Precursor Abundance Influences Divergent Antigen-Specific CD8+ T Cell Responses after Yersinia pseudotuberculosis Foodborne Infection.

Primary infection of C57BL/6 mice with the bacterial pathogen Yersinia pseudotuberculosis elicits an unusually large H-2Kb-restricted CD8+ T cell response to the endogenous and protective bacterial epitope YopE69-77. To better understand the basis for this large response, the model OVA257-264 epitope was inserted into YopE in Y. pseudotuberculosis and antigen-specific CD8+ T cells in mice were characterized after foodborne infection with the resulting strain. The epitope YopE69-77 elicited significantly larger CD8+ T cell populations in the small intestine, mesenteric lymph nodes (MLNs), spleen, and liver between 7 and 30 days postinfection, despite residing in the same protein and having an affinity for H-2Kb similar to that of OVA257-264. YopE-specific CD8+ T cell precursors were ∼4.6 times as abundant as OVA-specific precursors in the MLNs, spleens, and other lymph nodes of naive mice, explaining the dominance of YopE69-77 over OVA257-264 at early infection times. However, other factors contributed to this dominance, as the ratio of YopE-specific to OVA-specific CD8+ T cells increased between 7 and 30 days postinfection. We also compared the YopE-specific and OVA-specific CD8+ T cells generated during infection for effector and memory phenotypes. Significantly higher percentages of YopE-specific cells were characterized as short-lived effectors, while higher percentages of OVA-specific cells were memory precursor effectors at day 30 postinfection in spleen and liver. Our results suggest that a large precursor number contributes to the dominance and effector and memory functions of CD8+ T cells generated in response to the protective YopE69-77 epitope during Y. pseudotuberculosis infection of C57BL/6 mice.

Pentaplex real-time PCR for differential detection of Yersinia pestis and Y. pseudotuberculosis and application for testing fleas collected during plague epizootics.

Upon acquiring two unique plasmids (pMT1 and pPCP1) and genome rearrangement during the evolution from Yersinia pseudotuberculosis, the plague causative agent Y. pestis is closely related to Y. pseudotuberculosis genetically but became highly virulent. We developed a pentaplex real-time PCR assay that not only detects both Yersinia species but also differentiates Y. pestis strains regarding their plasmid profiles. The five targets used were Y. pestis-specific ypo2088, caf1, and pst located on the chromosome, plasmids pMT1 and pPCP1, respectively; Y. pseudotuberculosis-specific chromosomal gene opgG; and 18S ribosomal RNA gene as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1 fg to 100 fg, with Y. pestis-specific pst as the most sensitive target. Using the assay, Y. pestis strains were differentiated 100% by their known plasmid profiles. Testing Y. pestis and Y. pseudotuberculosis-spiked flea DNA showed there is no interference from flea DNA on the amplification of targeted genes. Finally, we applied the assay for testing 102 fleas collected from prairie dog burrows where prairie dog die-off was reported months before flea collection. All flea DNA was amplified by 18S rRNA; no Y. pseudotuberculosis was detected; one flea was positive for all Y. pestis-specific targets, confirming local Y. pestis transmission. Our results indicated the assay is sensitive and specific for the detection and differentiation of Y. pestis and Y. pseudotuberculosis. The assay can be used in field investigations for the rapid identification of the plague causative agent.

A Trimeric Autotransporter Enhances Biofilm Cohesiveness in Yersinia pseudotuberculosis but Not in Yersinia pestis.

Cohesion of biofilms made by Yersinia pestis and Yersinia pseudotuberculosis has been attributed solely to an extracellular polysaccharide matrix encoded by the hms genes (Hms-dependent extracellular matrix [Hms-ECM]). However, mutations in the Y. pseudotuberculosis BarA/UvrY/CsrB regulatory cascade enhance biofilm stability without dramatically increasing Hms-ECM production. We found that treatment with proteinase K enzyme effectively destabilized Y. pseudotuberculosiscsrB mutant biofilms, suggesting that cell-cell interactions might be mediated by protein adhesins or extracellular matrix proteins. We identified an uncharacterized trimeric autotransporter lipoprotein (YPTB2394), repressed by csrB, which has been referred to as YadE. Biofilms made by a ΔyadE mutant strain were extremely sensitive to mechanical disruption. Overexpression of yadE in wild-type Y. pseudotuberculosis increased biofilm cohesion, similar to biofilms made by csrB or uvrY mutants. We found that the Rcs signaling cascade, which represses Hms-ECM production, activated expression of yadE The yadE gene appears to be functional in Y. pseudotuberculosis but is a pseudogene in modern Y. pestis strains. Expression of functional yadE in Y. pestis KIM6+ weakened biofilms made by these bacteria. This suggests that although the YadE autotransporter protein increases Y. pseudotuberculosis biofilm stability, it may be incompatible with the Hms-ECM production that is essential for Y. pestis biofilm production in fleas. Inactivation of yadE in Y. pestis may be another instance of selective gene loss in the evolution of flea-borne transmission by this species.IMPORTANCE The evolution of Yersinia pestis from its Y. pseudotuberculosis ancestor involved gene acquisition and gene losses, leading to differences in biofilm production. Characterizing the unique biofilm features of both species may provide better understanding of how each adapts to its specific niches. This study identifies a trimeric autotransporter, YadE, that promotes biofilm stability of Y. pseudotuberculosis but which has been inactivated in Y. pestis, perhaps because it is not compatible with the Hms polysaccharide that is crucial for biofilms inside fleas. We also reveal that the Rcs signaling cascade, which represses Hms expression, activates YadE in Y. pseudotuberculosis The ability of Y. pseudotuberculosis to use polysaccharide or YadE protein for cell-cell adhesion may help it produce biofilms in different environments.

Outbreak of Yersinia pseudotuberculosis O:1 infection associated with raw milk consumption, Finland, 2014.

In March 2014, a Yersinia pseudotuberculosis (YP) outbreak was detected by a municipal authority in southern Finland. We conducted epidemiological, microbiological and traceback investigations to identify the source. We defined a case as a person with YP infection notified to the National Infectious Disease Registry between February and April 2014, or their household member, with abdominal pain and fever≥38 °C or erythema nodosum. Healthy household members were used as household-matched controls. We identified 43 cases and 50 controls. The illness was strongly associated with the consumption of raw milk from a single producer. The odds ratio of illness increased with the amount of raw milk consumed. Also previously healthy adults became infected by consuming raw milk. Identical YP strains were identified from cases' stool samples, raw milk sampled from a case's refrigerator and from the milk filter at the producer's farm. The producer fulfilled the legal requirements for raw milk production and voluntarily recalled the raw milk and stopped its production. We advised consumers to heat the raw milk to 72 °C for 15 s. Current legislation for raw milk producers should be reviewed and public awareness of health risks linked to raw milk consumption should be increased.

High frequency of reactive arthritis in adults after Yersinia pseudotuberculosis O:1 outbreak caused by contaminated grated carrots.

OBJECTIVE: We describe the epidemiological and microbiological process in the clearing of a foodborne outbreak of Yersinia pseudotuberculosis O:1 linked to raw carrots and frequency of the associated reactive extra-gastrointestinal manifestations. METHODS: The patient samples were investigated by routine culture or antibody testing methods. The real-time bacterial PCR was used to detect Y pseudotuberculosis in samples from the grated carrots and in those taken from the carrot storage. Genotype of bacterial isolates was determined by pulsed-field gel electrophoresis. For case identification, we retrospectively looked over the laboratory files of the central hospital focusing on the time period of the outbreak. RESULTS: Altogether 49 case patients were identified. Y pseudotuberculosis was detected by real-time PCR analysis in samples taken from grated carrots and from the carrot distributor. Bacterial isolates originating from the farm environment showed identical serotype (O:1) and genotype (S12) with the patients' isolates. Among 37 adults, reactive arthritis (ReA) was found in 8 (22%) and three adults had probable ReA. Six (67%) out of nine human leucocyte antigen (HLA) typed patients with ReA were HLA-B27 positive. Erythema nodosum was found in 42% of the 12 children, whereas none of them had definite ReA. CONCLUSIONS: In this outbreak, Y pseudotuberculosis was for the first time detected in both patient and food samples. ReA was more common than earlier reported in the outbreaks associated with this pathogen; the reason may be that the previous outbreaks have occurred among children. HLA-B27 frequency was higher than usually reported in single-source outbreaks of ReA.

Growth model of a plasmid-bearing virulent strain of Yersinia pseudotuberculosis in raw ground beef.

The growth kinetics of virulence plasmid-bearing Yersinia pseudotuberculosis (YPST) in sterile ground beef were studied at temperatures ranging from 0 to 30°C. In irradiated sterile ground beef, YPST replicated from 0 to 30°C, with corresponding growth rates (GR) ranging from 0.023 to 0.622 log CFU/h at 0-25°C, and the GR was 0.236 log CFU/h at 30°C. The maximum population densities (MPD) ranged from 8.7 to 11.0 log CFU/g. The growth and MPD of YPST were reduced significantly at 30°C. Models for GR and MPD of YPST in raw ground beef (RGB) as a function of storage temperatures were produced and displayed acceptable bias and accuracy. The models were validated with rifampicin-resistant YPST (rif-YPST) in sterile ground beef stored at 4, 10 and 25°C. The observed GR and MPD were within 95% of the predicted values. When compared to non-sterile retail ground beef, the growth of rif-YPST was not inhibited and displayed similar GR at 0, 10 and 25°C and MPDs as sterile ground beef at 10 and 25°C. Moreover, there was no loss of virulence plasmid in YPST during its growth in ground beef indicating that RGB contaminated with virulence plasmid-bearing YPST could cause disease due to refrigeration failure, temperature (10-25°C) abuse, and if the meat was not properly cooked.

Pathogenic Yersinia enterocolitica 2/O:9 and Yersinia pseudotuberculosis 1/O:1 strains isolated from human and non-human sources in the Plateau State of Nigeria.

Foodborne yersiniosis, caused by enteropathogenic Yersinia, especially Yersinia enterocolitica, is an important cause of diarrhea in developed countries, especially in temperate zones. Since studies concerning the presence of enteropathogenic Yersinia in humans and foods are rare in developing countries and tropical areas, human and non-human samples were studied in Plateau state of Nigeria to obtain information on the epidemiology of Y. enterocolitica and Yersinia pseudotuberculosis. Surprisingly, ail-positive Y. enterocolitica and inv-positive Y. pseudotuberculosis were isolated in Plateau state of Nigeria from several samples of human and non-human origin. Bioserotype 1/O:1 was the only Y. pseudotuberculosis type found. Y. enterocolitica belonging to bioserotype 2/O:9 was the dominating type found in most samples. Bioserotype 4/O:3 was isolated only from one pig and one sheep. Using PFGE, 5 genotypes were obtained among 45 Y. enterocolitica 2/O:9 strains with NotI, ApaI and XhoI enzymes and 3 among 20 Y. pseudotuberculosis 1/O:1 strains with NotI and SpeI enzymes. All human Y. pseudotuberculosis 1/O:1 strains were indistinguishable from pig, sheep or food strains. The dominating genotype of Y. enterocolitica 2/O:9 strains among humans was also found among strains isolated from pig, fermented cow milk and traditional intestine pepper soap samples.

Innate immune response during Yersinia infection: critical modulation of cell death mechanisms through phagocyte activation.

Yersinia pestis, the etiological agent of plague, is one of the most deadly pathogens on our planet. This organism shares important attributes with its ancestral progenitor, Yersinia pseudotuberculosis, including a 70-kb virulence plasmid, lymphotropism during growth in the mammalian host, and killing of host macrophages. Infections with both organisms are biphasic, where bacterial replication occurs initially with little inflammation, followed by phagocyte influx, inflammatory cytokine production, and tissue necrosis. During infection, plasmid-encoded attributes facilitate bacterial-induced macrophage death, which results from two distinct processes and corresponds to the inflammatory crescendo observed in vivo: Naïve cells die by apoptosis (noninflammatory), and later in infection, activated macrophages die by pyroptosis (inflammatory). The significance of this redirected cell death for the host is underscored by the importance of phagocyte activation for immunity to Yersinia and the protective role of pyroptosis during host responses to anthrax lethal toxin and infections with Francisella, Legionella, Pseudomonas, and Salmonella. The similarities of Y. pestis and Y. pseudotuberculosis, including conserved, plasmid-encoded functions inducing at least two distinct mechanisms of cell death, indicate that comparative studies are revealing about their critical pathogenic mechanism(s) and host innate immune responses during infection. Validation of this idea and evidence of similar interactions with the host immune system are provided by Y. pseudotuberculosis-priming, cross-protective immunity against Y. pestis. Despite these insights, additional studies indicate much remains to be understood concerning effective host responses against Yersinia, including chromosomally encoded attributes that also contribute to bacterial evasion and modulation of innate and adaptive immune responses.

Yersinia pseudotuberculosis in a closed colony of Egyptian fruit bats (Rousettus aegyptiacus).

An outbreak of Yersinia pseudotuberculosis (Yptb) occurred in a closed colony of Egyptian fruit bats (Rousettus aegyptiacus) and resulted in the death of seven bats over a 6-week period. An initial survey of the remaining bats revealed visceral abscessation characteristic of pseudotuberculosis in five of the 12 bats examined (41.7%), inciting depopulation of the colony. At necropsy, 70% of the 115 bats in the colony exhibited gross evidence suggestive of Yptb infection, including mesenteric lymphadenopathy (ML), hepatic abscessation (HA), and/or splenomegaly (SPM). Thirty of these bats (13 females and 17 males of various ages) were chosen at random and their tissues submitted for bacterial culture and histopathologic examination. Twenty-three of these 30 bats had one or more gross lesions considered consistent with Yptb, including ML, HA, and SPM. On histopathology, four of the 30 bats had necrotizing lesions containing Gram-negative bacteria in multiple organs, while 18 others exhibited mild mesenteric lymphadenitis and hepatitis. Four of the 30 bats had positive cultures for Yptb. Bats with gross evidence of mesenteric lymphadenopathy, splenomegaly, or histopathologic presence of demodicosis or bacteria in tissues were more likely (P < 0.05) to have a positive Yersinia culture. Examination of the correlation between population density and mortality rates of the colony revealed that the mortality rate of subadult bats increased dramatically at the time of the outbreak, when the population density was at its highest. It is suspected that stress, primarily from severe overcrowding, predisposed the bat colony to morbidity and mortality from this organism, which likely originated from a rodent reservoir.

Transmission of Yersinia pseudotuberculosis in the pork production chain from farm to slaughterhouse.

The transmission of Yersinia pseudotuberculosis in the pork production chain was followed from farm to slaughterhouse by studying the same 364 pigs from different production systems at farm and slaughterhouse levels. In all, 1,785 samples were collected, and the isolated Y. pseudotuberculosis strains were analyzed by pulsed-field gel electrophoresis. The results of microbial sampling were combined with data from an on-farm observation and questionnaire study to elucidate the associations between farm factors and the prevalence of Y. pseudotuberculosis. Following the same pigs in the production chain from farm to slaughterhouse, we were able to show similar Y. pseudotuberculosis genotypes in live animals, pluck sets (containing tongue, tonsils, esophagus, trachea, heart, lungs, diaphragm, liver, and kidneys), and carcasses and to conclude that Y. pseudotuberculosis contamination originates from the farms, is transported to slaughterhouses with pigs, and transfers to pluck sets and carcasses in the slaughter process. The study also showed that the high prevalence of Y. pseudotuberculosis in live pigs predisposes carcasses and pluck sets to contamination. When production types and capacities were compared, the prevalence of Y. pseudotuberculosis was higher in organic production than in conventional production and on conventional farms with high rather than low production capacity. We were also able to associate specific farm factors with the prevalence of Y. pseudotuberculosis by using a questionnaire and on-farm observations. On farms, contact with pest animals and the outside environment and a rise in the number of pigs on the farm appear to increase the prevalence of Y. pseudotuberculosis.

Distribution of virF/lcrF-positive Yersinia pseudotuberculosis serotype O:3 at farm level.

The distribution and persistence of pathogenic, virF/lcrF-positive Yersinia pseudotuberculosis were investigated in pigs and in the pig house environment during rearing to determine possible contamination routes of early infections. Based on Y. pseudotuberculosis-positive tonsils of slaughter pigs in our previous study, Y. pseudotuberculosis-positive animals were traced back to the farms. Eight farms were visited from 6-10 months later, and a total of 155 pooled and six individual faecal samples from pigs and 116 pooled environmental samples were collected for analysis by different culture methods. Four of the eight farms were found to be Y. pseudotuberculosis-positive. All positive faecal samples were obtained from fattening pigs, with prevalence varying from 5% to 71% on positive farms. Sows, boars and suckling piglets were Y. pseudotuberculosis-negative on all farms. Most Y. pseudotuberculosis-positive farms (three of four) were on a one-site production system, which had a higher prevalence of Y. pseudotuberculosis (5-26%) among fattening pigs than the all-in, all-out system (1-5%). All Y. pseudotuberculosis isolates belonged to serotype O:3 and carried the virF/lcrF gene on the virulence plasmid. Biotypes 2 and 3 were involved, the latter in one isolate and not being previously reported in pigs. Altogether 53 isolates from 16 positive samples were characterized with pulsed-field gel electrophoresis (PFGE). Using SpeI, NotI and XbaI enzymes, four, three and two different PFGE patterns were obtained respectively. A total of nine different genotypes were identified when the profiles of the enzymes were combined. The most common genotypes were gIV, found on three, and gXII, found on two of the four Y. pseudotuberculosis-positive farms. The same genotypes previously detected in pig tonsils were present in pig faeces from the same farm, indicating that some Y. pseudotuberculosis strains can persist in the pig house environment.

Oral inoculation with Type III secretion mutants of Yersinia pseudotuberculosis provides protection from oral, intraperitoneal, or intranasal challenge with virulent Yersinia.

The enteric pathogen Yersinia pseudotuberculosis (Yptb) causes gastroenteritis, mesenteric lymphadenitis, and systemic infections in humans, livestock, and wild animals. Yptb Type III secretion system (pTTSS) mutants efficiently colonize lymphoid tissues, but not the gastrointestinal tract, spleen, or liver. Here, we show that a single oral inoculation of pTTSS mutants prevents morbidity in almost 100% of mice challenged intragastrically with virulent Yptb. In addition, a single oral inoculation of a pTTSS mutant protected 50% of mice challenged intraperitoneally or intranasally with virulent Yptb. In addition, the intranasally challenged mice that succumbed to infection lived significantly longer than non-immunized mice. Thus, pTTSS mutants can function as live attenuated vaccine when delivered orally. Potential uses for these attenuated strains include use as a livestock vaccine, a rodent plague control reagent in endemic areas around the world, and a vector for delivery of other antigens to the mesenteric lymph nodes.

Serotype differences and lack of biofilm formation characterize Yersinia pseudotuberculosis infection of the Xenopsylla cheopis flea vector of Yersinia pestis.

Yersinia pestis, the agent of plague, is usually transmitted by fleas. To produce a transmissible infection, Y. pestis colonizes the flea midgut and forms a biofilm in the proventricular valve, which blocks normal blood feeding. The enteropathogen Yersinia pseudotuberculosis, from which Y. pestis recently evolved, is not transmitted by fleas. However, both Y. pestis and Y. pseudotuberculosis form biofilms that adhere to the external mouthparts and block feeding of Caenorhabditis elegans nematodes, which has been proposed as a model of Y. pestis-flea interactions. We compared the ability of Y. pestis and Y. pseudotuberculosis to infect the rat flea Xenopsylla cheopis and to produce biofilms in the flea and in vitro. Five of 18 Y. pseudotuberculosis strains, encompassing seven serotypes, including all three serotype O3 strains tested, were unable to stably colonize the flea midgut. The other strains persisted in the flea midgut for 4 weeks but did not increase in numbers, and none of the 18 strains colonized the proventriculus or produced a biofilm in the flea. Y. pseudotuberculosis strains also varied greatly in their ability to produce biofilms in vitro, but there was no correlation between biofilm phenotype in vitro or on the surface of C. elegans and the ability to colonize or block fleas. Our results support a model in which a genetic change in the Y. pseudotuberculosis progenitor of Y. pestis extended its pre-existing ex vivo biofilm-forming ability to the flea gut environment, thus enabling proventricular blockage and efficient flea-borne transmission.

The evolution of flea-borne transmission in Yersinia pestis.

Transmission by fleabite is a recent evolutionary adaptation that distinguishes Yersinia pestis, the agent of plague, from Yersinia pseudotuberculosis and all other enteric bacteria. The very close genetic relationship between Y. pestis and Y. pseudotuberculosis indicates that just a few discrete genetic changes were sufficient to give rise to flea-borne transmission. Y. pestis exhibits a distinct infection phenotype in its flea vector, and a transmissible infection depends on genes that are specifically required in the flea, but not the mammal. Transmission factors identified to date suggest that the rapid evolutionary transition of Y. pestis to flea-borne transmission within the last 1,500 to 20,000 years involved at least three steps: acquisition of the two Y. pestis-specific plasmids by horizontal gene transfer; and recruitment of endogenous chromosomal genes for new functions. Perhaps reflective of the recent adaptation, transmission of Y. pestis by fleas is inefficient, and this likely imposed selective pressure favoring the evolution of increased virulence in this pathogen.

[Mastitis caused by Yersinia pseudotuberculosis in a cow]. / Mastitis bij het rund door Yersinia pseudotuberculosis.

Subclinical mastitis with a raised somatic cell count was diagnosed in a cow in her fifth lactation. It was caused by Yersinia pseudotuberculosis, which can also infect humans. This is the first time that Yersinia pseudotuberculosis has been isolated from a mastitis sample in The Netherlands. Despite treatment with antibiotics in the dry period, Yersinia pseudotuberculosis was still present in the same quarter in the subsequent lactation. The somatic cell count was still high and milk production was much lower than in previous lactation. The pathogen did not spread to other quarters of the same cow or to herd mates on the farm over a 1-year period.

The Eyam plague revisited: did the village isolation change transmission from fleas to pulmonary?

Back in the 17th century the Derbyshire village of Eyam fell victim to the Black Death, which is thought to have arrived from London in some old clothes brought by a travelling tailor. The village population was 350 at the commencement of plague, of which only 83 survived. Led by the church leaders, the village community realized that the whole surrounding region was at risk from the epidemic, and therefore decided to seal themselves off from the other surrounding villages. In the first 275 days of the outbreak, transmission was predominantly from infected fleas to susceptible humans. From then onward, mortality sharply increased, which indicates a changing in transmission pattern. We hypothesize that the confinement facilitated the spread of the infection by increasing the contact rate through direct transmission among humans. This would be more consistent with pulmonary plague, a deadlier form of the disease. In order to test the above hypothesis we designed a mathematical model for plague dynamics, incorporating both the indirect (fleas-rats-humans) and direct (human-to-human) transmissions of the infection. Our results show remarkable agreement between data and the model, lending support to our hypotheses. The Eyam plague episode is celebrated as a remarkable act of collective self-sacrifice. However, to the best of our knowledge, there were no evidence before that the confinement actually increased the burden payed by the commoners. In the light of our results, it can be said that the hypothesis that confinement facilitated the spread of the infection by increasing the contact rate through direct transmission is plausible.

A widespread outbreak of Yersinia pseudotuberculosis O:3 infection from iceberg lettuce.

BACKGROUND: The vehicles and sources of Yersinia pseudotuberculosis infection are unknown. In Finland, clinical microbiology laboratories routinely report Y. pseudotuberculosis isolations and submit isolates for serotype analysis. In October 1998, the number of serotype O:3 infections increased markedly. METHODS: Case patients with culture-confirmed Y. pseudotuberculosis O:3 infection were identified by use of laboratory-based surveillance. We conducted a population-based case-control study. Healthy community control subjects were matched by age, sex, and postal code. Isolates were subtyped by pulsed-field gel electrophoresis (PFGE). RESULTS: Nationwide, 47 case patients were identified (age range, 2-77 years; median, 19 years). One patient with bacteremia died; 5 underwent appendectomies. We enrolled 38 case patients and 76 control subjects in the case-control study. Seventy-one percent of case patients and 42% of control subjects reported having eaten iceberg lettuce (matched odds ratio, 3.8; 95% confidence interval, 1.3-9.4); a dose-response relationship was found for increasing frequency of consumption. Of the 27 isolates obtained from case patients and tested in the analysis, all had indistinguishable PFGE patterns. Four lunch cafeterias that had served iceberg lettuce were associated with clusters of case patients. The lettuce was traced back to originating farms. CONCLUSIONS: Iceberg lettuce was implicated as the vehicle of a widespread foodborne Y. pseudotuberculosis outbreak. Ongoing laboratory-based surveillance and serotype analysis were essential in the rapid detection of infection. Cases of yersiniosis, which appear to be sporadic, may be part of unrecognized outbreaks caused by contaminated fresh produce.

Molecular relatedness between isolates Yersinia pseudotuberculosis from a patient and an isolate from mountain spring water.

A 40-yr-old buddhist monk was admitted to the hospital with abdominal pain, fever, and confusion. He had a history of drinking untreated mountain spring water in his temple, and experienced the above symptoms for several days before admission. In past medical history, he had suffered from hepatic cirrhosis. Yersinia pseudotuberculosis was isolated from his blood and ascitic fluid. The mountain spring water that he had ingested was cultivated and Y. pseudotuberculosis was also isolated. For identification of pathogenic Y. pseudotuberculosis, each isolate from the three sources (blood, ascitic fluid, and drinking water) was also analysed for the inv gene for Y. pseudotuberculosis and the virF gene for virulent plasmid by PCR. All strains were positive for both the virF and the inv genes and also positive for autoagglutination test. For relationship study, each isolate from the three sources was also analysed with serotyping and restriction endonuclease analysis of virulence plasmid DNA (REAP) using BamHI. All belonged to the serotype 4b and REAP pattern D. Thus, all these findings supported that the mountain spring water was the source of the Y. pseudotuberculosis infection in this case.