Yersiniae other than Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis: the ignored species

The genus Yersinia is composed of 11 species, of which three (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) have been exhaustively characterized. The remaining eight species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, and Y. aldovae) have not been studied extensively and, because of the absence of classical Yersinia virulence markers, are generally considered to be nonpathogenic. However, recent data suggest that some of these eight species may cause disease by virtue of their having virulence factors distinct from those of Y. enterocolitica. These data raise intriguing questions about the mechanisms by which these species interact with their host cells and elicit human disease.     

Yersinia genome diversity disclosed by Yersinia pestis genome-wide DNA microarray

The genus Yersinia includes 11 species, 3 of which (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) are pathogenic for humans. The remaining 8 species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, and Y. aldovae) are merely opportunistic pathogens found mostly in the environment. In this work, the genomic differences among Yersinia were determined using a Y. pestis-specific DNA microarray. The results revealed 292 chromosomal genes that were shared by all Yersinia species tested, constituting the conserved gene pool of the genus Yersinia. Hierarchical clustering analysis of the microarray data revealed the genetic relationships among all 11 species in this genus. The microarray analysis in conjunction with PCR screening greatly reduced the number of chromosomal genes (32) specific for Y. pestis to 16 genes and uncovered a high level of genomic plasticity within Y. pseudotuberculosis, indicating that its different serotypes have undergone an extensively parallel loss or acquisition of genetic content, which is likely to be important for its adaptation to changes in environmental niches.     

Identification and characterization of pathogenic Yersinia enterocolitica isolates by PCR and pulsed-field gel electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n=48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

Identification of Yersinia enterocolitica within the genus Yersinia

In this report we describe a PCR strategy for the unambigous identification of biochemically presumptive typed Yersinia (Y.) enterocolitica. A total of 269 isolates belonging to ten species of the genus Yersinia were investigated. In a first PCR only isolates classified as Y. enterocolitica (n = 113) gave rise to a specific amplification resulting in a sensitivity and a specificity of 100%. By sequencing the 269 amplicons of a second pan-Yersinia PCR spanning a distinct 16S rRNA gene region, 20 different sequence clusters could be identified within the genus. By this, Y. enterocolitica isolates of American and European origin could be distinguished safely and already described sequence clusters of the species Y. frederiksenii were confirmed. New 16S rRNA gene sequence clusters were detected for the species Y. frederiksenii, Y. intermedia, Y. mollaretii, Y. aldovae, Y. kristensenii, and Y. rohdei.     

Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes

The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage lambda-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.     

Characterization of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate and malate dehydrogenases of 192 strains of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gels. The six species were clearly separated from each other by their distinct enzyme electrophoretic polymorphism. For Y. enterocolitica, the strains of biotype 5 were differentiated from the other biotypes by the mobility of glutamate dehydrogenase. For Y. frederiksenii, six zymotypes were delineated by pI and by the mobility of the enzymes. Variation in number or mobility of esterases within each species could represent a marker for epidemiological and ecological analyses. A linear relationship was obtained between the mean genetic diversity coefficient of enzymes and the mean percentage DNA-DNA relatedness of Y. intermedia, Y. aldovae, Y. enterocolitica and Y. frederiksenii.     

Natural antimicrobial susceptibilities and biochemical profiles of Yersinia enterocolitica-like strains: Y. frederiksenii, Y. intermedia, Y. kristensenii and Y. rohdei

The natural susceptibility of 131 Yersinia strains of Y. frederiksenii (n=38), Y. intermedia (n=48), Y. kristensenii (n=26) and Y. rohdei (n=19) to 70 antibiotics was tested. Minimum inhibitory concentration (MIC) values were determined with a microdilution procedure in IsoSensitest broth (all strains) and cation-adjusted Mueller Hinton broth (some strains). All species were naturally sensitive or sensitive and of intermediate susceptibility to tetracyclines, aminoglycosides, acylureidopenicillins, numerous cephalosporins, carbapenems, aztreonam, quinolones, chloramphenicol, folate-pathway inhibitors, nitrofurantoin, and fosfomycin. Uniform natural resistance was found with penicillin G, oxacillin, several macrolides, lincosamides, streptogramins, glycopeptides, rifampicin and fusidic acid. Species-specific differences in susceptibility affecting clinical assessment criteria were seen with aminopenicillins (in the presence and absence of beta-lactamase inhibitors), ticarcillin and some cephalosporins. Major medium-dependent susceptibilities were found with fosfomycin. beta-Lactam MIC susceptibility patterns suggested that most strains of the species tested produce both class A and class C (AmpC) beta-lactamases that are characteristic for the species. The present study describes a database concerning the natural susceptibility of some Y. enterocolitica-like species to a wide range of antibiotics, which can be applied to validate forthcoming antibiotic susceptibility tests of these strains and might contribute to their identification. An evaluation of 30 biochemical tests that secured phenotypic identification to the Yersinia species level is presented.     

Studies on the pathogenicity of Yersinia enterocolitica. III. Comparative studies between Y. enterocolitica and Y. pseudotuberculosis

Comparative studies on pathogenicity between Yersinia enterocolitica and Yersinia pseudotuberculosis were performed using experimental infection systems in vivo and in vitro. All of thestra ins of both species successfully produced experimental enterocolitis in rabbits although the severity varied with the strains challenged. The changes were characterized by granulomatous lesions with necrobiotic centers in reticuloendothelial tissues of the intestine, mesenteric lymph nodes, liver and spleen. These strains uniformly had the ability to penetrate HeLa cells and to survive or multiply within cultured rabbit peritoneal macrophages. In addition, in infections with strain TP-2 or PST-III of Y. pseudotuberculosis, catarrhal inflammation all over the small intestine and/or focal necrosis and parenchymatous degeneration in the liver were observed, along with the granulomatous lesions. These strains, at the same time, exhibited cytotoxic effects on the cultured cells. The pathogenic factors of Y. enterocolitica are discussed in comparison with those of Y. pseudotuberculosis.     

Characterization of IS1541-like elements in Yersinia enterocolitica and Yersinia pseudotuberculosis

We characterized Yersinia enterocolitica and Yersinia pseudotuberculosis insertion sequences related to insertion sequence 1541, recently identified in Yersinia pestis. For each of the two species, two insertion sequence copies were cloned and sequenced. Genetic elements from Y. pseudotuberculosis were almost identical to insertion sequence 1541, whereas these from Y. enterocolitica were less related. Phylogenetic analysis of the putative transposases encoded by insertion sequences from the three pathogenic members of the genus Yersinia showed that they clustered with those encoded by Escherichia coli and Salmonella enterica elements belonging to the insertion sequence 200/insertion sequence 605 group. Insertion sequences originating from Y. pestis and Y. pseudotuberculosis constitute a monophyletic lineage distinct from that of Y. enterocolitica.     

Tapping the potential of intact cell mass spectrometry with a combined data analytical approach applied to Yersinia spp.: detection, differentiation and identification of Y. pestis

In the everyday routine of an analytic lab, one is often confronted with the challenge to identify an unknown microbial sample lacking prior information to set the search limits.In the present work, we propose a workflow, which uses the spectral diversity of a commercial database (SARAMIS) to narrow down the search field at a certain taxonomic level, followed by a refined classification by supervised modelling. As supervised learning algorithm, we have chosen a shrinkage discriminant analysis approach, which takes collinearity of the data into account and provides a scoring system for biomarker ranking. This ranking can be used to tailor specific biomarker subsets, which optimize discrimination between subgroups, allowing a weighting of misclassification.The suitability of the approach was verified based on a dataset containing the mass spectra of three Yersinia species Yersinia enterocolitica, Y. pseudotuberculosis and Yersinia pestis. Thereby, we laid the emphasis on the discrimination between the highly related species Yersinia pseudotuberculosis and Y. pestis.All three species were correctly identified at the genus level by the commercial database. Whereas Y. enterocolitica was correctly identified at the species level, discrimination between the highly related Y. pseudotuberculosis and Y. pestis strains was ambiguous. With the use of the supervised modelling approach, we were able to accurately discriminate all the species even when grown under different culture conditions.     

Ecological regularities of the existence of pathogenic Yersinia in soil ecosystems

In this review the data on the ecology of pathogenic Yersinia in soil ecosystems, based on prolonged observations, were analyzed and summarized. In contrast to saprophytic species, ubiquitously spread in nature, pathogenic representatives of the genus Yersinia occurred only in the soil of natural foci and of these, Y. pestis were found only in the soil of burrows of the main carriers. The complex of abiotic and biotic factors (temperature, humidity, chemical composition, interactions in biocenosis) which determined the possibility of the existence of Yersinia in the soil environment and the preservation of their pathogenic properties was considered. Special attention was paid to their geno-phenotypic variability as the main factor of the adaptation of the causative agents of plague, pseudotuberculosis and intestinal yersiniosis in the environment.     

Identification and Characterization of Pathogenic Yersinia enterocolitica Isolates by PCR and Pulsed-Field Gel Electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n = 48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

Pyrazinamidase activity of bacteria from Enterobacteriaceae family as characteristic for taxonomy

Pyrazinamidase activity in 330 strains of bacteria from Enterobacteriaceae family (14 genus, 27 species) has been assessed. Pyrazinamidase activity detected in species from following genuses: Citrobacter, Escherichia, Klebsiella, Kluyvera, Morganella, Providencia, Raourtella, Salmonella, Shigella, and also in Proteus mirabilis, and nonpathogenic serovars of Yersinia enterocolitica, Y. frederiksenii. Pirasinamidase was absent in Serratia (S. marcescens, S. liguefaciens), Hafnia alvei, P. vulgaris, P. penneri, Y. pseudotuberculosis and pathogenic serovars of Y. enterocolitica. Absence of pyrazinamidase activity in bacteria from Hafnia and Serratia genus is a key taxonomic characteristic for identification of enterobacteria with microvolume assay technology.     

Detection of pathogenic Yersinia enterocolitica using a digoxigenin labelled probe targeting the yst gene

A 145 base pair digoxigenin-d-UTP-labelled probe, specific for pathogenic Yersinia enterocolitica heat-stable enterotoxin yst gene, was prepared by PCR. The probe was used in DNA-DNA colony hybridization and dot-blot hybridization assays. The specificity of the probe was confirmed using 52 strains representing all Yersinia spp., except Y. pestis . Out of a total of 25 Y. enterocolitica strains screened, the probe correctly identified all 18 pathogenic strains. Among the other Yersinia spp. screened, only one strain of Y. kristensenii was positively detected by the yst probe but could be differentiated by its weak signal response as compared with that obtained by pathogenic strains of Y. enterocolitica .     

Simultaneous detection of Aeromonas salmonicida,Flavobacterium psychrophilum,and Yersinia ruckeri,three major fish pathogens,by multiplex PCR

A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.     

A highly specific one-step PCR - assay for the rapid discrimination of enteropathogenic Yersinia enterocolitica from pathogenic Yersinia pseudotuberculosis and Yersinia pestis

Based on differences within the yopT-coding region of Yersinia. enterocolitica, Y pseudotuberculosis and Y pestis, a rapid and sensitive one-step polymerase chain reaction assay with high specificity for pathogenic Y enterocolitica was developed. By this method pathogenic isolates of Y enterocolitica can be easily identified and discriminated from other members of this genus. The entire coding sequence of the yopT effector gene of Y. pseudotuberculosis Y36 was determined.     

Use of a single procedure for selective enrichment, isolation, and identification of plasmid-bearing virulent Yersinia enterocolitica of various serotypes from pork samples.

Many selective enrichment methods for the isolation of Yersinia enterocolitica from foods have been described. However, no single isolation procedure has been described for the recovery and identification of various plasmid-bearing serotypes. A single improved procedure for selective enrichment, isolation, identification, and maintenance of plasmid-bearing virulent serotypes of Y. enterocolitica from pork samples was developed. Enrichment at 12 degrees C in Trypticase soy broth containing yeast extract, bile salts, and Irgasan was found to be an efficient medium for the recovery of plasmid-bearing virulent strains of Y. enterocolitica representing O:3; O:8; O:TACOMA; O:5, O:27; and O:13 serotypes. MacConkey agar proved to be a reliable medium for the isolation of presumptive colonies, which were subsequently confirmed as plasmid-bearing virulent strains by Congo red binding and low calcium response. Further confirmation by multiplex PCR employed primers directed at the chromosomal ail and plasmid-borne virF genes, which are present only in pathogenic strains. The method was applied to pig slaughterhouse samples and was effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Strains isolated from ground pork and tongue expressed plasmid-associated phenotypes and mouse pathogenicity.     

Growth of Yersinia enterocolitica and Y. pseudotuberculosis in Yersinia selective enrichment broth according to Ossmer

The growth of Yersinia enterocolitica and Yersinia pseudotuberculosis in Yersinia enrichment broth according to Ossmer (YSEO) was investigated. Y. enterocolitica reached a higher concentration than Y. pseudotuberculosis but both always exceeded 10(6)CFU/ml. The medium may be useful for the detection of both species in foods.     

Immunochemical characteristics of synthetic peptides incorporating T- and B-cell epitopes nonspecific porins of pathogenic Yersinia

Multiple antigenic peptides (MAPs), a sequence which include common antigenic epitopes of outer membrane porins (OM) bacteria of the genus Yersinia (Y. pseudotuberculosis, Y. enterocolitica, Y. pestis), pathogenic for humans have been synthesized. After immunization of BALB/c mice the antiserum to the peptide have been obtained. With the help of ELISA we showed that these sera interact with porins isolated from OM pathogenic Yersinia, and MAP interact with antibodies in sera from rabbits immunized with individual porins, and with antibodies in sera of patients with intestinal yersiniosis and pseudotuberculosis.