Virulence Plasmid pYV Research Articles

Comparison of Yersinia enterocolitica DNA Methylation at Ambient and Host Temperatures.

Pathogenic bacteria recognize environmental cues to vary gene expression for host adaptation. Moving from ambient to host temperature, Yersinia enterocolitica responds by immediately repressing flagella synthesis and inducing the virulence plasmid (pYV)-encoded type III secretion system. In contrast, shifting from host to ambient temperature requires 2.5 generations to restore motility, suggesting a link to the cell cycle. We hypothesized that differential DNA methylation contributes to temperature-regulated gene expression. We tested this hypothesis by comparing single-molecule real-time (SMRT) sequencing of Y. enterocolitica DNA from cells growing exponentially at 22 °C and 37 °C. The inter-pulse duration ratio rather than the traditional QV scoring was the kinetic metric to compare DNA from cells grown at each temperature. All 565 YenI restriction sites were fully methylated at both temperatures. Among the 27,118 DNA adenine methylase (Dam) sites, 42 had differential methylation patterns, while 17 remained unmethylated regardless of the temperature. A subset of the differentially methylated Dam sites localized to promoter regions of predicted regulatory genes including LysR-type and PadR-like transcriptional regulators and a cyclic-di-GMP phosphodiesterase. The unmethylated Dam sites localized with a bias to the replication terminus, suggesting they were protected from Dam methylase. No cytosine methylation was detected at Dcm sites.

Prevalence, Genetic Homogeneity, and Antibiotic Resistance of Pathogenic Yersinia enterocolitica Strains Isolated from Slaughtered Pigs in Bulgaria.

Yersiniosis is the third most commonly reported foodborne zoonosis in the European Union. Here, we evaluated the prevalence of pathogenic Yersinia enterocolitica among healthy pigs (as a major reservoir) in a slaughterhouse in Bulgaria. A total of 790 tonsils and feces from 601 pigs were examined. Isolation and pathogenicity characterization was carried out by the ISO 10273:2003 protocol and Polymerase Chain Reaction (PCR), detecting the 16S rRNA gene, attachment and invasion locus (ail), Yersinia heat-stable enterotoxin (ystA), and Yersinia adhesion (yadA) genes. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE), and antimicrobial resistance by the standard disk diffusion method. Of all the pigs tested, 6.7% were positive for Y. enterocolitica. All isolates belonged to Y. enterocolitica bioserotype 4/O:3. ail, and ystA genes were detected in all positive strains (n = 43), while the plasmid Yersinia virulence plasmid (pYV) was detected in 41. High homogeneity was observed among the strains, with all strains susceptible to ceftriaxone, amikacin and ciprofloxacin, and resistant to ampicillin. In conclusion, a low prevalence of Y. enterocolitica 4/O:3 was found in healthy pigs slaughtered in Bulgaria, not underestimating possible contamination of pork as a potential risk to consumer health.

Cold Enrichment Methods for the Detection of Foodborne Yersiniosis: Friend or Foe?

Yersinia enterocolitica and Y. pseudotuberculosis are important causes of enteric illness worldwide. Rapid response to suspected foodborne outbreaks is hampered by the widespread use of cold enrichment methods that require incubation periods of 10–21 days. Although these species grow faster at elevated temperatures, part of the rationale for cold enrichment is that a key pathogenicity marker (pYV virulence plasmid) is said to be lost at elevated temperatures. Experimental data on this claim seems scarce. We previously described an approach involving an enrichment step at 37 °C for Yersinia detection, applied this approach to additional strains, and examined the presence of plasmids in reisolates, as well as those recovered in our original study. Plasmids were recovered from every reisolate examined; the presence of marker genes yadA and virF denoted the virulence plasmid in 10 of the 11 strains examined. Use of an enrichment step at 37 °C does not appear to promote loss of the pYV or other plasmids harboured by foodborne pathogenic Y. enterocolitica and Y. pseudotuberculosis; wider adoption of this approach may assist the development of more rapid detection methods.

Role of the Yersinia pseudotuberculosis Virulence Plasmid in Pathogen-Phagocyte Interactions in Mesenteric Lymph Nodes.

Yersinia pseudotuberculosis is an Enterobacteriaceae family member that is commonly transmitted by the fecal-oral route to cause infections. From the small intestine, Y. pseudotuberculosis can invade through Peyer's patches and lymph vessels to infect the mesenteric lymph nodes (MLNs). Infection of MLNs by Y. pseudotuberculosis results in the clinical presentation of mesenteric lymphadenitis. MLNs are important for immune responses to intestinal pathogens and microbiota in addition to their clinical relevance to Y. pseudotuberculosis infections. A characteristic of Y. pseudotuberculosis infection in MLNs is the formation of pyogranulomas. Pyogranulomas are composed of neutrophils, inflammatory monocytes, and lymphocytes surrounding extracellular microcolonies of Y. pseudotuberculosis. Key elements of the complex pathogen-host interaction in MLNs have been identified using mouse infection models. Y. pseudotuberculosis requires the virulence plasmid pYV to induce the formation of pyogranulomas in MLNs. The YadA adhesin and the Ysc-Yop type III secretion system (T3SS) are encoded on pYV. YadA mediates bacterial binding to host receptors, which engages the T3SS to preferentially translocate seven Yop effectors into phagocytes. The effectors promote pathogenesis by blocking innate immune defenses such as superoxide production, degranulation, and inflammasome activation, resulting in survival and growth of Y. pseudotuberculosis. On the other hand, certain effectors can trigger immune defenses in phagocytes. For example, YopJ triggers activation of caspase-8 and an apoptotic cell death response in monocytes within pyogranulomas that limits dissemination of Y. pseudotuberculosis from MLNs to the bloodstream. YopE can be processed as an antigen by phagocytes in MLNs, resulting in T and B cell responses to Y. pseudotuberculosis. Immune responses to Y. pseudotuberculosis in MLNs can also be detrimental to the host in the form of chronic lymphadenopathy. This review focuses on interactions between Y. pseudotuberculosis and phagocytes mediated by pYV that concurrently promote pathogenesis and host defense in MLNs. We propose that MLN pyogranulomas are immunological arenas in which opposing pYV-driven forces determine the outcome of infection in favor of the pathogen or host.

Evidence of Extensive Circulation of Yersinia enterocolitica in Rodents and Shrews in Natural Habitats from Retrospective and Perspective Studies in South Caucasus.

Yersinia enterocolitica culture-positive rodents and shrews were reported in different territories across Georgia during 14 of 17 years of investigations conducted for the period of 1981–1997. In total, Y. enterocolitica was isolated from 2052 rodents (15 species) and 33 shrews. Most isolates were obtained from Microtus arvalis, Rattus norvegicus, Mus musculus, and Apodemus spp. During the prospective study (2017−2019), isolates of Yersinia-like bacteria were cultured from 53 rodents collected in four parts of Georgia. All the Yersinia-like isolates were confirmed as Y. enterocolitica based on the API 20E and the BD Phenix50 tests. Whole-genome (WG) sequencing of five rodents and one shrew strain of Y. enterocolitica revealed that they possessed a set of virulence genes characteristic of the potentially pathogenic strains of biogroup 1A. All isolates lacked distinguished virulence determinants for YstA, Ail, TccC, VirF, and virulence plasmid pYV but carried the genes for YstB, YmoA, HemPR-HmuVSTU, YaxAB, PhlA, PldA, ArsCBR, and a flagellar apparatus. One strain contained a gene highly homologous to heat-labile enterotoxin, a chain of E. coli, a function not previously described for Y. enterocolitica. The WG single-nucleotide polymorphism-based typing placed the isolates in four distinct phylogenetic clusters.

Pathogenetic Value of pVM82 Plasmid of Yersinia Pseudotuberculosis, Causative Agent of Far Eastern Scarlet-Like Fever

The Far Eastern scarlet-like fever (FESLF) is the clinical-epidemic manifestation of pseudotuberculosis infection in humans is associated with a specific clonal lineage of the causative agent. Purpose of the study: to determine the pathogenetic value of the pVM82 plasmid of the FESLF causative agent, on the basis of a comparative analysis of the innate immunity cells metabolic activity and peculiarities of the inflammatory reaction during experimental infection, caused by different plasmid types of Yersinia pseudotuberculosis. Materials and Methods. The following Y. pseudotuberculosis plasmid types were used for the infection of mice: (1) H-5015 T+ strain (82+:48+) containing two plasmids pVM82 and pYV; (2) H-5015 T– strain (82+:48–) containing single pVM82 plasmid; (3) H-5013 T+ strain (48+) containing single pYV plasmid; (4) H-5013 T– plasmid-free strain (82–:48–). Peritoneal exudate cells and samples of liver, spleen and mesenteric lymph nodes were taken from infected animals at 1, 3, 5 and 7 days post infection. The functional state of the innate immunity cells was assessed by the activity of lactate dehydrogenase (LDH) and by the level of nitric oxide (NO) metabolites. The samples of target organs were embedded in paraffin, sections were stained with hematoxylin and eosin. Results. It was found that only Y. pseudotuberculosis strains containing pVM82 plasmid had a stimulating effect the innate immunity cells. The more pronounced increase in the LDH activity, an oxygen-dependent metabolism enzyme, and the level of NO metabolites occurred in response to infection of cells with bacteria containing single pVM82 plasmid, compared with plasmid type containing additional plasmid virulence pYV. In experimental animals infected with plasmid type pYV–: pVM82+ of Y. pseudotuberculosis, the disease pathology usually manifested less acute inflammation with a predominance of mononuclear cells and minor pathogen-associated destructive changes in organs. The mortality in animals infected with Y. pseudotuberculosis strains containing plasmid pVM82 (plasmid types 82+:48+ and 82+:48–) was lower compared to a strain carrying only the virulence plasmid pYV (plasmid type 48+),—40, 30 and 80%, respectively. Conclusions. Thus, the pVM82 plasmid of Y. pseudotuberculosis determined the effective immune defense of organism, mainly through nitroxide-dependent bactericidal mechanisms of innate immunity cells. Moreover, the restriction of a generalized inflammatory reaction and induction of granulomatous inflammation were associated with this plasmid.

Identification of a Novel Yersinia enterocolitica Strain from Bats in Association with a Bat Die-Off That Occurred in Georgia (Caucasus).

Yersinia entercolitica is a bacterial species within the genus Yersinia, mostly known as a human enteric pathogen, but also recognized as a zoonotic agent widespread in domestic pigs. Findings of this bacterium in wild animals are very limited. The current report presents results of the identification of cultures of Y. entercolitica from dead bats after a massive bat die-off in a cave in western Georgia. The growth of bacterial colonies morphologically suspected as Yersinia was observed from three intestine tissues of 11 bats belonging to the Miniopterus schreibersii species. These three isolates were identified as Y. enterocolitica based on the API29 assay. No growth of Brucella or Francisella bacteria was observed from tissues of dead bats. Full genomes (a size between 4.6–4.7 Mbp) of the Yersinia strains isolated from bats were analyzed. The phylogenetic sequence analyses of the genomes demonstrated that all strains were nearly identical and formed a distinct cluster with the closest similarity to the environmental isolate O:36/1A. The bat isolates represent low-pathogenicity Biotype 1A strains lacking the genes for the Ail, Yst-a, Ysa, and virulence plasmid pYV, while containing the genes for Inv, YstB, and MyfA. Further characterization of the novel strains cultured from bats can provide a clue for the determination of the pathogenic properties of those strains.

Function of pYV Plasmid on Biofilm Formation of Yersinia enterocolitica ERL032123 in the Presence of Ca2+

The effect of the virulence plasmid pYV and calcium ions on biofilm of Yersinia enterocolitica biofilm formation was determined using a microtiter plate assay. Loss of the pYV plasmid prevented biofilm formation and the presence of Ca2+ enhanced biofilm formation in cultures containing the pYV plasmid. Scanning electron microscopy supported the result from the microtiter plate assay showing that in the presence of Ca2+, the wild-type Y. enterocolitica strain formed a strong biofilm on a polycarbonate surface. The results implied that Ca2+ promotes Y. enterocolitica biofilm formation through the function of the pYV plasmid.

Inflammation induced by different plasmid types of russian <i>Yersinia pseudotuberculosis</i> strains

In the 2000s, a scientific interest to the Far Eastern scarlet-like fever (FESLF) mainly recorded In Russia and Japanwas remarkably increased. Such clinical and epidemic manifestation of human pseudotuberculosis is related to a certain bacterial clonal lineage characterized by a specific plasmid profile (pVM82, pYV48), sequence type (2ST) as well as the yadA gene allele (1st allele). In our study we examined features of inflammatory changes characterizing plasmidassociated pathogenicity of the FESLF pathogen. In addition, organ histopathology in experimental animals infected intraperitoneally with Y. pseudotuberculosis strains of the four plasmid types 48+:82+; 48+:82-; 48-:82+; 48-:82-; and 48-:82- was investigated. It was found that the mortality rate in animals infected with Y. pseudotuberculosis H-5015 strain (82+:48+) bearing two plasmids with a molecular weight of 82 and 48 MDa was 40%. A picture of diffuse pneumonia with moderate inflammatory infiltration in pulmonary tissue and changes in lymphoid organs characterizing immunodeficiency we observed as early as 3 days postinfection (p.i.). On the contrary, animals infected with Y. pseudotuberculosis H-5015 strain (82+:48) bearing a single plasmid 82 MDa pVM, mortality rate was 30%. A vascular reaction in the lungs and liver as well as deteriorated vascular destructive changes were revealed starting from day 3 and on day 57 days p.i., respectively, which was paralleled with perivascular infiltration mainly by mononuclear cells and focal pneumonia as well as a reaction of bronchialassociated lymphoid tissue and minimal organ destructive changes. On day 7 p.i., signs of granulomatous inflammation in the liver in a form of small mononuclear cell clusters and perivascular compact infiltrates were found. At all time points, lymphoid organ hyperplasia was noted. In case the infection caused by H-5013 strain (48+), the mortality rate was 80%. Inflammatory changes with dominant mononuclear cells were detected as early as 1 day p.i. associated with a picture of large focal bronchopneumonia, more pronounced in the non-survivor animals, also demonstrating signs of severe immunosuppression in the lymphoid organs. Starting from day 3 p.i., lymphoid hypoplasia in the spleen and lymph nodes was detected in all infected animals paralleled with pathogen-associated tissue damage in various organs. Animals infected with the plasmid-free H-5013 strain 48-) resulted in 25% mortality rate. Moreover, starting from day 3 p.i., a slight mononuclear inflammatory infiltration in the lungs and liver, a moderate giant cell reaction in the splenic pulp, and loose perinodal inflammatory infiltration in the lymph nodes were observed. Thus, while modeling infection caused by different plasmid types of Y. pseudotuberculosis, the data on differences in histopathology of changes in diverse organs regarding dynamics and generalization of the inflammatory response, as well as the severity of pathogen-associated damage in the lymphoid tissue were obtained. In case Y. pseudotuberculosis strains contained pVM82 plasmid with or without virulence plasmid pYV vs. single pYV-positive strains, an area of the inflammatory response as well as severity of immunosuppression manifested by splenic and lymph node delymphatization were decreased. It allowed to suggest that pVM82 plasmid found In Russia-originating Y. pseudotuberculosis strains might be implicated in limiting intensity of inflammatory response, bacterial dissemination and severity of lymphoid organ damage.

An Experimental Pipeline for Initial Characterization of Bacterial Type III Secretion System Inhibitor Mode of Action Using Enteropathogenic Yersinia.

Dozens of Gram negative pathogens use one or more type III secretion systems (T3SS) to disarm host defenses or occupy a beneficial niche during infection of a host organism. While the T3SS represents an attractive drug target and dozens of compounds with T3SS inhibitory activity have been identified, few T3SS inhibitors have been validated and mode of action determined. One issue is the lack of standardized orthogonal assays following high throughput screening. Using a training set of commercially available compounds previously shown to possess T3SS inhibitory activity, we demonstrate the utility of an experiment pipeline comprised of six distinct assays to assess the stages of type III secretion impacted: T3SS gene copy number, T3SS gene expression, T3SS basal body and needle assembly, secretion of cargo through the T3SS, and translocation of T3SS effector proteins into host cells. We used enteropathogenic Yersinia as the workhorse T3SS-expressing model organisms for this experimental pipeline, as Yersinia is sensitive to all T3SS inhibitors we tested, including those active against other T3SS-expressing pathogens. We find that this experimental pipeline is capable of rapidly distinguishing between T3SS inhibitors that interrupt the process of type III secretion at different points in T3SS assembly and function. For example, our data suggests that Compound 3, a malic diamide, blocks either activity of the assembled T3SS or alters the structure of the T3SS in a way that blocks T3SS cargo secretion but not antibody recognition of the T3SS needle. In contrast, our data predicts that Compound 4, a haloid-containing sulfonamidobenzamide, disrupts T3SS needle subunit secretion or assembly. Furthermore, we suggest that misregulation of copy number control of the pYV virulence plasmid, which encodes the Yersinia T3SS, should be considered as a possible mode of action for compounds with T3SS inhibitory activity against Yersinia.

EFFECT OF YERSINIA PSEUDOTUBERCULOSIS TOXINS ON THE BIOFILM FORMATION

Aim. To study the effect of heat-labile (HLTY) and heat-stable (HSTY) lethal toxins of the of Yersinia pseudotuberculosis on the formation of biofilms by these bacteria. Materials and methods. For the isolation of toxins and the investigation their ability to effect on the biofilm formation there were used the strain of Y. pseudotuberculosis 512 (pYV48Mjl. рУМ82МД) and strain 2517, carrying virulence plasmid pYV and lost it, correspondingly. Results. The stimulation of biofilm formation at 20°C by the strain 2517 (pYV+), carrying virulence plasmid as well as the strain 2517 (pYV-) without plasmid were observed In the presence of HLTY. At low positive temperature (6 - 8°C) HLTY reduces the amount of the formed biofilm. HSTY inhibited the biofilm formation by the both strains of Y. pseudotuberculosis tested during the incubation for 3 days at 20°C and 6 - 8°C. Moreover the extent of inhibition was decreased with temperature decreasing. Conclusion. The both of the Y. pseudotuberculosis protein toxins has been revealed to affect on the biofilm formation by Y. pseudotuberculosis bacteria, however, the impact of HLTY and HSTY in the processes of biofilm formation was shown to be different, and the mechanism of such action of toxinsis under way.

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

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

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

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

PLASMID-ASSOCIATED VIRULENCE OF YERSINIA PSEUDOTUBERCULOSIS AND INFECTIOUS PROCESS

Literature data regarding genetically-determined pathogenicity factors of Y pseudotuberculo- sis and associated manifestations of this infection caused by various plasmid types of the causative agent are generalized. Principal attention is given to features of cell-tissue alterations mediated by virulence plasmid pYV, as well as effects of pathogenicity of an understudied pVM82 plasmid present only in Y pseudotuberculosis sttains causing clinical-epidemic manifestation of the infec- tions as Far East scarlet-like fever (FESLF). The data obtained on the ability of far-eastern strains to produceYPMa super-antigenj Ypseudotuberculosis-derivative mitogenA, probablygive evidence on its key role in FESLF pathogenesis. Variability of damage of innate immunity cells and target- organs caused by various plasmid types of Y pseudotuberculosis by virulence could determine polymorphism of clinical-morphological manifestations of this infection. In-depth understanding of dependency of immune pathogenesis mechanisms of the disease on molecular characteristics of the causative agent opens up-perspectives of enhancement of diagnostics and prognosis of the severity of the course of pseudotuberculosis and yersiniosis in human in general.

All Yersinia enterocolitica are pathogenic: virulence of phylogroup 1 Y. enterocolitica in a Galleria mellonella infection model.

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.

Characterisation of Yersinia pseudotuberculosis isolated from animals with yersiniosis during 1996–2013 indicates the presence of pathogenic and Far Eastern strains in Italy

Yersinia pseudotuberculosis is a pathogen that infects both animals and humans worldwide. The epidemiology of infection caused by Y. pseudotuberculosis is poorly understood; however, its outbreaks have been traced back to a probable source in wildlife. This study aimed to characterise Y. pseudotuberculosis isolates collected from animals with yersiniosis. This study included 90 isolates of Y. pseudotuberculosis collected from different animals with yersiniosis between 1996 and 2013 in Italy. The isolates were tested for antimicrobial susceptibility and were biotyped. Genes associated with virulence plasmid pYV and those encoding O-antigen, high pathogenicity island (HPI), and superantigenic toxin (YPM) were determined by performing PCR. Pulsed-field gel electrophoresis (PFGE) was performed using NotI and SpeI enzymes, and 3 dendrograms were generated. No antibiotic resistance was found. The presence of pYV was shown in 57 out of 90 isolates. Virulence profiles of majority of the isolates indicated that they belonged to O:1a and O:1b serotypes, biotype 1, and genetic type 2. Isolates belonging to O:2a serotype were detected in sheep and cattle and were found to be associated (for the first time) with septicemia in hares. Y. pseudotuberculosis isolates belonging to O:5a and O:12–O13 serotypes were also detected in hares. To our knowledge, this is the first study to detect Y. pseudotuberculosis isolates belonging to the O:12–O13 serotype from a clinical case in Europe. Results of PFGE indicated that it was a reliable method for investigating the genetic relatedness of Y. pseudotuberculosis isolates. Thus, characterisation of Y. pseudotuberculosis infection in animals should be considered a possible tool for the surveillance of pseudotuberculosis.

Absence of YbeY RNase compromises the growth and enhances the virulence plasmid gene expression of Yersinia enterocolitica O:3.

YbeY was recently recognized as an endoribonuclease playing a role in ribosome biosynthesis. In Escherichia coli it functions as a single-strand-specific RNase that processes the 3' end of the 16S rRNA and is crucial for the late-stage 70S ribosome quality control system. Here we report that YbeY is not essential in Yersinia enterocolitica serotype O:3, yet its absence strongly compromised the bacterium. The lack of YbeY resulted in misprocessing of 16S rRNA and a severe decrease of growth rate with complete growth arrest observed at elevated temperatures. Moreover, a ybeY mutation severely disturbed regulation of the Yersinia virulence plasmid (pYV) genes and affected the expression of regulatory small RNA species. Transcription of the pYV genes was upregulated in the ybeY mutant at 22 °C; the same genes were repressed in the wild-type bacterium. Furthermore, ybeY inactivation impaired many virulence-related features, such as resistance to elevated temperature and acid, and hindered utilization of different carbohydrates. In addition, the ybeY mutant strain showed decreased infectivity in a tissue culture infection model, especially at the stage of cell adhesion. Taken together, this study demonstrates the crucial role of YbeY in Y. enterocolitica O:3 physiology and pathogenicity.

High prevalence of pathogenic Yersinia enterocolitica in pig cheeks

Samples from pork cuts for minced meat and cheeks from processing plants and a slaughterhouse, and modified atmosphere (MA) packaged pork from retail were studied to estimate the prevalence of pathogenic, i.e. virulence plasmid bearing, Yersinia enterocolitica and Yersinia pseudotuberculosis in pork, as well as to quantify pathogenic Y. enterocolitica in pork cuts. Pathogenic (virF-positive) Y. enterocolitica was isolated from 17 pig cheeks (23%) but not from any of the MA-packaged 54 retail pork samples and only from one of the 155 pork cut (0.6%). Most (16/17) of the cheek samples were contaminated with pathogenic Y. enterocolitica 4/O:3 and one with bioserotype 2/O:9. No Y. pseudotuberculosis was isolated. The prevalence of pathogenic Y. enterocolitica was clearly higher (39%) in 155 pork cuts when studied with nested PCR targeting yadA on the virulence plasmid pYV although the contamination level was low varying between 0.1 and 1.6 MPN/g. Raw pork cuts and especially pig cheeks may serve as possible sources for yersiniosis caused by pathogenic Y. enterocolitica.

Radiation resistance and loss of crystal violet binding activity in Yersinia enterocolitica suspended in raw ground pork exposed to gamma radiation and modified atmosphere.

Virulence of many foodborne pathogens is directly linked to genes carried on self-replicating extra-chromosomal elements, which can transfer genetic material, both vertically and horizontally, between bacteria of the same and different species. Pathogenic Yersinia enterocolitica harbors a 70-kb virulence plasmid (pYV) that encodes genes for low calcium response, crystal violet (CV) binding, Congo red uptake, autoagglutination (AA), hydrophobicity (HP), type III secretion channels, host immune suppression factors, and biofilm formation. Ionizing radiation and modified atmosphere packaging (MAP) are used to control foodborne pathogens and meat spoilage. In this study, the effect of gamma radiation and modified atmosphere (air, 100% N2 , 75% N2 : 25% CO2 , 50% N2 : 50% CO2 , 25% N2 : 75% CO2 , 100% CO2 ) were examined by using the CV binding phenotype, for the presence or absence of pYV in Y. enterocolitica, suspended in raw ground pork. All Y. enterocolitica serovars used (O:3, O:8, and O5,27) were more sensitive to radiation as the CO2 concentration increased above 50%. Crystal violet binding following a radiation dose of 1.0 kGy, which reduced the Y. enterocolitica serovars >5 log, was greatest in the presence of air (ca. 8%), but was not affected by N2 or CO2 concentration (ca. 5%). Following release from modified atmosphere after irradiation, the loss of CV binding rose from 5% to 8% immediately following irradiation to >30% after outgrowth at 25 °C for 24 h. These results, using Y. enterocolitica as a model system, indicate that the risk of foodborne illness could be affected by the loss of virulence factors when postprocess intervention technologies are used. Provides gamma radiation D10 data for inactivation data for Y. enterocolitica irradiated under modified atmosphere and information to risk assessors regarding the difference between pathogen presence versus actual virulence.

LcrQ Blocks the Role of LcrF in Regulating the Ysc-Yop Type III Secretion Genes in Yersinia pseudotuberculosis

Pathogenic Yersinia species employ the Ysc-Yop type III secretion system (T3SS) encoded by a highly conserved pYV virulence plasmid to export the virulence effectors into host cells. The Ysc-Yop T3SS is tightly regulated by multiple contributing proteins that function at different levels. However, systematic transcriptional regulation analysis of Ysc-Yop T3SS is lacking and the detailed mechanism under this regulation process is still elusive. Aimed at systematically characterizing transcriptional regulations of all T3SS genes in Y. pseudotuberculosis, we amplified 97 non-coding fragments from the pYV plasmid and analyzed transcriptional responses of the T3SS genes under different growth conditions. Transcriptions of T3SS genes were induced at 37°C and genes encoding T3SS effectors were highly induced by further depletion of Ca2+. The temperature induced gene transcription process is mediated by modules encoded on the chromosome, while the Ca2+ depletion-induced process is controlled by the positive regulatory protein LcrF as well as the negative regulatory protein LcrQ. In this process, LcrQ shares the same targets with LcrF and the effect of LcrQ is dependent on the presence of LcrF. Furthermore, over-expression of LcrF showed the same phenotype as that of the lcrQ mutant strain and intracellular amount balance of LcrQ and LcrF is important in T3SS regulation. When the expression level of LcrF exceeds LcrQ, expression of the Ysc-Yop T3SS genes is activated and vice versa. Together, these data support a model in which LcrQ blocks the activation role of LcrF in regulating the transcription of T3SS genes in Yersinia.