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New bacteriophage typing system for Yersinia enterocolitica, Yersinia kristensenii, Yersinia frederiksenii, and Yersinia intermedia: correlation with serotyping, biotyping, and antibiotic susceptibility.

Yersinia enterocolitica is listed as a single species in Bergey's Manual of Determinative Bacteriology, but has recently been split into "true" Y. enterocolitica, Y. kristensenii, Y. intermedia, and Y. frederiksenii. From 48 bacteriophages isolated from raw sewage, 24 were chosen as being the most useful for differentiating strains within the four Yersinia species. The composite set of 24 phages typed 92% of 236 Y. enterocolitica strains, 100% of 16 Y. kristensenii strains, 97% of 29 Y. frederiksenii strains, and 90% of 20 Y. intermedia strains. The most common phage type in any of the groups contained 22% of the strains tested, but most of the phage types contained greater than 5% of the strains. The new typing schema was tested in three outbreaks of Y. enterocolitica, and the results agreed well with serotyping and epidemiological findings. In the same outbreaks, biotyping (API 20E profiles; Analytab Products, Plainview, N.Y.) and antibiograms were less reliable markers and probably should be used only in conjunction with serotyping or phage typing or both. Caution should be used in identifying cultures of Y. frederiksenii and Y. intermedia with the API 20E system, since the tests at 37 degrees C for L-rhamnose and melibiose fermentation are often delayed past 24 h, which is the cut-off point for the final reading in the API system. There were distinct differences in the susceptibilities of Y. enterocolitica and Y. kristensenii to ampicillin, carbenicillin, and cephalothin, which adds further support for classifying the latter as a separate species.

Anti-Bacterial Agents↗

Yersinia pseudotuberculosis and Yersinia pestis are more resistant to bactericidal cationic peptides than Yersinia enterocolitica.

The action of bactericidal polycationic peptides was compared in Yersinia spp. by testing peptide binding to live cells and changes in outer membrane (OM) morphology and permeability. Moreover, polycation interaction with LPS was studied by measuring the dependence of dansylcadaverine displacement and zeta potential on polycation concentration. When growth at 37 degrees C, Yersinia pestis and Yersinia pseudotuberculosis bound less polymyxin B (PMB) than pathogenic or non-pathogenic Yersinia enterocolitica, regardless of virulence plasmid expression. Y. pseudotuberculosis OMs were unharmed by PMB concentrations causing extensive OM blebbing in Y. enterocolitica. The permeability to lysozyme caused by PMB was greater in Y. enterocolitica than in Y. pseudotuberculosis or Y. pestis and differences increased at 37 degrees C. Similar observations were made with other polycations using a polymyxin/novobiocin permeability assay. With LPS of cells grown at 26 degrees C, polycation binding was highest for Y. pseudotuberculosis and lowest for Y. pestis, with Y. enterocolitica yielding intermediate results which were lower for pathogenic than for non-pathogenic strains. With LPS of cells grown at 37 degrees C, polycation binding remained unchanged for Y. pestis and pathogenic Y. enterocolitica, increased for non-pathogenic Y. enterocolitica and decreased for Y. pseudotuberculosis to Y. pestis levels. Polycation binding related in part to differences in charge density (zeta potential) of LPS aggregates, suggesting similar effects at bacterial surfaces. It is suggested that species and temperature differences in polycation resistance relate to infection route, invasiveness and intracellular multiplication of Yersinia spp.

Anti-Bacterial Agents↗

Serological crossreactivity between Brucella abortus and Yersinia enterocolitica 0:9 II the use of Yersinia outer proteins for the specific detection of Yersinia enterocolitica infections in ruminants.

Yersinia outer protein (YOP) preparations from Y. enterocolitica and Y. pseudotuberculosis were used as antigens in immunoblots for the detection of Yersinia infections in experimentally and naturally infected ruminants. Sera from 9 groups of animals were used: (1) 51 sera from cattle which were false-positive in the standard brucellosis serological tests, (2) 52 sera from brucellosis-negative cattle, (3) 51 sera from a deer herd in which 16 animals were positive in the brucellosis tests and Yersina species were isolated from 5 animals, (4) 50 sera from a deer herd in which sera from all animals were negative in the brucellosis tests, (5) 107 sera from brucellosis-negative cattle which were received from throughout New Zealand, (6) 30 sera from cattle naturally infected with B. abortus and from which B. abortus was isolated, (7) 55 sera from cattle naturally infected with B. abortus, (8) 26 sera from cattle experimentally infected with B. abortus, with mostly high titres in the conventional brucellosis tests, and (9) sera taken weekly from 3 cattle experimentally infected with Y. enterocolitica 0:9. In all 3 Y. enterocolitica 0:9 experimentally infected animals the antibody reactivity against major YOPs in the Y. enterocolitica and in the Y. pseudotuberculosis YOP preparation correlated well with the strength in the classical brucellosis tests and with the staining of smooth lipopolysaccharides (SLPS) in blots, thus confirming the usefulness of YOPs for the detection of Yersinia infections. Sera from naturally infected cattle and deer herds, regardless of whether they were false positive or negative in the brucellosis tests, showed high frequencies of staining in YOP blots (53-58% in cattle and 80-100% in deer), indicating a high prevalence of field infections with Yersinia species in New Zealand. In two of the three sera groups from B. abortus infected animals, antibodies against YOPs were detected with high frequency, showing that dual infections may be common and may interfere with differential serological testing.

Animals↗

Immunity against Yersinia enterocolitica by vaccination with Yersinia HSP60 immunostimulating complexes or Yersinia HSP60 plus interleukin-12.

Microbial heat shock proteins (HSP) are dominant antigens for the host immune response. Because of the high sequence homology between mammalian and microbial HSP, their value as component of a subunit vaccine has been the subject of controversy. Previous work from this laboratory, however, demonstrated for the first time that the adoptive transfer of HSP60-reactive CD4+ alphabeta T-cell clones confers protection against bacterial infection in mice but does not induce autoimmunity. In the present study, we have therefore evaluated the potential role of Yersinia HSP60 (Y-HSP60) as a vaccine in the Yersinia enterocolitica mouse infection model. For this purpose, immunostimulating complexes (ISCOM) which included Y-HSP60 were constructed. Parenteral administration of this vaccine induced high Y-HSP60-specific serum antibody responses as well as T-cell responses. This reaction was parallelled by immunity against a lethal challenge with Y. enterocolitica. In contrast, mucosal application of Y-HSP60-ISCOM failed to induce systemic Y-HSP60-specific T-cell responses and thus failed to induce immunity against yersiniae. Likewise, vaccination with purified recombinant Y-HSP60 induced antibody responses but only weak T-cell responses. Therefore, this vaccination protocol was not protective. However, when interleukin-12 was used as an adjuvant, purified Y-HSP60 induced significant Y-HSP60-specific T-cell responses and thus induced protection against subsequent challenge with yersiniae. These studies suggest that (i) microbial HSP might be promising candidates for the design of subunit vaccines and (ii) interleukin-12 is an efficient alternative adjuvant to ISCOM particles for induction of protective CD4 Th1-cell-dependent immune responses against bacterial pathogens.

Adjuvants, Immunologic↗

Genotyping of human and porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis.

In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.

Animals↗

The 102-kilobase pgm locus of Yersinia pestis: sequence analysis and comparison of selected regions among different Yersinia pestis and Yersinia pseudotuberculosis strains.

We report the complete 119,443-bp sequence of the pgm locus from Yersinia pestis and its flanking regions. Sequence analysis confirms that the 102-kb unstable pgm locus is composed of two distinct parts: the pigmentation segment and a high-pathogenicity island (HPI) which carries virulence genes involved in iron acquisition (yersiniabactin biosynthetic gene cluster). Within the HPI, three genes coding for proteins related to phage proteins were uncovered. They are located at both extremities indicating that the entire HPI was acquired en bloc by phage-mediated horizontal transfer. We identified, within the pigmentation segment, two novel loci that may be involved in virulence: a fimbriae gene cluster and a locus probably encoding a two component regulatory system similar to the BvgAS regulatory system of Bordetella pertussis. Three genes containing frameshift mutations and two genes interrupted by insertion element insertion were found within this region. To investigate diversity among different Y. pestis and Yersinia pseudotuberculosis strains, the sequence of selected regions of the pgm locus and flanking regions were compared from 20 different Y. pestis and 10 Y. pseudotuberculosis strains. The results showed that the genes interrupted in Y. pestis are intact in Y. pseudotuberculosis. However, one of these mutations, in the bvgS homologue, is only present in Y. pestis strains of biovar Orientalis and not in those of the biovars Antiqua and Medievalis. The results obtained by analysis of variable positions in the sequence are in accordance with historical records, confirming that biovar Orientalis is the most recent lineage. Furthermore, sequence comparisons among 29 Yersinia strains suggest that Y. pestis is a recently emerged pathogen that is probably entering the initial phase of reductive evolution.

Bacteriophages↗

A cloned chromosomal DNA fragment which differentiates Yersinia pestis from Yersinia pseudotuberculosis and Yersinia enterocolitica.

Chromosomal DNA from reference Yersinia strains was digested individually with 9 restriction endonucleases. DNA fragments were separated and analyzed by electrophoresis through agarose gels. The clearest fragment patterns were obtained when EcoRI was employed. The Y. pestis fragment pattern obtained after the use of this enzyme showed the presence of a unique DNA fragment with molecular mass 1400 bp. This DNA fragment was cloned, purified, labeled with 32P and then used to probe EcoRI digests of all three Yersinia species. A strong hybridization signal was obtained with Y. pestis strain. No such signal was found with Y. pseudotuberculosis or Y. enterocolitica. These results indicate that the DNA fragment is species specific and could be used as a diagnostic DNA probe for Y. pestis.

Cloning, Molecular↗

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.

Animals↗

Characterization of the synovial T cell response to various recombinant Yersinia antigens in Yersinia enterocolitica-triggered reactive arthritis. Heat-shock protein 60 drives a major immune response.

OBJECTIVE: In Yersinia enterocolitica-triggered reactive arthritis (Yersinia ReA), the synovial T cell response is primarily directed against bacterial components, which are mostly unknown. This study was performed to investigate the synovial proliferative T cell response to a panel of recombinant Yersinia antigens in patients with Yersinia ReA and in controls. METHODS: Synovial fluid mononuclear cells (SFMC) were obtained from 4 patients with Yersinia ReA and from 14 patients with arthritides of different etiology. SFMC were stimulated with 5 recombinant Yersinia antigens (the 19-kd urease beta subunit, 13-kd ribosomal L23 protein, 32-kd ribosomal L2 protein, 18-kd outer membrane protein H, and Y. enterocolitica heat-shock protein 60 [hsp60]), and with human, Chlamydia trachomatis, and Borrelia burgdorferi hsp60. Three T cell clones specific for Y. enterocolitica hsp60 were generated from 1 patient with Yersinia ReA. Antigen-induced cytokine release was measured by enzyme-linked immunosorbent assay. RESULTS: SFMC from all 4 patients with Yersinia ReA responded to each of the Yersinia antigens except the 13-kd protein. These antigens were also recognized by SFMC from a subgroup of patients with undifferentiated arthritis (n = 4), but not by SFMC from other patients with arthritis of different etiology (n = 10). Y. enterocolitica hsp60 induced the strongest proliferative response in all cases. Two types of hsp60-reactive T cell clones could be obtained. One clone responded to all hsp60 variants, including the human variant, and showed a type 2 T helper (Th2)-like cytokine-secretion pattern. In contrast, another clone with specificity for the bacterial hsp60 proteins, but not the human equivalent, reacted with a more Th1-like pattern. CONCLUSION: In Y. enterocolitica-triggered ReA, at least 4 immunodominant T cell antigens exist, which might be used in lymphocyte proliferation assays to identify patients with Yersinia ReA. The hsp60 is a strong antigen, inducing both bacteria-specific and potentially autoreactive CD4+ T cells of both the Th1 and Th2 type.

Adolescent↗

Turning Yersinia pathogenesis outside in: subversion of macrophage function by intracellular yersiniae.

Three bacterial species within the genus Yersinia are causative agents of human disease. Yersinia pestis is transmitted by fleas or in aerosols, infects regional lymph nodes or lungs, and causes the highly lethal disease known as plague. Yersinia enterocolitica and Yersinia pseudotuberculosis are enteric pathogens most commonly associated with self-limiting infections of the mesenteric lymph nodes. Although Y. pestis and the enteropathogenic Yersinia species utilize different modes of transmission and cause different diseases, they rely on a common set of "core" virulence determinants to successfully infect a mammalian host. These virulence factors are encoded on the bacterial chromosome and on an approximately 70-kb plasmid. Once established in lymphoid tissue, all three Yersinia species replicate as aggregates of extracellular bacteria within necrotic lesions or abscesses. At this stage of the infectious process, the bacteria resist phagocytosis by neutrophils, which are able to destroy the bacteria if they are internalized. A type III secretion system encoded on the 70-kb plasmid functions to export multiple proteins (the Yops and LcrV) that are delivered to the extracellular milieu, the plasma membrane, or the cytosol of a host target cell. The Yops and LcrV act in concert to inhibit phagocytosis and downregulate inflammation. Although it is clear that the bulk of bacterial multiplication occurs in an extracellular phase, there is also evidence that all three pathogenic Yersinia survive and multiply in macrophages. Survival and replication of Yersinia in macrophages may occur throughout the infection, but is likely to be of greatest importance at early stages of colonization. That macrophages can serve as permissive sites for bacterial replication in vivo is supported by in vitro experiments, which demonstrate that Y. pestis, Y. peudotuberculosis, and Y. enterocolitica share the ability to survive and multiply in macrophage phagosomes. There is also evidence that the bacteria can subvert the functions of macrophages from within, by inhibiting phagosome acidification (Y. pseudotuberculosis) and the production of nitric oxide (Y. pestis and Y. pseudotuberculosis). Although considerable attention has been focused on how Yersinia subverts the functions of phagocytes from the outside, the study of how these bacteria subvert macrophage functions from the inside will lead to a better overall understanding of Yersinia pathogenesis.

Animals↗

[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.

Adhesins, Bacterial↗

The role of Yersinia enterocolitica and Yersinia pseudotuberculosis in granulomatous appendicitis: a histologic and molecular study.

Granulomatous appendicitis is an enigmatic entity. Purported causes include Crohn's disease, foreign body reactions, sarcoidosis, and infectious agents; however, most cases remain idiopathic. Yersinia enterocolitica (YE) and Y. pseudotuberculosis (YP) have been implicated as causes of appendicitis, ileocolitis, and mesenteric adenitis. The authors examined the potential role of YE and YP in granulomatous appendicitis using histologic and molecular methods. Forty cases of granulomatous appendicitis were evaluated for histologic features including transmural inflammation, number and character of granulomas, and mucosal changes. Twort Gram, Grocott methenamine-silver (GMS), and Ziehl-Neelsen stains were evaluated, and polymerase chain reaction (PCR) analysis was performed to identify pathogenic YP and YE. Twenty-five percent (10 of 40) of the cases were positive for pathogenic Yersinia by PCR (four YE, four YP, and two with both species). Prominent histologic features included epithelioid granulomas with lymphoid cuffing, transmural inflammation with lymphoid aggregates, mucosal ulceration, and cryptitis. One Yersinia-positive case contained mural Gram-negative bacilli; fungal and acid-fast bacilli stains were all negative. Except for one culture-negative case, serologies and cultures were not done or results were unavailable. Two Yersinia-positive patients were diagnosed subsequently with Crohn's disease, suggesting a possible relationship between the two entities. No other patients developed significant sequelae. YE and YP are important causes of granulomatous appendicitis, and Yersinia infection may mimic Crohn's disease. No histologic features distinguish reliably between Yersinia species, or between Yersinia-positive and Yersinia-negative cases. Because special stains and cultures are often not diagnostic, PCR analysis is an excellent technique for the diagnosis of Yersinia.

Adolescent↗

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India.

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.

Animals↗

A single nonamer from the Yersinia 60-kDa heat shock protein is the target of HLA-B27-restricted CTL response in Yersinia-induced reactive arthritis.

The reason for the high association of HLA-B27 with diseases such as ankylosing spondylitis and reactive arthritis is not clear. In reactive arthritis, the triggering bacteria are known, thus allowing investigation of their interaction with HLA-B27. CTL lines derived from five patients with Yersinia-induced reactive arthritis were raised by repeated stimulation in vitro with either Yersinia-infected autologous macrophages (four patients) or pooled peptides (three patients) having the HLA-B27-binding motif. The peptides were derived from five Yersinia proteins and from the chlamydial 57-kDa heat shock protein (hsp). Cytotoxicity of T cell lines was then tested against these peptides. Lytic activity was obtained with T cells stimulated with viable Yersinia or pooled peptides. Targets successfully used for lysis were cells pulsed with peptides from the Yersinia 60-kDa hsp, but not cells pulsed with peptides from other Yersinia proteins or the chlamydial hsp. T cell lines raised with 60-kDa peptides also lysed targets infected with Yersinia. Most interestingly, all three CTL lines tested (one raised with Yersinia; two with pool of peptides) recognized only one single peptide (321-329) of seven tested from the Yersinia hsp60. Cytotoxicity occurred only when target cells were matched for HLA-B27. This identification of an immunogenic peptide derived from an arthritogenic bacterium and presented by HLA-B27 opens the way for future investigation of the role of T cells specific for this peptide or cross-reacting peptides, in the immunopathology of HLA-B27-associated diseases.

Arthritis, Infectious↗