Population structure and evolution of pathogenicity of Yersinia pseudotuberculosis

Yersinia pseudotuberculosis is an enteric human pathogen but is widespread in the environment. Pathogenicity is determined by a number of virulence factors, including the virulence plasmid pYV, the high-pathogenicity island (HPI), and the Y. pseudotuberculosis-derived mitogen (YPM), a superantigen. The presence of the 3 virulence factors varies among Y. pseudotuberculosis isolates. We developed a multilocus sequence typing (MLST) scheme to address the population structure of Y. pseudotuberculosis and the evolution of its pathogenicity. The seven housekeeping genes selected for MLST were mdh, recA, sucA, fumC, aroC, pgi, and gyrB. An MLST analysis of 83 isolates of Y. pseudotuberculosis, representing 19 different serotypes and six different genetic groups, identified 61 sequence types (STs) and 12 clonal complexes. Out of 26 allelic changes that occurred in the 12 clonal complexes, 13 were mutational events while 13 were recombinational events, indicating that recombination and mutation contributed equally to the diversification of the clonal complexes. The isolates were separated into 2 distinctive clusters, A and B. Cluster A is the major cluster, with 53 STs (including Y. pestis strains), and is distributed worldwide, while cluster B is restricted to the Far East. The YPM gene is widely distributed on the phylogenetic tree, with ypmA in cluster A and ypmB in cluster B. pYV is present in cluster A only but is sporadically absent in some cluster A isolates. In contrast, an HPI is present only in a limited number of lineages and must be gained by lateral transfer. Three STs carry all 3 virulence factors and can be regarded as high-pathogenicity clones. Isolates from the same ST may not carry all 3 virulence factors, indicating frequent gain or loss of these factors. The differences in pathogenicity among Y. pseudotuberculosis strains are likely due to the variable presence and instability of the virulence factors.     

The superantigen gene ypm is located in an unstable chromosomal locus of Yersinia pseudotuberculosis

Yersinia pseudotuberculosis produces YPM (Y. pseudotuberculosis-derived mitogen), a superantigenic toxin that exacerbates the virulence of the bacterium in vivo. To date, three alleles of the superantigen gene (ypmA, ypmB, and ypmC) have been described. These genes are not found in all Y. pseudotuberculosis strains and have a low GC content, suggesting their location on mobile genetic elements. To elucidate this question, the genetic environment of the superantigen-encoding genes was characterized and 11 open reading frames (ORFs) were defined. Sequence analysis revealed that the ypm genes were not associated with plasmids, phages, transposons, or pathogenicity islands and that the superantigen genes were always located in the chromosome between ORF3 and ORF4. Nonsuperantigenic strains exhibited the same genetic organization of the locus but lacked the ypm gene between ORF3 and ORF4. A new insertion sequence, designated IS1398, which displays features of the Tn3 family, was characterized downstream of the ypmA and ypmC genes. A 13.3-kb region containing the ypm genes was not found in the genome of Y. pestis (CO92 and KIM 5 strains). We experimentally induced deletion of the ypm gene from a superantigen-expressing Y. pseudotuberculosis: using the association of aph(3')-IIIa and sacB genes, we demonstrated that when these reporter genes were present in the ypm locus, deletion of these genes was about 250 times more frequent than when they were located in another region of the Y. pseudotuberculosis chromosome. These results indicate that unlike other superantigenic toxin genes, the Yersinia ypm genes are not associated with mobile genetic elements but are inserted in an unstable locus of the genome.     

The Yersinia high-pathogenicity island.

A pathogenicity island present only in highly pathogenic strains of Yersinia (Y. enterocolitica 1B, Y. pseudotuberculosis I and Y. pestis) has been identified on the chromosome of Yersinia spp. and has been designated High-Pathogenicity Island (HPI). The Yersinia HPI carries a cluster of genes involved in the biosynthesis, transport and regulation of the siderophore yersiniabactin. The major function of this island is thus to acquire iron molecules essential for in vivo bacterial growth and dissemination. The presence of an integrase gene and att sites homologous to those of phage P4, together with a G + C content much higher than the chromosomal background, suggests that the HPI is of foreign origin and has been acquired by chromosomal integration of a phage. The HPI can excise from the chromosome of Y. pseudotuberculosis and is found inserted into any of the three copies of the asn tRNA loci present in this species. A unique characteristic of the HPI is its wide distribution in various enterobacteria. Although first identified in Yersinia spp., it has subsequently been detected in other genera such as E. coli, Klebsiella and Citrobacter.     

The plasmid composition and pathogenetic characteristics of Yersinia pseudotuberculosis strains isolated from human subjects against a background of epidemic and sporadic morbidity

112 newly isolated clinical cultures of Y. pseudotuberculosis have been studied. The strains have been characterized by the presence of plasmids and pathogenicity signs associated with plasmids. The results of the study have confirmed the decisive role of the plasmid with a molecular weight of 44-48 MD in the virulence of Y. pseudotuberculosis. The plasmid with a molecular weight of 82 MD, previously attributed the role of an epidemic marker, has also been found to be widely spread. Our study has revealed no specific features in the plasmid composition of the strains isolated under the conditions of sporadic and epidemic pseudotuberculosis morbidity. The results of the study of the pathogenicity of isogenic derivatives differing by the presence of pXV indicate that the role of plasmids with molecular weights of 3.8 and 82 MD in this process is not essential in the model systems, traditional for enteroinvasive Yersinia.     

Conjugative R-plasmid resistance of the causative agents of Yersinia infection

Nature, structure, occurrence and drug resistance of 160 strains of Y. pseudotuberculosis and 60 strains of Y. enterocolitica isolated from various sources within 1986-1988 were studied. In the strains of Y. pseudotuberculosis, the cell composition with respect to the requirements in calcium ions as well as the plasmid profiles with determination of the molecular weights of the plasmids in the antibiotic sensitive and resistant pathogens and R(+)-transconjugants were investigated. Some molecular genetic properties of the Yersinia R plasmids were also investigated. Antibiotic polyresistant strains of Y. enterocolitica were the most frequent donors of the R plasmids while the strains of Y. pseudotuberculosis were less frequently the donors, in the resistance pattern of which there were more frequent streptomycin and tetracycline resistance determinants. The conjugative R plasmids of Y. pseudotuberculosis were characterized by strict control of replication, repressed frequency of transfers, and a molecular weight of about 47 MD. Their replicones as a rule contained streptomycin and tetracycline markers determining resistance to streptomycin and tetracycline at the levels of 1250 and 156 micrograms/ml, respectively.     

The high-pathogenicity island of Yersinia pseudotuberculosis can be inserted into any of the three chromosomal asn tRNA genes

Pathogenicity islands (PAIs) have been identified in several bacterial species. A PAI called high-pathogenicity island (HPI) and carrying genes involved in iron acquisition (yersiniabactin system) has been previously identified in Yersinia enterocolitica and Yersinia pestis . In this study, the HPI of the third species of Yersinia pathogenic for humans, Y. pseudotuberculosis , has been characterized. We demonstrate that the HPI of strain IP32637 has a physical and genetic map identical to that of Y. pestis . A gene homologous to the bacteriophage P4 integrase gene is located downstream of the asn tRNA locus that borders the HPI of strain IP32637. This int gene is at the same position on the HPI of all three pathogenic Yersinia species. However, in contrast to Y. pestis 6/69, the HPI of Y. pseudotuberculosis IP32637 is not invariably adjacent to the pigmentation segment and can be inserted at a distance ≥ 190 kb from this segment. Also, in contrast to Y. pestis and Y. enterocolitica , the HPI of Y. pseudotuberculosis IP32637 can precisely excise from the chromosome, and, strikingly, it can be found inserted in any of the three asn tRNA loci present on the chromosome of this species, one of which is adjacent to the pigmentation segment. The pigmentation segment, which is present in Y. pestis but not in Y. enterocolitica , is also present and well conserved in all strains of Y. pseudotuberculosis studied. In contrast, the presence and size of the HPIs vary depending on the serotype of the strain: an entire HPI is found in strains of serotypes I only, a HPI with a 9 kb truncation in its left-hand part that carries the IS 100 sequence and the psn and ybtE genes characterizes the strains of serotype III, and no HPI is found in strains of serotypes II, IV and V.     

A novel integrative and conjugative element (ICE) of Escherichia coli: the putative progenitor of the Yersinia high-pathogenicity island

Diversification of bacterial species and pathotypes is largely caused by horizontal transfer of diverse DNA elements such as plasmids, phages and genomic islands (e.g. pathogenicity islands, PAIs). A PAI called high-pathogenicity island (HPI) carrying genes involved in siderophore-mediated iron acquisition (yersiniabactin system) has previously been identified in Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica IB strains, and has been characterized as an essential virulence factor in these species. Strikingly, an orthologous HPI is a widely distributed virulence determinant among Escherichia coli and other Enterobacteriaceae which cause extraintestinal infections. Here we report on the HPI of E. coli strain ECOR31 which is distinct from all other HPIs described to date because the ECOR31 HPI comprises an additional 35 kb fragment at the right border compared to the HPI of other E. coli and Yersinia species. This part encodes for both a functional mating pair formation system and a DNA-processing region related to plasmid CloDF13 of Enterobacter cloacae. Upon induction of the P4-like integrase, the entire HPI of ECOR31 is precisely excised and circularised. The HPI of ECOR31 presented here resembles integrative and conjugative elements termed ICE. It may represent the progenitor of the HPI found in Y. pestis and E. coli, revealing a missing link in the horizontal transfer of an element that contributes to microbial pathogenicity upon acquisition.     

virF-positive Yersinia pseudotuberculosis and Yersinia enterocolitica found in migratory birds in Sweden

During spring and autumn migrations, 468 fecal samples from 57 different species of migratory birds were collected in Sweden. In total, Yersinia spp. were isolated from 12.8% of collected samples. The most commonly found species was Yersinia enterocolitica, which was isolated from 5.6% of all collected samples, followed by Y. intermedia (3.8%), Y. frederiksenii (3.0%), Y. kristensenii (0.9%), Y. pseudotuberculosis (0.6%), and Y. rohdei (0.4%). The pathogenic, virF-positive Y. pseudotuberculosis strains were recovered from three thrushes. These strains belonged to the same bioserotype, 1/O:2, but had two different profiles as determined by pulsed-field gel electrophoresis with NotI and SpeI enzymes. In addition, 10 Y. enterocolitica strains, all from barnacle geese, belonged to bioserotype 3/O:3, which is associated with human disease. Two of the strains were pathogenic, carrying the virF gene on their plasmids. All pathogenic Y. pseudotuberculosis and Y. enterocolitica strains were recovered during the spring, and as the birds were caught during active migration they likely became infected at an earlier stage of the migration, thus potentially transporting these bacterial pathogens over long geographical distances.     

Yersinia pseudotuberculosis plasmids and their significance in the realization of the epidemic process in pseudotuberculosis

The results obtained in the electrophoretic study of the plasmid spectra of 190 Y. pseudotuberculosis strains, isolated from different sources, in 0.6% agarose gel are presented. 11 types of plasmids differing in their molecular weight have been detected. Plasmids with a molecular weight of 45 MD determine Ca2+ dependence, bacterial virulence for white mice and autoagglutination. The presence of differences in Y. pseudotuberculosis strains of serovars I, III and IV has been established, which is manifested by their differing plasmid spectra. The relationship between the presence of plasmids with a molecular weight of 75 and 45 DM in the strains and the character of pseudotuberculosis morbidity in the population has been demonstrated. The epidemic course of infection correlates with the presence of both these plasmids and the sporadic course of infection, with the presence of the plasmid with a molecular weight of 45 MD only.     

BamHI restriction endonuclease analysis of Yersinia ruckeri plasmids and their relatedness to the genus Yersinia 42- to 47-megadalton plasmid

The plasmid profile and BamHI restriction pattern of 17 sorbitol-negative and 1 sorbitol-positive French Yersinia ruckeri strain of the American type strain were studied. The 17 sorbitol-negative strains and the American strain harbored a 62-megadalton (MDa) plasmid with an identical BamHI restriction pattern. Southern hybridization indicated that this 62-MDa plasmid is common among these various strains. The sorbitol-positive strain had four plasmid bands (70, 62, 32, and 25 MDa), and there was no comigration of the DNA fragments of these cleaved plasmids with the fragments of the 62-MDa plasmid. Hybridization of these restricted plasmids with the common 62-MDa plasmid showed a weak DNA homology. The Y. ruckeri plasmid (62 MDa) had a different molecular weight than the virulence plasmid (42 to 47 MDa) of the genus Yersinia, and they had different BamHI restriction patterns. Furthermore, no sequence of the Y. ruckeri plasmid DNA was recognized after Southern hybridization when the 47-MDa plasmid of Y. enterocolitica was used as a probe.     

BamHI restriction endonuclease analysis of Yersinia ruckeri plasmids and their relatedness to the genus Yersinia 42- to 47-megadalton plasmid.

The plasmid profile and BamHI restriction pattern of 17 sorbitol-negative and 1 sorbitol-positive French Yersinia ruckeri strain of the American type strain were studied. The 17 sorbitol-negative strains and the American strain harbored a 62-megadalton (MDa) plasmid with an identical BamHI restriction pattern. Southern hybridization indicated that this 62-MDa plasmid is common among these various strains. The sorbitol-positive strain had four plasmid bands (70, 62, 32, and 25 MDa), and there was no comigration of the DNA fragments of these cleaved plasmids with the fragments of the 62-MDa plasmid. Hybridization of these restricted plasmids with the common 62-MDa plasmid showed a weak DNA homology. The Y. ruckeri plasmid (62 MDa) had a different molecular weight than the virulence plasmid (42 to 47 MDa) of the genus Yersinia, and they had different BamHI restriction patterns. Furthermore, no sequence of the Y. ruckeri plasmid DNA was recognized after Southern hybridization when the 47-MDa plasmid of Y. enterocolitica was used as a probe.     

Characteristics of Yersinia spp. isolated from wild and zoo animals

Thirteen strains of Yersinia spp. were isolated at the Rome zoo and at Castelporziano, a game preserve near Rome. The strains were tested for calcium dependency, autoagglutination, heat-stable toxin production, 50% minimum lethal dose in mice (LD50), pyrazinamidase activity and content of plasmids by electrophoresis in agarose gel. The former three tests were negative for all strains, the LD50 was always greater than or equal to 1 X 10(7.6) CFU/ml and pyrazinamidase activity was positive for all strains. Electrophoresis revealed the presence of two plasmids of 27 and 66 megadaltons (MDa) in the two strains of Y. enterocolitica of serotype 027 isolated from animals in the zoo. The two strains of the same species and serotype, isolated from wild animals harboured a 42-MDa plasmid. A small plasmid of 2 MDa was found in two strains of Y. enterocolitica of serotype 07.8 from two subsequent samples of a zoo animal.     

A new pathogenic trait encoded by Yersinia pseudotuberculosis pVM82 plasmid

The strains of Yersinia pseudotuberculosis isolated from patients in the course of outbreaks of infection (epidemic strains) were found to possess at least two plasmids with molecular masses of 45 and 82 MD. In contrast, the strains obtained in sporadic cases harbored different sets of plasmids, but never the 82 MD plasmids. These plasmids designated pVM82 and isolated from strains of different geographic regions of the country were identical. pVM82 have no homology with Y. pestis plasmids of the similar size coding for the FraI antigen. The pVM82 DNA was found to be composed of the 57 MD plasmid DNA and the 25 MD fragment of Y. pseudotuberculosis DNA. Using Western blot hybridization technique it was shown that the presence of pVM82 suppressed formation of antibody against some major antigenic determinants of Y. pseudotuberculosis. Immunosuppression took place when the animals were infected with bacteria grown below 20 but not at 37 degrees C. The 57 MD plasmid failed to produce immunosuppression. It was concluded that the 25 MD fragment of pFN82 encoded a novel pathogenic factor responsible for immunosuppression.     

Horizontal transfer of the high-pathogenicity island of Yersinia pseudotuberculosis

The horizontal transfer of genetic elements plays a major role in bacterial evolution. The high-pathogenicity island (HPI), which codes for an iron uptake system, is present and highly conserved in various Enterobacteriaceae, suggesting its recent acquisition by lateral gene transfer. The aim of this work was to determine whether the HPI has kept its ability to be transmitted horizontally. We demonstrate here that the HPI is indeed transferable from a donor to a recipient Yersinia pseudotuberculosis strain. This transfer was observable only when the donor and recipient bacteria were cocultured at low temperatures in a liquid medium. When optimized conditions were used (bacteria actively growing in an iron-deprived medium at 4 degrees C), the frequency of HPI transfer reached approximately 10(-8). The island was transferable to various serotype I strains of Y. pseudotuberculosis and to Yersinia pestis, but not to Y. pseudotuberculosis strains of serotypes II and IV or to Yersinia enterocolitica. Upon transfer, the HPI was inserted almost systematically into the asn3 tRNA locus. Acquisition of the HPI resulted in the loss of the resident island, suggesting an incompatibility between two copies of the HPI within the same strain. Transfer of the island did not require a functional HPI-borne insertion-excision machinery and was RecA dependent in the recipient but not the donor strain, suggesting that integration of the island into the recipient chromosome occurs via a mechanism of homologous recombination. This lateral transfer also involved the HPI-adjacent sequences, leading to the mobilization of a chromosomal region at least 46 kb in size.     

Occurrence of yersiniosis and listeriosis in wild boars in Japan

From December 1994 to February 1995, 131 wild boars (Sus scrofa leucomysta) living in a mountainous area in Japan were examined for yersiniosis and listeriosis. Of 131 wild boars, 76 (58%) were males and 55 (42%) were females. Four Yersinia spp. including Y. pseudotuberculosis, Y. enterocolitica, Y. frederiksenii, and Y. aldovei, were isolated from 49 (37%) of 131 wild boars. Yersinia pseudotuberculosis was isolated from five (4%) of 131 wild boars. All Y. pseudotuberculosis isolates were serotype 4b and harbored virulence plasmids. Yersinia pseudotuberculosis was isolated only from boars under 2-yr-old. No human pathogenic Y. enterocolitica was isolated. Listeria monocytogenes was isolated from two (1%) of the wild boars and both isolates were serotype 4b. These findings indicated that wild boar could be a reservoir of Y. pseudotuberculosis and L. monocytogenes in Japan.     

The effect of Yersinia pestis EV76 6 MD plasmid on the composition of outer membrane proteins of Yersinia pseudotuberculosis YPIII]

On the basis of Yersinia pseudotuberculosis strain YPIII the isogenic variants containing the different combinations of 47 Md plasmids from Yersinia pestis or Yersinia pseudotuberculosis cells with the 6 Md pYP plasmid from Yersinia pestis EV (intact or having impaired the pla gene determining the synthesis of plasmocoagulase). The degradation of the secreted proteins encoded by the 47 Md plasmids of Yersinia pestis and Yersinia pseudotuberculosis in the cells harbouring the 6Md pYP plasmid has been registered. Yersinia pseudotuberculosis strain YPIII carrying its own 47Md and pYP plasmids also contained no YOP1 protein, in contract to the parent strain. The damage of the pla gene eliminated the destructive effect on the outer membrane proteins. Imposition of the 47Md and 6Md plasmids from Yersinia pestis in Yersinia pseudotuberculosis cells may be used for obtaining and study of the physiological role of low molecular mass proteins resulting from proteolysis of proteins encoded by the 47Md virulence plasmid of Yersinia.     

Virulence of Yersinia pseudotuberculosis isolated from pork and from the throats of swine

Yersinia pseudotuberculosis was isolated from retail pork and from healthy swine throats. These wild-type strains and their representative cured isogenic strains were tested for the presence of plasmids and several virulence factors, and these characteristics were compared with those of virulent strains from humans. Two pork isolates (serotype IVB) and four swine isolates (serotypes IIB, IIC, III, and IVB) harbored a 42- to 48-megadalton plasmid which had similar fragmentation patterns resulting from digestion with restriction endonuclease. These six strains were lethal for mice via oral challenge and were positive in autoagglutination and calcium dependency tests. They also invaded HeLa cells and induced cytotoxicity. Histopathological examination and indirect fluorescent-antibody staining provided definite evidence of the pathogenicity of these strains when tissue sections from orally infected mice were used. The virulence factors of wild-type pork and swine isolates with the 42- to 48-megadalton plasmid were identical to those of two human isolates (serotypes IVB and VB). Hence, these pork and swine isolates should be considered potentially pathogenic for humans. The finding suggests that retail pork and swine may play an important role in the epidemiology of human infections caused by Y. pseudotuberculosis.     

Virulence of Yersinia pseudotuberculosis isolated from pork and from the throats of swine.

Yersinia pseudotuberculosis was isolated from retail pork and from healthy swine throats. These wild-type strains and their representative cured isogenic strains were tested for the presence of plasmids and several virulence factors, and these characteristics were compared with those of virulent strains from humans. Two pork isolates (serotype IVB) and four swine isolates (serotypes IIB, IIC, III, and IVB) harbored a 42- to 48-megadalton plasmid which had similar fragmentation patterns resulting from digestion with restriction endonuclease. These six strains were lethal for mice via oral challenge and were positive in autoagglutination and calcium dependency tests. They also invaded HeLa cells and induced cytotoxicity. Histopathological examination and indirect fluorescent-antibody staining provided definite evidence of the pathogenicity of these strains when tissue sections from orally infected mice were used. The virulence factors of wild-type pork and swine isolates with the 42- to 48-megadalton plasmid were identical to those of two human isolates (serotypes IVB and VB). Hence, these pork and swine isolates should be considered potentially pathogenic for humans. The finding suggests that retail pork and swine may play an important role in the epidemiology of human infections caused by Y. pseudotuberculosis.     

Isolation of Yersinia group in human infections, animals and environment factors

The presence of Y. enterocolitica and Y. pseudotuberculosis was studied in 4479 enteritis cases, 430 children, presenting appendicular syndrome, and 60 hospitalized patients with arthritis and Reiter syndrome. Y. enteritis was detected in 41 (0.9%) enteritis cases, 15 (3.4%) appendectomized children and 5 (8.3%) arthritis cases. Antibodies to Y. pseudotuberculosis were detected in 2 (3.3%) arthritis patients. Y. enterocolitica was isolated in swine, fish and environment factors (water, soil, food). Y. pseudotuberculosis was isolated in soil. The isolated strains belonged to biotypes 1, 2, 4 and serotypes 0:3; 0:5; 0:5.27; 0:5, 6, 7, 8; 0:6; 0:9; some were non-typable and polyagglutinable. The strains were sensitive to bacteriophages for Yersinia, obtained in our laboratory.     

Evaluation of usefulness for selected virulence markers for identifying pathogenic Yersinia enterocolitica strains. IV. Genes myfA and ureC

We check by polymerase chain reaction (PCR) the presence of gene ureC and myfA, encoding subunits of urease and Myf fimbriae, among clinical and food-originated strains of Yersinia to determine their usefulness as molecular virulence markers of Y. enterocolitica. The examinations were done on 130 clinical strains of Y. enterocolitica O:3/4 isolated in Poland from humans. All strains were obtained from stool and possessed the virulence plasmid pYV. In addition 40 isogenic, plasmid-cured strains were tested. The 52 strains including Y. enterocolitica (biotype 1A, 4, 2 and 1B), Y. pseudotuberculosis, Y. intermedia, Y. frederiksenii, Y. kristensenii, E. coli, Citrobatcer, Shigella and Salmonella were used as controls. The PCR assay resulted in detection of genes: ureC and myfA in genomic DNA of all 130 tested clinical strains of Y. enterocolitica pYV+, as well as in plasmid cured strains. Furthermore, ureC was found in all tested strains of Y. enterocolitica biotype A1 and in one strain of Y. intermedia and Y. kristensenii. In contrast to ureC, myfA was detected only in strains of Y. enterocolitica considered as pathogenic. Obtained results show, gene myfA seems to be the reliable virulence marker of Y. enterocolitica, whereas ureC is not recommended for identification of pathogenic strains of this species.