pathogenic isolates, lineage 4 was the most common (87.2%) followed by lineages 2/3-9b (10.6%), 2/3-5a (1.2%), 2/3-9a (0.6%), 3-3b, 3-3c, 1B, and 3-3d (0.1% per each). Importantly, we developed a routine surveillance system based on a new typing method consisting of a 1,727-genes core genome Multilocus Sequence Typing (cgMLST) specific to the species Y. enterocolitica followed by isolate clustering. Thresholds of allelic distances (AD) were determined and fixed for the clustering of isolates: AD ≤ 5 for lineages 4, 2/3-5a, and 2/3-9a, and AD ≤ 3 for lineage 2/3-9b. Clustering programs were implemented in 2019 in routine surveillance to detect genomic clusters of pathogenic isolates. In total, 419 clusters with at least 2 isolates were identified, representing 2,504 of the 3,503 isolates characterized between 2019 and 2021. Most clusters ( n = 325) comprised 2 to 5 isolates. The new typing method proved to be useful for the molecular investigation of unusual grouping of cases as well as for the detection of genomic clusters in routine surveillance., Importance: We describe here the new typing method used for molecular surveillance of Yersinia enterocolitica infections in France based on a novel core genome Multilocus Sequence Typing (cgMLST) specific to Y. enterocolitica species. This method can reliably identify the pathogenic Y. enterocolitica subspecies and compare the isolates with a high discriminatory power. Between 2017 and 2021, 5,145 pathogenic isolates belonging to 8 lineages were characterized and lineage 4 was by far the most common followed by lineage 2/3-9b. A clustering program was implemented, and detection thresholds were cross-validated by the molecular and epidemiological investigation of three unusual groups of Y. enterocolitica infections. The routine molecular surveillance system has been able to detect genomic clusters, leading to epidemiological investigations., Competing Interests: The authors declare no conflict of interest.

Yersinia enterocolitica bio-serotype 4/O:3 was previously identified in a pork production chain in Brazil and the obtained isolates presented high identity by pulsed-field gel electrophoresis (PFGE, XbaI). For the current study, an additional 147 porcine samples (tonsils = 100, palate = 30, head meat = 17) were collected from the same pork production chain 2-years later and 14 (9.5%) tested positive for Y. enterocolitica. Isolates (n = 24, 1 to 2 per positive sample) were bio-serotype 4/O:3 and harbored virulence genes ail, inv, wbbU, virF, myfA, ystA, ymoA, hreP and sat, and the multidrug resistance related genes emrD, marC and yfhD. PFGE (XbaI) demonstrated no differences among isolates (100% similarity) and were identical to some Y. enterocolitica isolates (n = 13) obtained previously from the same pork chain. A second PFGE analysis (NotI) confirmed the high degree of similarity among isolates obtained over time, demonstrating the persistence of an apparent clonal Y. enterocolitica bio-serotype 4/O:3 in this particular pork production chain in Brazil., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Conventional methods for Yersinia enterocolitica detection in food samples are generally considered inadequate. Problems arise from the presence of the so-called "background flora", coupled to the low contamination level of the pathogen. Since, data on the microbial ecology occurring in competitive microflora are still lacking, MALDI TOF MS was used for strains 'identification after enrichment in PSB or ITC broths, and after plating on selective CIN medium at different incubation times. SYBR Green Real time PCR was used for the Y. enterocolitica strains' detection (4/O:3, 1A/O:5) in experimentally contaminated foods, as well as in naturally contaminated samples. A higher number of different bacterial genera (10 on CIN and 18 on PCA) was recorded after enrichment in PSB, whilst enrichment in ITC led to recovery of 6 and 10 genera on CIN and PCA, respectively. Yersiniaceae was the dominant family on the first day of incubation, but on the second day the percentage of isolation considerably decreased. By testing experimentally contaminated samples, substantial difficulties were encountered. The biotype 1A was always detected, whereas strain 4/O:3 proved to be poorly competitive. Based on the data, the enrichment media PSB and ITC, currently proposed for Y. enterocolitica detection, need to be improved to promote a successful pathogen's recovery., (Copyright © 2020. Published by Elsevier Ltd.)

Food-associated outbreaks linked to enteropathogenic Yersinia enterocolitica are of concern to public health. Pigs and their meat are recognized risk factors for transmission of Y. enterocolitica . This study aimed to describe the comparative genomics of Y. enterocolitica along with a number of misclassified Yersinia isolates, now constituting the recently described Yersinia hibernica . The latter was originally cultured from an environmental sample taken at a pig slaughterhouse. Unique features were identified in the genome of Y. hibernica, including a novel integrative conjugative element (ICE), denoted as ICE Yh-1 contained within a 255 kbp region of plasticity. In addition, a zebrafish embryo infection model was adapted and applied to assess the virulence potential among Yersinia isolates including Y. hibernica .

This study is aimed at offering an overview of the prevalence of Yersinia enterocolitica and related species in San Luis, Argentina, from samples of diverse origin received in our laboratory between 1984 and 2014, and providing an analysis of the distribution of Yersinia isolates according to their isolation sources, highlighting bioserotypes and potential reservoirs and vehicles of transmission to humans. From a total of 4572 samples of human, animal, food and environmental origins analyzed by traditional culture methods and molecular techniques, 229 (5%) samples were Yersinia positive. The highest frequency of Yersinia isolates was observed in environmental specimens (14.3%), followed by animal (9.2%), food (5%) and human (0.6%) samples. A total of 255 Yersinia isolates were characterized, including 183 Y. enterocolitica and 72 isolates of other Yersinia species. Biotype 1A associated to several serotypes was identified in Y. enterocolitica isolates from environment (100%), animals (95.5%), foods (71.7%) and human samples (40%); bioserotype 2/O:9 was identified in isolates from foods (25.5%), and biotype 3 was associated with strains from humans (60%), animals (4.5%) and foods (2.8%). This biotype included three strains O:3 and six strains O:5. The data highlight animals and foods as the main Y. enterocolitica sources in our region., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Gut is often subject to infection by different pathogens like Y. enterocolitica. To date, biotypes (BTs) 1A have been considered as non-pathogenic, because they do not express plasmid of virulence pYV; however, BTs 1A strains present other chromosomic virulence genes and recent studies suggest an implication of this microorganism in reactive arthritis. Although many studies highlighted the molecular basis of pathogenesis of Ye infection, scanty data are available about several environmental BTs 1A strains, often isolated in cases of foodborne disease but not included in pathogenicity studies. The aim of our work was to verify the ability of different Ye 1A strains to adhere and penetrate IPEC-J2 cells and to modulate intestinal innate immunity. Our results showed that all strains under study were able to adhere and penetrate enterocytes, causing inflammatory responses. Indeed, adhesion and invasion of enterocytes is an essential step in Ye pathogenesis (Fàbrega and Vila, 2012). Moreover, our data suggest the possible involvement of strains Ye2/O:9 in reactive arthritis, due to their ability (i) to penetrate enterocytes as pathogenic Ye1/O:8 strains do, and (ii) to increase IL-6, IL-8, IL-12 and IL-18 release. Lastly, our results confirm that IPEC-J2 cells are a very good model to evaluate host-pathogen interaction, and indicate IL-8, TNF-α, TLRs1 and 4 as possible markers of the ability of Ye strains to penetrate enterocytes. Moreover, we showed that Ye strains differently affect the host's innate immune responses., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

A thorough understanding of the mechanisms of Rcs and EnvZ/OmpR phosphorelay systems that allow Yersinia enterocolitica to thrive in various environments is crucial to prevent and control Y. enterocolitica infections. In this study, we showed that RcsB and OmpR have the ability to function differently in modulating a diverse array of physiological processes in Y. enterocolitica. The rcsB mutant stimulated flagella biosynthesis and increased motility, biofilm formation and c-di-GMP production by upregulating flhDC, hmsHFRS and hmsT. However, mutation in ompR exhibited a non-motile phenotype due to the lack of flagella. Biofilm formation was reduced and less c-di-GMP was produced through the downregulation of flhDC, hmsHFRS and hmsT expression when Y. enterocolitica was exposed to low osmolarity conditions. Furthermore, OmpR was identified to be important for Y. enterocolitica to grow in extreme temperature conditions. Importantly, ompR mutations in Y. enterocolitica were more sensitive to polymyxin B and sodium dodecyl sulfate than rcsB mutations. Since motility, biofilm formation and environmental tolerance are critical for bacterial colonization of the host, these findings indicated that OmpR is more critical than RcsB in shaping the pathogenic phenotype of Y. enterocolitica., (© FEMS 2019.)

Descriptive epidemiology of Yersinia enterocolitica infection in an area of Castellón (Spain) between 2006 and 2013 from Yersinia enterocolitica strains isolated in the area and confirmed by the Spanish national reference laboratory. There were a total of 144 cases. The estimated incidence was 9.7 cases per 10 5 person-year. The age group most affected was 0-4 years (rate 110.3 per 10 5 p-y), with a maximum in infants aged 6 to 11 months of age (190.4 per 10 5 p-y). The average duration of the disease was 15.5 days. 7% of the patients were hospitalised. Only 2 outbreaks of a family nature related to the consumption of pork were detected. The temporal evolution reflects higher incidence during the winter season (January). The most common exposure factor among the cases was the consumption of dried pork sausage (50% of the cases interviewed). The 58 typed strains were all of the biotype 4, serotype O:3, except one O:9. We distinguished 21 pulsotypes grouped in 8 clusters with a similarity of 97%. Over a number of years, a substitution of some pulsotypes for others was observed. Yersiniosis has a high incidence in our area, with a clear seasonality of winter predominance. It affects very young children, in particular. The strains are of the same serotype, but the variety of pulsotypes changed over time. As an exposure factor for further analytical studies, the consumption of some pork products is proposed, without ruling out other factors., (Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The pig is one of the main reservoirs of Yersinia enterocolitica strains pathogenic to humans. A description of the Y. enterocolitica population in this reservoir, and accurate discriminatory techniques for typing isolates are needed for prevention, outbreak investigation, and surveillance. This study investigates the genetic diversity of pathogenic Y. enterocolitica isolates obtained from pig tonsils in a French pig slaughterhouse in 2009 (S1) and 2010 (S2). The use of Pulsed-Field Gel Electrophoresis (PFGE) and MLVA as typing techniques was also compared and evaluated. First, a total of 167 isolates (12 of biotype 3 recovered during S1, and 155 of biotype 4 recovered during S1 and S2) were typed by PFGE using the XbaI enzyme. MLVA was then tested on all the biotype 3 isolates in addition to 70 selected biotype 4 isolates recovered over the 2 years. PFGE generated two specific XbaI-PFGE profiles for biotype 3 isolates. Nine XbaI profiles were obtained for biotype 4, with a higher diversity (ID = 0.599) than biotype 3 (ID = 0.167). Two out of the nine XbaI profiles were reported during both surveys and at different months. MLVA improved the differentiation between isolates; the index of diversity reached 0.621 and 0.958, respectively, for biotype 3 (three MLVA types) and biotype 4 (32 MLVA types). The MLVA types for biotype 4 differed over the two surveys, but some isolates with different MLVA types were genetically closely related. This study provides an initial evaluation of the genetic diversity of Y. enterocolitica strains isolated from pigs in France. We show that some PFGE profiles are maintained in the pig production sector, and, through MLVA, that part of the Y. enterocolitica population remained genetically close over the two years. MLVA proved its effectiveness as a tool for investigating pathogenic Y. enterocolitica strains isolated from pigs., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Inducible 'AmpC-type' chromosomal cephalosporinases have been reported to be differentially expressed in different biotypes of Yersinia entercolocolitica. AmpD amidases are key regulators of the expression of ampC genes in Y. entercolocolitica as their inactivation results in hyper production of AmpC. To understand the differences in regulation of ampC expression in different biotypes of Y. enterocolitica, characteristics of ampD homologs were studied in strains of Y. enterocolitica belonging to five biotypes namely 1A, 1B, 2, 3 and 4. Our results indicated that the mechanisms which regulate expression of ampC might differ in different biotypes. While a three-step regulation mechanism seemed to be functional in biotypes 2, 3 and 4, a two-step regulation mechanism using other AmiD like proteins might be functional in biotypes 1A and 1B. The existence of ampD homolog(s)-mediated expression of ampC in other members of the family Enterobacteriaceae may provide further credence to our findings., (Copyright © 2019. Published by Elsevier B.V.)

PurposeandMethodology. Epidemiological and microbiological data on Yersinia enterocolitica (n=699) and Yersinia pseudotuberculosis (n=35) isolated from human clinical specimens in England between April 2004 and March 2018 were reviewed. Traditional biochemical species identification and serological typing results were compared with species identifications and serotypes derived from whole-genome sequencing (WGS) data for a sub-set of these isolates (n=179).Results. Most Y. enterocolitica isolates were from faecal specimens (74.4%) from adults (80.7%) and 50.7  % of isolates were from male patients. Most Y. pseudotuberculosis isolates were from blood cultures (68.6%) from adults (91%) and 60.0  % of isolates were from male patients. All sequenced isolates of Y. enterocolitica (n=158) and Y. pseudotuberculosis (n=21), as well as isolates belonging to other Yersinia species (n=21), were correctly identified from genomic data using a kmer-based identification approach. Traditional phenotypic serotyping typed 82/158 and 12/21 isolates of Y. enterocolitica and Y. pseudotuberculosis, respectively, while 118/158 and 21/21 isolates of Y. enterocolitica and Y. pseudotuberculosis, respectively, were typed by the genome-derived serotyping method. In addition, WGS data provided a multi-locus sequence type profile and virulence gene profile for all isolates.Conclusion. The use of WGS for typing Y. enterocolitica and Y. pseudotuberculosis at Public Health England will facilitate the monitoring of animal-to-human transmission of these important foodborne pathogens in the UK and improve public health surveillance of the pathogenic lineages.

Objective: To investigate the antimicrobial resistance and molecular epidemiology of foodborne Yersinia ( Y .) enterocolitica in Pudong New District of Shanghai. Methods: Four kinds of raw food samples were collected in retail circulation sites in Pudong from 2012 to 2016. Cold enrichment method was used to isolate Y. enterocolitica and further detection of biotype, serotype, virulent genes, antimicrobial susceptibility of the isolates and pulsed field gel electrophoresis (PFGE) were conducted. Results: A total of 3 900 raw food samples were collected during this period, including poultry product ( n =590), livestock product ( n =1 074), aquatic product ( n =1 488), vegetable ( n =748), in which 111 (2.8 % ) were contaminated by Y. enterocolitica . The detection rates of Y. enterocolitica in poultry product samples (5.3 % , 31/590) and livestock product samples (4.5 % , 48/1 074) were higher than those in aquatic product samples (1.6 % , 24/1 488) and vegetable samples (1.1 % , 8/748). The predominant biotype was 1A (95.5 % ) and predominant serotype was O∶8 (42.3 % ). All the strains lacked ail , ystA, yadA and virF genes, which encoded pathogenic Y. enterocolitica . Seventy six (68.5 % ) strains harbored ystB gene, in which 35 (31.5 % ) belonged to 1A/O∶8/ ystB pattern. Most strains were resistant to ampicillin (74.8 % ) and amoxicillin/clavulanic acid (70.3 % ), and non-sensitive rate to Cefoxitin was over 50.0 % . No third generation cephalosporin or fluoroquinolone resistant strains were detected, but 38.7 % (43/111) strains were multidrug resistant (MDR). Serotype O∶8 and O∶5 strains had 44 and 18 PFGE patterns, respectively. Conclusions: The main foodborne exposure sources of Y. enterocolitica in raw food were poultry and livestock products in Pudong New District. 1A/O∶8/ ystB was the predominant pattern with potential pathogenicity despite lacks of typical pathogenic virulent genes. The antimicrobial resistant rates of Y. enterocolitica were at a low level, but MDR strains still existed. Molecular types of the isolates showed highly genetic diversity.

Yersinia enterocolitica, a member of the Enterobacteriaceae family, is a zoonotic agent that causes gastrointestinal diseases and some extraintestinal disorders in humans. Y. enterocolitica ssp. palearctica bioserotype 4/O:3 is the primary pathogenic bioserotype in Europe, where it has a high public health relevance. The isolation and identification of Y. enterocolitica from various sources on selective media have been seldom successful due to several reasons. In an attempt to overcome the problems associated with traditional culture-based methods, we developed a single duplex PCR assay for the detection of Y. enterocolitica ssp. palearctica bioserotype 4/O:3 using DNA extracted from a source. We combined the primer for tufA (elongation factor Tu) with the primer for rfbC (the biosynthesis of the O side chain) in one single reaction, which showed good results when we analyzed 88 Yersinia strains and when it was tested in the DNA from stool samples of two groups of pregnant women, one comprising HIV-positive women and the other comprising of HIV-negative women. Furthermore, the duplex PCR assay was found to be 16 times better in detecting Yersinia spp. in stool samples than the culture-based method. In addition, it was found to be a rapid screening method for the detection of Y. enterocolitica serotype O:3, and it could still detect other Y. enterocolitica serotypes and Yersinia species as well. We anticipate that the duplex PCR assay could be a useful tool for hospital and veterinary surveillance studies on Yersinia worldwide., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The aims of the present study were to determine Yersinia enterocolitica prevalence in finishing pigs and piglets at slaughter and to characterize the isolates in terms of bioserotype, virulence profile, antimicrobial susceptibility and genetic diversity. During the years 2013-2014, nine pig slaughterhouses placed in Sardinia (Italy) were visited twice, in order to collect animal samples and scalding water. Overall, 609 samples respectively of tonsils (126), colon content (161), mesenteric lymph nodes (161) and carcass surfaces (161) were collected from 126 finishing pigs and 35 piglets. Moreover, 18 scalding water samples were collected. Samples were analyzed for the detection of Y. enterocolitica according to ISO 10273-2003 standard (with some modifications). With regard to finishing pigs, Y. enterocolitica was detected in 11.9% of colon content samples, 3.2% of tonsils and 2.4% of lymph nodes. In piglets, Y. enterocolitica prevalence was 8.6% in colon content and 2.8% lymph nodes samples. Y. enterocolitica was not detected from carcass surface samples of both finishing pigs and piglets and from scalding water samples. Isolates were bio- and serotyped, tested for the presence of four virulence genes by PCR (ail, ystA, ystB and inv) and for antimicrobial resistance by disc-diffusion method. Among 47 confirmed isolates, 33 (70.2%) belonged to bio-serotype 4:O3, 7 (14.9%) to bio-serotype 2/O:5 and 7 (14.9%) to bio-serotype 1A. Bio-serotype 1A was detected only in isolates of piglets' samples. In bio-serotype 4/O:3 isolates the most common virulence genes were ystA (97.0%), ail (84.8%) and inv (78.8%). In bio-serotype 2/O:5, ail, inv and ystA genes were detected in all of the isolates. All bio-serotype 1A isolates were ystB positive (lacking ail, inv and ystA). All isolates were susceptible to cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, enrofloxacin, gentamicin, nalidixic acid, sulphonamide, tetracycline and trimethoprim-sulphametoxazole. Resistances to ampicillin and cefalothin were the most common (100%), followed by amoxicillin/clavulanic acid (83.0%) and streptomycin (4.3%). Resistance to amoxicillin/clavulanic acid was detected in 57% of bio-serotype 4/O:3 isolates, 71% of bio-serotype 1A and 100% of bio-serotype 2/O:5 isolates. Two bio-serotype 4/O:3 isolates (6%) were resistant to streptomycin. Thirty-two pathogenic Y. enterocolitica isolates were tested by NotI-PFGE, which identified 5 patterns among bio-serotype 4/O:3 isolates and 2 patterns among bio-serotype 2/O:5 isolates. This study provides epidemiological data about human pathogenic Y. enterocolitica and highlight the role of pigs as a potential source of infection for the consumers in Sardinia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We show that Yersinia enterocolitica strain Ye9 (bio-serotype 2/O:9) utilizes heme-containing molecules as an iron source. The Ye9 genome contains two multigenic clusters, hemPRSTUV -1 and hemPRST -2, encoding putative heme receptors HemR1 and HemR2, that share 62% amino acid identity. Expression of these proteins in an Escherichia coli mutant defective in heme biosynthesis allowed this strain to use hemin and hemoglobin as a source of porphyrin. The hemPRSTUV -1 and hemPRST -2 clusters are organized as operons, expressed from the p hem-1 and weaker p hem-2 promoters, respectively. Expression of both operons is negatively regulated by iron and the iron-responsive transcriptional repressor Fur. In addition, OmpR, the response regulator of two component system (TCSs) EnvZ/OmpR, represses transcription of both operons through interaction with binding sequences overlapping the -35 region of their promoters. Western blot analysis of the level of HemR1 in ompR, fur , and ompRfur mutants, showed an additive effect of these mutations, indicating that OmpR may regulate HemR expression independently of Fur. However, the effect of OmpR on the activity of the p hem-1 promoter and on HemR1 production was observed in both iron-depleted and iron-replete conditions, i.e., when Fur represses the iron-regulated promoter. In addition, a hairpin RNA thermometer, composed of four uracil residues (FourU) that pair with the ribosome-binding site in the 5'-untranslated region (5'-UTR) of hemR1 was predicted by in silico analysis. However, thermoregulated expression of HemR1 could not be demonstrated. Taken together, these data suggest that Fur and OmpR control iron/heme acquisition via a complex mechanism based on negative regulation of hemR1 and hemR2 at the transcriptional level. This interplay could fine-tune the level of heme receptor proteins to allow Y. enterocolitica to fulfill its iron/heme requirements without over-accumulation, which might be important for pathogenic growth within human hosts.

Background: Yersinia enterocolitica is widespread within the humans, pigs and wild boars. The low isolation rate of Y. enterocolitica from food or environmental and clinical samples may be caused by limited sensitivity of culture methods. The main goal of present study was identification of presumptive Y. enterocolitica isolates using MALDI TOF MS. The identification of isolates may be difficult due to variability of bacterial strains in terms of biochemical characteristics. This work emphasizes the necessity of use of multiple methods for zoonotic Y. enterocolitica identification., Results: Identification of Y. enterocolitica isolates was based on MALDI TOF MS, and verified by VITEK ® 2 Compact and PCR. There were no discrepancies in identification of all human' and pig' isolates using MALDI TOF MS and VITEK ® 2 Compact. However three isolates from wild boars were not decisively confirmed as Y. enterocolitica. MALDI TOF MS has identified the wild boar' isolates designated as 3dz, 4dz, 8dz as Y. enterocolitica with a high score of matching with the reference spectra of MALDI Biotyper. In turn, VITEK ® 2 Compact identified 3dz and 8dz as Y. kristensenii, and isolate 4dz as Y. enterocolitica. The PCR for Y. enterocolitica 16S rDNA for these three isolates was negative, but the 16S rDNA sequence analysis identified these isolates as Y. kristensenii (3dz, 4dz) and Y. pekkanenii (8dz). The wild boar' isolates 3dz, 4dz and 8dz could not be classified using biotyping. The main bioserotype present within pigs and human faeces was 4/O:3. It has been shown that Y. enterocolitica 1B/O:8 can be isolated from human faeces using ITC/CIN culturing., Conclusion: The results of our study indicate wild boars as a reservoir of new and atypical strains of Yersinia, for which protein and biochemical profiles are not included in the MALDI Biotyper or VITEK ® 2 Compact databases. Pigs in the south-west Poland are the reservoir for pathogenic Y. enterocolitica strains. Four biochemical features included in VITEK ® 2 Compact known to be common with Wauters scheme were shown to produce incompatible results, thus VITEK ® 2 Compact cannot be applied in biotyping of Y. enterocolitica.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

Contamination of food by chemicals or pathogenic bacteria may cause particular illnesses that are linked to food consumption, commonly referred to as foodborne diseases. Bacteria are present in/on various foods products, such as fruits, vegetables and ready-to-eat products. Bacteria that cause foodborne diseases are known as foodborne pathogens (FBPs). Accurate detection methods that are able to reveal the presence of FBPs in food matrices are in constant demand, in order to ensure safe foods with a minimal risk of causing foodborne diseases. Here, a multiplex PCR-based Illumina sequencing method for FBP detection in food matrices was developed. Starting from 25 bacterial targets and 49 selected PCR primer pairs, a primer collection called foodborne pathogen - panel (FPP) consisting of 12 oligonucleotide pairs was developed. The FPP allows a more rapid and reliable identification of FBPs compared to classical cultivation methods. Furthermore, FPP permits sensitive and specific FBP detection in about two days from food sample acquisition to bioinformatics-based identification. The FPP is able to simultaneously identify eight different bacterial pathogens, i.e. Listeria monocytogenes, Campylobacter jejuni, Campylobacter coli, Salmonella enterica subsp. enterica serovar enteritidis, Escherichia coli, Shigella sonnei, Staphylococcus aureus and Yersinia enterocolitica, in a given food matrix at a threshold contamination level of 10 1 cell/g. Moreover, this novel detection method may represent an alternative and/or a complementary approach to PCR-based techniques, which are routinely used for FBP detection, and could be implemented in (parts of) the food chain as a quality check., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

Pathogenic Yersinia enterocolitica is widely distributed in China where the primary bio-serotypes are 3/O: 3 and 2/O: 9. Recently, the distribution of 2/O: 9 strains are being gradually replaced by 3/O: 3 strains where presently 3/O: 3 strains are the major pathogenic Y. enterocolitica in China. To identify the growth conditions and cytokines induced by Y. enterocolitica and providing some clues for this shift, we performed competitive growth in vitro and in vivo for these two bio-serotype strains; and we also compared the cytokines induced by them in infected BALB/C mice. We found 2/O: 9 strains grew more in vitro , while 3/O: 3 strains grew more in vivo regardless of using single cultures or mixed cultures. The cytokines induced by the two strains were similar: interleukin-6 (IL-6), IL-9, IL-13, granulocyte colony-stimulating factor (G-CSF), chemokines (KC), monocyte chemotactic protein 1 (MCP-1), macrophage inflammation protein-1α (MIP-1α), tumor necrosis factor-α (TNF-α), and RANTES were statistically up-regulated upon activation of normal T cells compared to the control. The cytokine values were higher in mixed infections than in single infections except for IL-6, G-CSF, and KC. The data illustrated the different growth of pathogenic Y. enterocolitica bio-serotype 3/O: 3 and 2/O: 9 in vitro and in vivo , and the cytokine changes induced by the two strains in infected BALB/C mice. The growth comparisons of two strains maybe reflect the higher pathogenic ability or resistance to host immune response for Y. enterocolitica bio-serotype 3/O: 3 and maybe it as one of the reason for bacteria shift.

In the spring of 2015, we experienced a cluster of 4 sporadic cases of yersiniosis in children in Nagano prefecture, a rural area of Japan. Two patients developed appendicitis-like episodes; one had acute gastroenteritis, and the other had bacteremia associated with liver abscess. The causative agent of these infections was Yersinia enterocolitica serogroup O:8. None of the patients had an underlying illness, and all have recovered completely. The patients were neither socially nor geographically related to each other. These 4 consecutive cases suggest that Y. enterocolitica O:8 has spread substantially in the middle part of Japan, and that this virulent strain might be more common than previously reported in our country.

In bacteria, the RNA chaperone Hfq enables pairing of small regulatory RNAs with their target mRNAs and therefore is a key player of post-transcriptional regulation network. As a global regulator, Hfq is engaged in the adaptation to external environment, regulation of metabolism and bacterial virulence. In this study we used RNA-sequencing and quantitative proteomics (LC-MS/MS) to elucidate the role of this chaperone in the physiology and virulence of Yersinia enterocolitica serotype O:3. This global approach revealed the profound impact of Hfq on gene and protein expression. Furthermore, the role of Hfq in the cell morphology, metabolism, cell wall integrity, resistance to external stresses and pathogenicity was evaluated. Importantly, our results revealed that several alterations typical for the hfq-negative phenotype were due to derepression of the transcriptional factor RovM. The overexpression of RovM caused by the loss of Hfq chaperone resulted in extended growth defect, alterations in the lipid A structure, motility and biofilm formation defects, as well as changes in mannitol utilization. Furthermore, in Y. enterocolitica RovM only in the presence of Hfq affected the abundance of RpoS. Finally, the impact of hfq and rovM mutations on the virulence was assessed in the mouse infection model., (© 2016 John Wiley & Sons Ltd.)

Background: Enteropathogenic 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 Findings: From 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/significance: This 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., Competing Interests: The authors have declared that no competing interests exist.

Yersinia enterocolitica is an important foodborne pathogen, but the prevalence in food is underestimated due to drawbacks in the detection methods. Problems arise from the low concentration of pathogenic strains present in food samples, similarities with other Enterobacteriaceae and Y. enterocolitica-like species and the heterogeneity of Y. enterocolitica as it comprises both pathogenic and non-pathogenic isolates. New rapid, cost-effective and more sensitive culture media and molecular techniques have been developed to overcome the drawbacks of conventional culture methods. Recent molecular subtyping methods have been applied to Y. enterocolitica strains to track infection sources and to investigate phylogenetic relationships between different Yersinia strains. Further application of modern subtyping tools such as WGS in a variety of bioserotypes, and comparison with other members of the genus will help to better understanding of the virulence determinants of pathogenic Y. enterocolitica, its mechanisms to cope in the host environments, and can contribute to the development of more specific detection and typing strategies.

Yersiniosis is the third most common reported zoonoses in Europe, with Y. enterocolitica and Y. pseudotuberculosis responsible for 98.66% and 0.94% of the confirmed human cases in 2013. From June 2013 to October 2014, 201 pigs at slaughter belonging to 67 batches were tested for Y. enterocolitica and Y. pseudotuberculosis in tonsils. Diaphragm muscle samples were tested for antibodies against Yersinia by a commercially available ELISA test. Y. enterocolitica 4/O:3 was detected in 55/201 pig tonsils (27.4%; 95% CI 23.1-37.1). The positive pigs came from 38/67 batches (56.7%) and were reared in 36/61 farms (59.0%). There was no statistical difference between farrow-to-finish and finishing farms. The mean count of Y. enterocolitica was 3.56±0.85log10CFU/g with a minimum of 2.0log10CFU/g and a maximum of 4.78log10CFU/g. Y. pseudotuberculosis was isolated from 4/201 pig tonsils (2.0%; 95% CI 0.0-4.5). Three isolates belonged to serotype O:3 and one to serotype O:1. The positive pigs belonged to 4/67 batches (6.0%) and came from finishing farms only. Y. pseudotuberculosis could be enumerated in one sample only (4.27log10CFU/g). The ELISA test demonstrated that 56.1% of the meat juice samples were positive for Yersinia antibodies. Serological positivity was found in 67.9% (36/53) of the Y. enterocolitica- and 75.0% (3/4) of the Y. pseudotuberculosis positive pigs. A significant association was found between serological results and the presence of Y. enterocolitica in tonsils (OR=1.97, p=0.044). All the Y. enterocolitica 4/O:3 isolates were susceptible to amoxicillin-clavulanic acid, gentamicin, ceftazidime, ertapenem and meropenem, 94.5% to cefotaxime, 89.1% to kanamycin and 78.2% to tetracycline. The highest resistance rates were observed for ampicillin (100%), sulphonamides (98.2%) and streptomycin (78.2%). Y. pseudotuberculosis strains were sensitive to all the antimicrobials tested, i.e. amoxicillin, amoxicillin/clavulanic acid, azithromycin, cephalothin, cefoxitin, ceftriaxone, ciprofloxacin, nalidixic acid, sulphonamide, tetracycline and ticarcillin. The study shows that Italian fattening pigs are frequently infected with human pathogenic Y. enterocolitica 4/O:3. Although the isolation rate is slightly lower than in other European countries, the serological test demonstrates that the infection is widespread among pig population. In fact, seroprevalence is similar to other EU countries. The detection of Y. pseudotuberculosis serotypes O:1 and O:3 in pig tonsils is of concern. Since tonsils may represent a contamination source for pig meat at slaughter, further studies regarding human infections by both microbial species are strongly recommended., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Yersinia enterocolitica is currently divided into two subspecies: subsp. enterocolitica including highly pathogenic strains of biotype 1B and subsp. palearctica including nonpathogenic strains of biotype 1A and moderately pathogenic strains of biotypes 2-5. In this work, we characterized 162 Y. enterocolitica strains of biotype 1A and 50 strains of biotypes 2-4 isolated from human, animal and food samples by restriction fragment length polymorphism using the HindIII restriction enzyme. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). In all the analyses, biotype 1A strains formed a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and from the strains of biotypes 2-4 of Y. enterocolitica subsp. palearctica. Based on these results, biotype 1A strains considered nonpathogenic should not be included in subspecies palearctica containing pathogenic strains of biotypes 2-5., Significance and Impact of the Study: Yersinia enterocolitica strains are currently divided into six biotypes and two subspecies. Strains of biotype 1A, which are phenotypically and genotypically very heterogeneous, are classified as subspecies palearctica. In this study, European Y. enterocolitica 1A strains isolated from both human and nonhuman sources were characterized using restriction fragment length polymorphism and multilocus sequence analysis. The European biotype 1A strains formed a separate group, which differed from strains belonging to subspecies enterocolitica and palearctica. This may indicate that the current division between the two subspecies is not sufficient considering the strain diversity within Y. enterocolitica., (© 2016 The Society for Applied Microbiology.)

Yersinia enterocolitica is the most diverse species among the Yersinia genera and shows more polymorphism, especially for the non-pathogenic strains. Individual non-pathogenic Y. enterocolitica strains are wrongly identified because of atypical phenotypes. In this study, we isolated an unusual Y. enterocolitica strain LC20 from Rattus norvegicus. The strain did not utilize urea and could not be classified as the biotype. API 20E identified Escherichia coli; however, it grew well at 25 °C, but E. coli grew well at 37 °C. We analyzed the genome of LC20 and found the whole chromosome of LC20 was collinear with Y. enterocolitica 8081, and the urease gene did not exist on the genome which is consistent with the result of API 20E. Also, the 16 S and 23 SrRNA gene of LC20 lay on a branch of Y. enterocolitica. Furthermore, the core-based and pan-based phylogenetic trees showed that LC20 was classified into the Y. enterocolitica cluster. Two plasmids (80 and 50 k) from LC20 shared low genetic homology with pYV from the Yersinia genus, one was an ancestral Yersinia plasmid and the other was novel encoding a number of transposases. Some pathogenic and non-pathogenic Y. enterocolitica-specific genes coexisted in LC20. Thus, although it could not be classified into any Y. enterocolitica biotype due to its special biochemical metabolism, we concluded the LC20 was a Y. enterocolitica strain because its genome was similar to other Y. enterocolitica and it might be a strain with many mutations and combinations emerging in the processes of its evolution.

In May 2014, a cluster of Yersinia enterocolitica (YE) O9 infections was reported from a military base in northern Norway. Concurrently, an increase in YE infections in civilians was observed in the Norwegian Surveillance System for Communicable Diseases. We investigated to ascertain the extent of the outbreak and identify the source in order to implement control measures. A case was defined as a person with laboratory-confirmed YE O9 infection with the outbreak multilocus variable-number tandem repeat analysis (MLVA)-profile (5-6-9-8-9-9). We conducted a case-control study in the military setting and calculated odds ratios (OR) using logistic regression. Traceback investigations were conducted to identify common suppliers and products in commercial kitchens frequented by cases. By 28 May, we identified 133 cases, of which 117 were linked to four military bases and 16 were civilians from geographically dispersed counties. Among foods consumed by cases, multivariable analysis pointed to mixed salad as a potential source of illness (OR 10.26; 95% confidence interval (CI): 0.85-123.57). The four military bases and cafeterias visited by 14/16 civilian cases received iceberg lettuce or radicchio rosso from the same supplier. Secondary transmission cannot be eliminated as a source of infection in the military camps. The most likely source of the outbreak was salad mix containing imported radicchio rosso, due to its long shelf life. This outbreak is a reminder that fresh produce should not be discounted as a vehicle in prolonged outbreaks and that improvements are still required in the production and processing of fresh salad products., Competing Interests: Conflicts of Interest: None declared., (This article is copyright of The Authors, 2016.)

Yersinia enterocolitica is a zoonotic pathogen and a common cause of gastroenteritis in humans. The species is composed of six diverse phylogroups, of which strains of phylogroup 1 are considered non-pathogenic to mammals due to the lack of the major virulence plasmid pYV, and their lack of virulence in a mouse infection model. In the present report we present data examining the pathogenicity of strains of Y. enterocolitica across all six phylogroups in a Galleria mellonellla model. We have demonstrated that in this model strains of phylogroup 1 exhibit severe pathogenesis with a lethal dose of as low as 10 c.f.u., that this virulence is an active process and that flagella play a major role in the virulence phenotype. We have also demonstrated that the complete lack of virulence in Galleria of the mammalian pathogenic phylogroups is not due to carriage of the pYV virulence plasmid. Our data suggest that all Y. enterocolitica can be pathogenic, which may be a reflection of the true natural habitat of the species, and that we may need to reconsider the eco-evo perspective of this important bacterial species.

Aims: 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., Methods and Results: 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., Conclusions: 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., Significance and Impact of the Study: 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., (© 2016 The Society for Applied Microbiology.)

A total of 253 multiple-locus variable-number tandem-repeat analysis (MLVA) types among 634 isolates were discovered while studying the genetic diversity of porcine Yersinia enterocolitica 4/O:3 isolates from eight different European countries. Six variable-number tandem-repeat (VNTR) loci V2A, V4, V5, V6, V7, and V9 were used to study the isolates from 82 farms in Belgium (n = 93, 7 farms), England (n = 41, 8 farms), Estonia (n = 106, 12 farms), Finland (n = 70, 13 farms), Italy (n = 111, 20 farms), Latvia (n = 66, 3 farms), Russia (n = 60, 10 farms), and Spain (n = 87, 9 farms). Cluster analysis revealed mainly country-specific clusters, and only one MLVA type consisting of two isolates was found from two countries: Russia and Italy. Also, farm-specific clusters were discovered, but same MLVA types could also be found from different farms. Analysis of multiple isolates originating either from the same tonsils (n = 4) or from the same farm, but 6 months apart, revealed both identical and different MLVA types. MLVA showed a very good discriminatory ability with a Simpson's discriminatory index (DI) of 0.989. DIs for VNTR loci V2A, V4, V5, V6, V7, and V9 were 0.916, 0.791, 0.901, 0.877, 0.912, and 0.785, respectively, when studying all isolates together, but variation was evident between isolates originating from different countries. Locus V4 in the Spanish isolates and locus V9 in the Latvian isolates did not differentiate (DI 0.000), and locus V9 in the English isolates showed very low discriminatory power (DI 0.049). The porcine Y. enterocolitica 4/O:3 isolates were diverse, but the variation in DI demonstrates that the well discriminating loci V2A, V5, V6, and V7 should be included in MLVA protocol when maximal discriminatory power is needed.

The prevalence of human pathogenic Yersinia enterocolitica was determined in tonsil and intestinal content samples from 388 healthy fattening pigs at the four biggest Finnish slaughterhouses. These slaughterhouses process 73% of pigs in Finland. Tonsil samples were tested by PCR targeted for yadA, and intestinal samples were cultured. All pathogenic Y. enterocolitica isolates represented bioserotype 4/O:3. The prevalence of Y. enterocolitica in tonsil samples was 60% (95% confidence limit, 55.4 to 65.1%), and its prevalence in intestinal samples was 26% (95% confidence limit, 22.1 to 31.2%). The prevalence of Y. enterocolitica in tonsil and intestinal samples varied between the four slaughterhouses. The tonsil prevalence of Y. enterocolitica was higher in slaughterhouse B, and the prevalence in intestinal content was higher in slaughterhouse C. There were more positive results in both tonsil and intestinal samples in pigs coming from fattening farms than in pigs coming from farrowing-and-fattening farms. A seasonal variation was observed in the prevalence of Y. enterocolitica in intestinal samples, with the highest prevalence during July and August, but no seasonal variation was detected in tonsil samples.

The aim of the study was to monitor Y. enterocolitica strains from free-living animals captured during 2011-2014 hunting seasons in Poland using warm (ITC) and cold (PSB) enrichment and molecular examination. Over 1600 samples have been cultured. After ITC/PSB enrichment 237 strains presenting features characteristic for Y. enterocolitica were isolated. Molecular examination using multiplex PCR revealed 140 isolates from PSB and 78 from ITC. The concentration of pathogenic Yersinia in asymptomatic carriers is low and the PCR detection should be preceded by bacteriological examination.

Yersinia enterocolitica is a common zoonotic pathogen and facultative intracellular bacterium which can survive within blood cells. Cattle and horses are considered a reservoir of Y. enterocolitica which often causes several serious syndromes associated with yersiniosis such as abortions, premature births or infertility. The aim of our investigation was to determine the vitality of Y. enterocolitica O:9 strains (Ye9) in bovine and horse sera (NBS and NHrS) and explain the role of outer membrane proteins (OMPs) in serum resistance of these bacteria. Our previous studies demonstrated moderate human serum (NHS) resistance of the wild type Ye9 strain, whereas mutants lacking YadA, Ail or OmpC remained sensitive to the bactericidal activity of NHS. The present study showed that the wild type of Ye9 strain was resistant to the bactericidal activity of both NHrS and NBS, while Ye9 mutants lacking the YadA, Ail and OmpC proteins were sensitive to NHrS and NBS as well as to NHS. The mechanisms of complement activation against Ye9 strains lacking Ail and YadA were distinguished, i.e. activation of the classical/lectin pathways decisive in the bactericidal mechanism of complement activation of NBS, parallel activation of the classical/lectin and alternative pathways of NHrS. In this research the mechanism of independent activation of the classical/lectin or the alternative pathway of NBS and NHrS against Ye9 lacking OmpC porin was also established. The results indicate that serum resistance of Ye9 is multifactorial, in which extracellular structures, i.e. outer membrane proteins (OMPs) such as Ail, OmpC or YadA, play the main role.

The outer membrane protein A (OmpA) is one of the intra-species conserved proteins with immunogenicity widely found in the family of Enterobacteriaceae. Here we first confirmed OmpA is conserved in the three pathogenic Yersinia: Yersinia pestis, Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica, with high homology at the nucleotide level and at the amino acid sequence level. The identity of ompA sequences for 262 Y. pestis strains, 134 Y. pseudotuberculosis strains and 219 pathogenic Y. enterocolitica strains are 100%, 98.8% and 97.7% similar. The main pattern of OmpA of pathogenic Yersinia are 86.2% and 88.8% identical at the nucleotide and amino acid sequence levels, respectively. Immunological analysis showed the immunogenicity of each OmpA and cross-immunogenicity of OmpA for pathogenic Yersinia where OmpA may be a vaccine candidate for Y. pestis and other pathogenic Yersinia., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Neutrophil extracellular trap (NET) formation is described as a tool of the innate host defence to fight against invading pathogens. Fibre-like DNA structures associated with proteins such as histones, cell-specific enzymes and antimicrobial peptides are released, thereby entrapping invading pathogens. It has been reported that several bacteria are able to degrade NETs by nucleases and thus evade the NET-mediated entrapment. Here we studied the ability of three different Yersinia serotypes to induce and degrade NETs. We found that the common Yersinia enterocolitica serotypes O:3, O:8 and O:9 were able to induce NETs in human blood-derived neutrophils during the first hour of co-incubation. At later time points, the NET amount was reduced, suggesting that degradation of NETs has occurred. This was confirmed by NET degradation assays with phorbol-myristate-acetate-pre-stimulated neutrophils. In addition, we found that the Yersinia supernatants were able to degrade purified plasmid DNA. The absence of Ca(2+) and Mg(2+) ions, but not that of a protease inhibitor cocktail, completely abolished NET degradation. We therefore postulate that Y. enterocolitica produces Ca(2+)/Mg(2+)-dependent NET-degrading nucleases as shown for some Gram-positive pathogens., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Yersinia enterocolitica is a foodborne pathogen that is very rarely encountered in Turkey. In this work, several human, porcine, and environmental samples collected from Izmir region in Turkey were examined for the presence of Y. enterocolitica using different cultivation and enrichment methods. A total of nine pathogenic Y. enterocolitica strains were isolated; five strains from pig stool and manure samples and four strains from waste water samples. On the other hand, no Y. enterocolitica was isolated from human diarrheal stool samples (n = 102) and from 12 gulf, canal, municipal pool, and well water samples. Biochemical and serological characterization of the nine Y. enterocolitica strains revealed that they belonged to three different bioserotypes: 4/O:3, 2/O:9, and 2/O:5,27. All the strains were deemed pathogenic based on virulence factor-specific PCR analysis. Detection of pathogenic Y. enterocolitica strains from the pig and waste water samples from the Izmir region indicates that Y. enterocolitica is a potential risk for public health.

Possession of mechanisms for iron acquisition and its storage enhances the ability of the bacteria to survive in the iron-limiting environment of the host. In this study, 81 strains of Yersinia enterocolitica biovar 1A isolated from various clinical (n = 51) and non-clinical (n = 30) sources were investigated for the presence of the genes related to iron acquisition and storage. Important genes which were present in more than 85% of the strains included hasA, foxA, bfr, bfd, ftnA, and hmsT as well as the fhuCDB, fepBDGCfesfepA, feoAB, yfuABCD, hemPRSTUV, and hmsHFRS gene clusters. Majority of these genes is being reported for the first time in biovar 1A strains and showed significant homology with genes present in the known pathogenic biovars of Y. enterocolitica. However, no significant difference was observed in the distribution of iron acquisition and storage-related genes among clinical and non-clinical biovar 1A strains. Thus, it may be suggested that the presence of iron acquisition and storage-related genes per se might not be responsible for the supposedly better ability of clinical biovar 1A strains to cause infections in humans. However, in the backdrop of this data, the need to undertake functional studies are highly recommended., (© 2015 APMIS. Published by John Wiley & Sons Ltd.)

A cross-sectional survey was undertaken to determine the overall prevalence of enteropathogenic Yersinia spp. in the tonsils, feces and on carcasses of pigs at slaughter. Moreover, factors associated with Yersinia contamination of freshly eviscerated pig carcasses were studied. Yersinia enterocolitica serotype O:3 was isolated from the tonsils and feces of 55.3% and 25.6% of pigs, and Y. pseudotuberculosis from 1.4% and 0.6%, respectively. The pathogens were also recovered from 39.7% of carcass surfaces post-evisceration. The highest prevalence was found at the mandibular region (28.9%), followed by the sternal region (16.4%), pelvic duct (7.8%), and split surface near the sacral vertebrae (6.9%). Regarding the quantification of the pathogen, the median concentration of pathogenic Y. enterocolitica was 4.14l og10 CFU/g in tonsils with countable numbers (n=143) and 2.80 log10 CFU/g for fecal samples with countable numbers (n=26). The quantitative load on the carcass surface was generally low as the majority of the carcass samples (97.0%) had Yersinia concentrations below the detection limit of enumeration (<1.30 log10 CFU/100 cm(2)). The initial presence of Y. enterocolitica in the tonsils and/or feces was significantly associated with carcass contamination at all sampled areas. Other risk factors for carcass contamination are the splitting of the head together with the carcass, and incision of the tonsils during removal of the pluck. Small adaptations in slaughter practices and the training of slaughterhouse personnel to respect basic hygienic instructions may diminish carcass contamination with enteropathogenic Yersinia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Yersiniosis is strongly associated with the consumption of pork contaminated with enteropathogenic Yersinia enterocolitica, which is harbored by domestic pigs without showing clinical signs of disease. In contrast to data on Y. enterocolitica isolated from conventionally reared swine, investigations into the occurrence of Y. enterocolitica in wild boars in Germany are rare. The objectives of the study were to get knowledge about these bacteria and their occurrence in wild boars hunted in northern Germany by isolation of the bacteria from the tonsils, identification of the bioserotypes, determination of selected virulence factors, macrorestriction analysis, multilocus sequence typing (MLST), and testing of antimicrobial susceptibility. Altogether, tonsils from 17.1% of 111 tested wild boars were positive for Y. enterocolitica by culture methods. All but two isolates belonged to biotype (BT) 1A, with the majority of isolates bearing a ystB nucleotide sequence which was revealed to have 85% identity to internal regions of Y. enterocolitica heat-stable enterotoxin type B genes. The remaining Y. enterocolitica isolates were identified to be BT 1B and did not carry the virulence plasmid. However, two BT 1A isolates carried the ail gene. Macrorestriction analysis and results from MLST showed a high degree of genetic diversity of the isolates, although the region where the samples were taken was restricted to Lower Saxony, Germany, and wild boars were shot during one hunting season. In conclusion, most Y. enterocolitica isolates from wild boars investigated in this study belonged to biotype 1A. Enteropathogenic Y. enterocolitica bioserotypes 4/O:3 and 2/O:9, usually harbored by commercially raised pigs in Europe, could not be identified., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)