A novel insertion sequence element, IS Yen2, as an epidemiological marker for weakly pathogenic bioserotypes of Yersinia enterocolitica

The structure and distribution of IS Yen2, a new Yersinia enterocolitica insertion sequence element, were investigated. ISYen2 is related to IS elements of the IS21 family and is present in two isoforms in Y. enterocolitica serotype O:3. Analysis of all copies of both isoforms, IS Yen2A and IS Yen2B, by PCR and sequencing indicated that they are not flanked by direct repeats which are typical of the IS21 family of repetitive elements. IS Yen2 is present in multiple copies in Y. enterocolitica O:3 and O:9 and in a single copy in Y. enterocolitica O:1 and O:2 serotypes. The probe for IS Yen2 efficiently detected all weakly pathogenic Y. enterocolitica bioserotypes investigated, whereas it did not hybridize with other strains. This indicates that IS Yen2 can serve as an additional tool for Y. enterocolitica differentiation and epidemiological studies. Distribution of the different groups of IS elements in two Y. enterocolitica pathotypes is in favor of the parallel evolution of American and European Y. enterocolitica strains.     

Distribution of pathogenic Yersinia enterocolitica in China

Yersinia enterocolitica (1,295 strains) was isolated from diarrhea patients, livestock, poultry, wild animals, insect vectors, food, and the environment. They were studied for epidemiology distribution using bacterial biochemical metabolism tests, their virulence genes, and pulsed-field gel electrophoresis (PFGE) sub-typing. The data showed that 416 of the 1,295 strains were pathogenic, where the pathogenic Chinese isolates were of serotypes O:3 and O:9. These two serotypes were found in livestock and poultry, with swine serving as the major reservoir. The geographic distribution of pathogenic isolates was significantly different, where most of the strains were isolated from the cold northern areas, whereas some serotype O:3 strains were recovered from the warm southern areas. By the analysis of the data of the Ningxia Hui Autonomous Region, we find the phenomenon of ‘concentric circle distribution’ around animal reservoirs and human habitation. The clustering of PFGE showed that the patterns of the pathogenic strains isolated from diarrhea patients were identical compared to those from the animals in the same area, thus, suggesting that the human infection originated from the animals.     

Detection of pathogenic Yersinia enterocolitica by using congo red-magnesium oxalate agar medium.

A Congo red-magnesium oxalate agar medium was developed to detect expression of virulence-associated calcium dependency and Congo red absorption in Yersinia enterocolitica. Of the 157 pathogenic serotypes tested, 119 (75.8%) were positive; 98% of nonpathogenic serotypes and strains of three other Yersinia species were negative.     

The pathogenic potential of Yersinia enterocolitica 1A

Yersinia enterocolitica 1A strains are generally considered apathogenic. However, besides environmental sources, foods and animals, they are repeatedly isolated from patients with gastrointestinal symptoms typical to those evoked by Yersinia of the virulent 1B and 2–4 biotypes. Also, at least 2 gastrointestinal outbreaks associated with 1A strains have been reported. There is a general controversy concerning the pathogenic potential of 1A isolates of clinical and non-clinical origin. To address the 1A puzzle, we have determined the genome sequences of 2 1A strains, a nosocomial O:5 and environmental O:36 isolates, and compared them to each other and to O:8/1B and O:3/4 representatives of the virulent serobiotypes.1A isolates have mosaic genomes and share genes both with serobiotypes O:8/1B and O:3/4 that implies their common descent. Besides the pYV virulence plasmid, 1A strains lack the classical virulence markers, like the Ail adhesin, the YstA enterotoxin, and the virulence-associated protein C. However, they still possess genes encoding such known and suspect virulence-associated determinants like the YstB enterotoxin, the InvA invasin, the mucoid Yersinia factor MyfA, and the enterochelin utilisation fepBDGC/fepA/fes gene cluster. In contrast to previous studies, we have found that the strains of the 1A group possess the MyfA antigen although with limited similarity to the highly conserved MyfA in the virulent serobiotypes. In turn, the MyfB chaperone coevolved with the MyfA fibrillae, while the MyfC usher retains 90% identity to its MyfC counterparts in O:3/O:8 group. The only notable difference between clinical and non-clinical 1A strains was the presence of a truncated Rtx toxin-like gene cluster and remnants of a P2-like prophage in the hospital O:5 isolate.Taken together, Y. enterocolitica BT 1A group represents opportunistic pathogens whose opportunity to establish infection seems to rely mainly on the state of the host defence system. However, presence of known and putative virulence-associated features shared with the pathogenic serobiotypes compels to reconsider properly the pathogenic potential of this group of emerging pathogens.     

Detection of pathogenic Yersinia enterocolitica by polymerase chain reaction and digoxigenin-labeled polynucleotide probes.

Yersinia enterocolitica is widespread in nature, but only a few bioserotypes are involved in human infections. Pigs are considered to be the major reservoirs of pathogenic strains. It is essential to have an accurate and rapid method for the detection of pathogenic yersiniae. To achieve this objective, 19-base synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) to detect the ail gene (which is conserved only in pathogenic strains) in strains of Y. enterocolitica and related species originating from pigs or pork products. Digoxigenin-labeled probes derived from the ail, inv, and yst genes were also evaluated on these strains. The PCR amplified a 273-bp fragment of the ail gene involved in eukaryotic cell invasion and serum resistance. The PCR detected template DNA only in strains of Y. enterocolitica traditionally classified as human pathogens but not in biotype 1A strains and related species. Other members of the family Enterobacteriaceae were also negative for the target gene. The digoxigenin-labeled ail probe gave identical results to the PCR. By use of this nonisotopic method, inv-homologous DNA was detected only among yersiniae, except for Y. ruckeri. Although all pathogenic serotypes of Y. enterocolitica were positive for the heat-stable enterotoxin yst gene, two strains of biotype 1A, one Y. intermedia strain, and six other species of the Enterobacteriaceae were also positive. Our results support the notion that pigs constitute an important reservoir of pathogenic Y. enterocolitica and that the inv-homologous sequence is Yersinia specific.     

Detection and identification of Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica by an improved polymerase chain reaction method.

We developed a polymerase chain reaction method in order to detect and identify both Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica. Polymerase chain reaction was performed by using a mixture of primers against the inv gene from Y. pseudotuberculosis and the ail gene from pathogenic Y. enterocolitica. Further addition of primers against the plasmid-coded virF gene from Y. enterocolitica made it possible to detect a virulence-associated gene of both species at the same time. This method was proved to be an adequate and convenient procedure for routine detection and identification of these bacilli.     

Isolation and use of eight phages for typing Yersinia enterocolitica O3

Subdivision of 137 isolates of Yersinia enterocolitica O3 into eight phagovars has been achieved. Some geographical differences were found in the sources of these phagovars and also of two biovars.     

Atypical Yersinia enterocolitica: clinical and epidemiological parameters.

Infections due to biochemically typical Yersinia enterocolitica usually present as gastroenteritis, mesenteric lymphadenitis, terminal ileitis, and septicemia often with visceral abscesses. In these instances, the isolates have been biochemically typical and of well-established serotypes, namely 0:3 or 0:9 and, in the United States, 0:5 or 0:8. The recovery, recognition, and significance of biochemically and serologically atypical Y. enterocolitica in human infections has proceeded more slowly. From an analysis of the clinical histories of 20 patients infected with 21 such aberrant Y. enterocolitica, it appears that these strains are of restricted pathogenic potential, producing various clinical entities such as localized skin abscesses, conjunctivitis, self-limiting enteritis, and wound and urinary tract infections in hosts with predisposing factors. Epidemiologically, whereas episodic acquisition of atypical strains by hospitalized patients is indicative of nosocomial transmission, in the present series sporadic isolations over a 4-year period, mainly from ambulatory patients, suggest an occult reservoir in the community serviced by The Mount Sinai Hospital. In contrast to typical Y. enterocolitica, which has become well adapted in animal and human hosts, it appears that environmental strains may be in the evolutionary process of becoming adapted to humans.     

Evaluation of enzyme immunoassay for the detection of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis strains.

To determine the virulence plasmid-harboring strains of Yersinia enterocolitica, we prepared antiserum against plasmid-encoded proteins of Y. enterocolitica serotype O3 and carried out an enzyme immunoassay (EIA) against temperature-inducible released proteins. This serum reacted with proteins released from not only a Y. enterocolitica serotype O3 strain but also Y. enterocolitica serotype O5:27, O8, and O9 and Y. pseudotuberculosis serotype 1b, 2a, 2b, 2c, 3, 4a, 4b, 5a, 5b, 6, 7, and 8 strains, which all harbored plasmids. Plasmid-cured Y. enterocolitica and Y. pseudotuberculosis strains did not react in the EIA, nor did nonpathogenic Y. enterocolitica strains or Y. frederiksenii, Y. intermedia, and Y. kristensenii strains. These observations demonstrated that this EIA was useful for determining whether the isolated Yersinia strains were pathogenic or not.     

Characterization of a novel porin involved in systemic Yersinia enterocolitica infection

Yersinia enterocolitica is an enteric pathogen capable of causing systemic disease in a murine model. We have identified a novel protein, systemic factor protein A (SfpA), conserved in other pathogenic bacteria that is involved in systemic disease. Analysis of bacterial colonization revealed that a DeltasfpA strain is defective in mesenteric lymph node colonization. Bioinformatics and functional studies suggest that SfpA is a porin.     

Multilocus sequence typing for studying genetic relationships among Yersinia species

The intra- and interspecies genetic relationships of 58 strains representing all currently known species of the genus Yersinia were examined by multilocus sequence typing (MLST), using sequence data from 16S RNA, glnA, gyrB, recA, and Y-HSP60 loci. Yersinia aldovae, Y. bercovieri, Y. intermedia, Y. pestis, Y. pseudotuberculosis, Y. rohdei, and Y. ruckeri were genetically more homogeneous than were Y. enterocolitica, Y. frederiksenii, Y. kristensenii, and Y. mollaretii. The MLST data concerning the genetic relatedness within and among various species of Yersinia support the idea that Y. pestis and Y. pseudotuberculosis are two lineages within the same species rather than two distinct species. Y. ruckeri is the genetically most distant species within the genus. There was evidence of O-antigen switching and genetic recombination within and among various species of Yersinia. The genetic relatedness data obtained by MLST of the four housekeeping genes and 16S RNA agreed in most, but not all, instances. MLST was better suited for determining genetic relatedness among yersiniae than was 16S RNA analysis. Some strains of Y. frederiksenii and Y. kristensenii are genetically less related to other strains within those species, compared to strains of all other species within the genus. The taxonomic standing of these strains should be further examined because they may represent currently unrecognized Yersinia species.     

O:13a,13b, a new pathogenic serotype of Yersinia enterocolitica

Forty strains of a new Yersinia enterocolitica serotype isolated in the United States from nonhuman primates and humans were characterized as serotype O:13a,13b. Most of the human strains were isolated from a large multistate outbreak of milkborne enteritis. A common antigenic factor between this new serotype and four previously described Y. enterocolitica (O:7,13; O:18; O:44; and O:44,45) led to recharacterization of the latter as serotypes O:7,13a,13b; O:18,13b; O:44,13a; and O:44,13a,45.     

Polymerase chain reaction-gene probe detection system specific for pathogenic strains of Yersinia enterocolitica.

The polymerase chain reaction technique was used to develop a rapid diagnostic assay for detection of pathogenic Yersinia enterocolitica strains. The assay targeted a stretch of 163 bp of the yst gene and could be applied to both pure cultures and crude DNA extracted from feces. The defined primer pair amplified the targeted sequence from only pathogenic strains and fecal samples seeded with the serotype O:3 strain of Y. enterocolitica, whereas neither nonpathogenic strains nor normal stools yielded any amplified fragments. Of the other Yersinia species and non-Yersinia species tested, only two strains of Y. kristensenii yielded the same amplified product. A 20-mer oligonucleotide probe specifically hybridized within the amplified yst fragment of Y. enterocolitica but did not hybridize with the amplified yst fragment of Y. kristensenii by Southern and dot blot hybridizations. This confirms the reliability of this diagnostic assay in both clinical and epidemiological studies. The availability of the extracted DNA for the polymerase chain reaction was checked by simultaneous amplification of a part of the 16S rDNA and the yst gene. The entire diagnostic assay, including a simplified technique for DNA extraction, the amplification process, and gel electrophoresis, could be completed within 1 working day, which is better than the time required for the time-consuming traditional techniques used in clinical laboratories.     

Comparison of restriction endonuclease analysis and phenotypic typing methods for differentiation of Yersinia enterocolitica isolates.

Restriction endonuclease analysis of chromosomal DNA (REAC) was used to study polymorphism in restriction fragment patterns among Yersinia enterocolitica isolates belonging to serogroups O3, O5,27, O8, O9, O13, and O21. Using the enzyme HaeIII and electrophoresis on thin (0.75-mm) vertical 5% polyacrylamide gels, we were able to distinguish at least 22 DNA fragment patterns among the 72 strains examined. The method showed the greatest discriminatory power with regard to serogroup O8, within which as many as 10 different DNA fragment patterns were detected among the 16 strains examined. Compared with O8, serogroups O3 and O9 were relatively homogeneous with regard to REAC patterns. The discriminatory power of the method was compared with H-antigen typing, biotyping, phage typing, antimicrobial susceptibility typing, and restriction enzyme analysis of the virulence plasmid (REAP), by means of Simpson's index of diversity. The results showed that REAC and REAP constitute an effective supplement or alternative to conventional phenotypic methods for tracing epidemiologically related isolates of Y. enterocolitica. Our finding that human and porcine isolates exhibited the same REAC, REAP, and H-antigen patterns provides additional support for the hypothesis that pigs play an important role in the epidemiology of human Y. enterocolitica infection.     

Molecular biogrouping of pathogenic Yersinia enterocolitica: development of a diagnostic PCR assay with histologic correlation

Yersinia enterocolitica (YE) is associated with several inflammatory gastrointestinal disorders. Pathogenic YE organisms are classified as biogroup 1B (high-virulence [HV] serovars) or biogroups 2 through 5 (low virulence [LV]). We developed the first molecular assay designed to distinguish between these groups and correlated the molecular results with histologic patterns of inflammation. Eleven known pathogenic YE culture isolates (6 biogroup 1B and 5 biogroups 2-5) and 6 YE-positive archival cases were subjected to polymerase chain reaction analysis using primer pairs targeting a strain-dependent variable region, allowing discrimination between biogroups with a single assay. All 11 known culture isolates were confirmed. Of the 6 archival cases, 4 were LV, and 2 were HV. Histologic correlation revealed granulomatous inflammation in the LV cases and suppurative inflammation in the HV cases. This novel assay is useful for diagnosis using culture samples and archival tissues. It also could yield important information correlating YE epidemiology, pathogenesis, and morphology because these preliminary data suggest that LV strains may be associated with chronic granulomatous processes and HV strains with suppurative inflammation.     

Detection of Pathogenic Yersinia enterocolitica by a Rapid and Sensitive Duplex PCR Assay

A duplex PCR assay targeting the ail and 16S rRNA genes of Yersinia enterocolitica was developed to specifically identify pathogenic Y. enterocolitica from pure culture. Validation of the assay was performed with 215 clinical Yersinia strains and 40 strains of other bacterial species. Within an assay time of 4 h, this assay offers a very specific, reliable, and inexpensive alternative to the conventional phenotypic assays used in clinical laboratories to identify pathogenic Y. enterocolitica.     

Detection of Yersinia enterocolitica serogroup O:3 by a PCR method.

Yersinia enterocolitica is the etiologic agent of a range of clinical situations in humans, but only a small number of serotypes are involved. Among these, Y. enterocolitica O:3 is the most frequently implicated. A PCR method was developed to detect Y. enterocolitica O:3. For this purpose, two pairs of primers were designed to amplify two fragments of the rfb cluster of Y. enterocolitica O:3: a 253-bp fragment of the rfbB gene and a 405-bp fragment of the rfbC gene. A specific detection was obtained only with rfbC primers, which yielded a PCR product of the expected size exclusively with pathogenic Y. enterocolitica of serotype O:3. This pair of primers was combined with the ail, inv, and virF primers previously described (H. Nakajima, M. Inoue, T. Mori, K.-I. Itoh, E. Arakawa, and H. Watanabe, J. Clin. Microbiol. 30:2484-2486, 1992) to allow both the detection and the differentiation between Y. pseudotuberculosis, pathogenic Y. enterocolitica of serotype O:3 and other pathogenic Y. enterocolitica.