The gene identification of bacterial species and serovariants by the polymerase chain reaction with universal oligonucleotides: the reidentification of earlier isolated strains of Yersinia pseudotuberculosis]

Genetic analysis of 19 standard strains belonging to 6 Yersinia species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. kirstensenii, Y. frederiksenii, Y. intermedia) revealed that gene typing by the method of polymerase chain reaction (PCR) with the use of universal primers permitted the identification of species in bacterial cultures by PCR patterns and the determination of Y. pseudotuberculosis serovars within 4 hours. By this method 23 Y. pseudotuberculosis strains (serovar 1), earlier isolated in different regions of the USSR from humans and rodents, were studied. The study showed that out of 14 strains of human origin only two strains could actually be classified with serovar 1, while the remaining strains were reidentified as belonging to serovar 5. Among 9 strains isolated from rodents those of serovar 1 prevailed (8 strains). The authors suppose that strains of serovar 5 cause outbreaks and sporadic cases of pseudotuberculosis, occurring considerably more often than it is commonly believed in the USSR.     

Characterization of Yersinia pseudotuberculosis heat stable serovar specific polypeptides with the use of monoclonal antibodies

The capacity of Y. pseudotuberculosis to express serovar specific polypeptides with different specificity of antigenic determinants was proved with the use of monoclonal antibodies (McAb). For the first time Y. pseudotuberculosis O antigens were found to have heat stable protein components carrying linear epitopes complementary to serovar specific MaAb and ensuring the serological specificity of the infective agent. The possibility of improving intraspecific classification of Y. pseudotuberculosis and their differentiation from other pathogenic Yersinia on the basis of the capacity of these bacteria for synthesizing species and serovar specific proteins is substantiated.     

Immunoelectrophoretic analysis of the antigenic composition of Yersinia

The antigenic composition of 24. Y. pseudotuberculosis newly isolated and reference strains, 7 Y. enterocolitica strains, as well as Y. pestis vaccine strain EV, has been studied by the method of immunoelectrophoresis in agar. The antigenic composition of these bacteria has been found to be complicated and to comprise not less than 8-11 antigens, and among them nonspecific protein antigens common for enterobacteria, the common generic antigen, the antigen common with Y. pestis, as well as O-antigens specific for each serovar are identified. Immunoelectrophoretic study has shown the possibility of Y. pseudotuberculosis O-antigen, serovar I, with Salmonella sera, serogroup A, and Y. enterocolitica 09 with brucellar and cholera sera.     

The lytic activity of Yersinia pestis phage P 3d serovar

The lytic activity of plague phage II, serovar 3, with respect to 1,800 bacterial strains has been studied: 760 Yersinia pestis strains, 262 Y. pseudotuberculosis strains, 252 Y. enterocolitica strains, 166 Escherichia coli strains, 90 Shigella strains and 270 strains of other species. The phage has been found to lyse 81.8% of Y. pestis strains, 1 Y. pseudotuberculosis strain and 1 Y. enterocolitica strain. The representatives of other 19 bacterial species have proved to be resistant to the phage. Though having a wide range of action within Y. pestis, the phage does not lyse most of the strains of the causative agent of plague, isolated in certain natural foci. This fact offers promise for using the phage for the differentiation of Y. pestis.     

The cultural properties and virulence of Yersinia]

Study of the cultivation properties of 82 enterobacterial strains has revealed that the colonies of virulent Y. enterocolitica (serovars O3, O9) and Y. pseudotuberculosis (serovar I) are temperature-sensitive. This sign, closely connected with the presence and expression of the virulence plasmid with a molecular weight of 44-48 MD, is not characteristic of other strains. Virulent Yersinia grown in nutrient agar for 48 hours at 37 degrees C form colonies which are smaller in diameter than those formed during cultivation at 26 degrees C (with the significance of differences t greater than or equal to 4), their diameter at 37 degrees C not exceeding 1.0 mm. The test for the determination of the temperature-sensitive morphology of Yersinia colonies, along with the tests for other virulence markers, is probably suitable for the detection of the causative agents of yersiniosis or pseudotuberculosis.     

The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.     

A survey for Yersinia pseudotuberculosis in migratory birds in coastal Japan

Yersinia pseudotuberculosis was isolated from three specimens of two species of birds, the black-faced bunting (Emberiza spodocephala) and pied wagtail (Motacilla alba), of 528 specimens of birds examined from coastal regions in Japan. The two isolated strains of Y. pseudotuberculosis were identified as serovar 4b and serovar 3. This is the first isolation of Y. pseudotuberculosis from birds in Japan. Yersinia enterocolitica was isolated from three specimens of the pied wagtail, one specimen of the reed bunting (Emberiza schoeniclus) and one specimen of the rustic bunting (Emberiza rustica). Yersinia frederiksenii was isolated from two specimens of the gray-rumped sandpiper (Heteroscelus brevipes). Yersinia intermedia was isolated from one specimen of the pied wagtail.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat.

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

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.     

The prevalence of Yersinia and its serologic variants in patients with acute intestinal diseases of undetermined etiology in Leningrad

Among Yersinia enterocolitica strains of 32 serovars, proposed as typing strains, some strains were found to belong to new species. Y. enterocolitica sensu stricto was represented by 21 serovars in the collection of typing strains. The occurrence of different Yersinia serovars in patients with acute enteric diseases of unknown etiology in Leningrad in 1983-1986 was determined with the use of the set of monoreceptor to 21 serovars. Out of 2,947 cultures studied by biochemical and serological methods, 81% were typed. Among them 18 Y. enterocolitica serovars were determined. Their characteristic feature was the prevalence of serovar O3 and an insignificant proportion of serovar O9. More frequently Yersinia were detected in patients with the primary diagnosis of acute enteric diseases (93.5%). The overwhelming majority (two-thirds) of Yersinia strains were isolated from children. A great number of strains detected in this study (70%) was isolated on days 10-15 of the bacteriological examination. In 927 cultures the following biovars were determined: the strains of serovar O3 belonged to biovar 4 and all other strains, to biovar 1.     

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

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

Evidence for two evolutionary lineages of highly pathogenic Yersinia species.

Sensitivity to Yersinia pestis bacteriocin pesticin correlates with the existence of two groups of human pathogenic yersiniae, mouse lethal and mouse nonlethal. The presence of the outer membrane pesticin receptor (FyuA) in mouse-lethal yersiniae is a prerequisite for pesticin sensitivity. Genes that code for FyuA (fyuA) were identified and sequenced from pesticin-sensitive bacteria, including Y. enterocolitica biotype 1B (serotypes O8; O13, O20, and O21), Y. pseudotuberculosis serotype O1, Y. pestis, two known pesticin-sensitive Escherichia coli isolates (E. coli Phi and E. coli CA42), and two newly discovered pesticin-sensitive isolates, E. coli K49 and K235. A 2,318-bp fyuA sequence was shown to be highly conserved in all pesticin-sensitive bacteria, including E. coli strains (DNA sequence homology was 98.5 to 99.9%). The same degree of DNA homology (97.8 to 100%) was established for the sequenced 276-bp fragment of the irp2 gene that encodes high-molecular-weight protein 2, which is also thought to be involved in the expression of virulence by Yersinia species. Highly conserved irp2 was also found in all pesticin-sensitive E. coli strains. On the basis of the fyuA and irp2 sequence homologies, two evolutionary groups of highly pathogenic Yersinia species can be established. One group includes Y. enterocolitica biotype 1B strains, while the second includes Y. pestis, Y. pseudotuberculosis serotype O1, and irp2-positive Y. pseudotuberculosis serotype O3 strains. E. coli Phi, CA42, K49, and K235 belong to the second group. The possible proximity of these two iron-regulated genes (fyuA and irp2), as well as their high levels of sequence conservation and similar G+C contents (56.2 and 59.8 mol%), leads to the assumption that these two genes may represent part of an unstable pathogenicity island that has been acquired by pesticin-sensitive bacteria as a result of a horizontal transfer.     

Molecular genetic monitoring of Yersinia pseudotuberculosis using PCR O-genotyping

Irkutsk PCR O-genotyping of 117 Yersinia pseudotuberculosis serotype O:1-O:4 strains isolated in Siberia and Far East was performed. These methods allowed both O-genotype and its variants (a, b, c) to be detected. It was demonstrated that three Y. pseudotuberculosis O-genotypes were circulated in Siberia (O:1a, O:1b and 0:3) and six genotypes (O:1a, O:1b, O:1c, O:2a, O:3 and O:4b) were circulated at Far East. Genotype O:1b dominates at both regions (87.8%). PCR-algorithm for identification of Y. pseudotuberculosis O-genotypes having epidemic significance was developed. The identification of the etiological agent O-genotype without bacteriological isolation of the stimulus is possible by PCR analysis of the clinical material.     

The genetics and structure of the O-specific polysaccharide of Yersinia pseudotuberculosis serotype O:10 and its relationship with Escherichia coli O111 and Salmonella enterica O35

The O-specific polysaccharide (OPS) is a variable constituent of the lipopolysaccharide of Gram-negative bacteria. The polymorphic nature of OPSs within a species is usually first defined serologically, and the current serotyping scheme for Yersinia pseudotuberculosis consists of 21 O serotypes of which 15 have been characterized genetically and structurally. Here, we present the structure and DNA sequence of Y. pseudotuberculosis O:10 OPS. The O unit consists of one residue each of d-galactopyranose, N-acetyl-d-galactosamine (2-amino-2-deoxy-d-galactopyranose) and d-glucopyranose in the backbone, with two colitose (3,6-dideoxy-l-xylo-hexopyranose) side-branch residues. This structure is very similar to that shared by Escherichia coli O111 and Salmonella enterica O35. The gene cluster sequences of these serotypes, however, have only low levels of similarity to that of Y. pseudotuberculosis O:10, although there is significant conservation of gene order. Within Y. pseudotuberculosis, the O10 structure is most closely related to the O:6 and O:7 structures.     

The sensitivity of Yersinia to bacteriophage Mu

Various representatives of the genus Yersinia were found to differ in their sensitivity to the lytic action of bacteriophage Mu cts62, which could serve as an auxiliary test for the differentiation of Y. pestis and Y. pseudotuberculosis. Among the strains under study, the causative agents of plague (34 strains) were sensitive to phage Mu cts62, while the causative agents of enteric yersiniosis (42 strains) and pseudotuberculosis (73 strains), except 3 strains with the properties of Y. pestis, were resistant to this phage.     

Comparative evaluation of Yersinia interaction with HEp-2 cells

In experiments on HEp-2 cells, the comparative characterization of the mechanism of invasion and the cytotoxic action of a number of Y. pseudotuberculosis strains, serovar 1, and Y. enterocolitica strains 03 and 09 (24 strains newly isolated from human patients and rodents and 5 laboratory strains at different degrees of attenuation) has been made on the basis of data obtained by optical and electron microscopy, as well as by cytoenzymological analysis. As revealed in these experiments, the invasion of Yersinia into epithelial cells and their capacity for intracellular multiplication depend on the cytotoxicity of the bacteria, most pronounced in Y. pseudotuberculosis, serotype 1, considerably less pronounced in Y. enterocolitica 09, and poorly pronounced (or absent) in Y. enterocolitica 03. Cytopathic changes are manifested by vacuolization, the exocytosis (clasmatosis) of the peripheral cytoplasm, impoverishment in organellae, the formation of autophagosomes, the shriveling of the nuclei and the perinuclear cytoplasm, the rounding of the cells and their separation from the glass surface. The development of these changes is accompanied by the increased activity of phosphatases diffusing into the cytoplasm and by the inhibition of cell respiration and dehydrogenase activity.     

A heat-modifiable outer membrane protein carries an antigen specific for the species Yersinia enterocolitica and Yersinia pseudotuberculosis

The outer membranes of gram-negative bacteria are considered to be of importance in host-bacteria interaction, in protective immunity, and occasionally in subclassification within a species. In this study, the outer membranes of several strains of Yersinia enterocolitica and Y. pseudotuberculosis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the appearance of the major proteins depended on the temperature at which they were solubilized in SDS. A protein was identified with the use of two-dimensional gels and preparative SDS-PAGE, which was equivalent to the "heat-modifiable protein" (protein II) of other Enterobacteriaceae species. A monoclonal antibody, 4G1, was generated against an isolated preparation of this Y. enterocolitica protein. This antibody was tested with whole cell bacterial antigens of 46 individual bacterial strains. The reactive strains included only Y. enterocolitica and Y. pseudotuberculosis. In addition, the reactivity of the 4G1 monoclonal antibody preparation could be absorbed only with Y. enterocolitica and Y. pseudotuberculosis, and not with other strains of bacteria. The reactivity of this 4G1 monoclonal antibody was also tested by the Western Blot technique. Six individual strains were tested: a Y. enterocolitica serotype 0:3, a Y. enterocolitica serotype 0:9, an Escherichia coli, a Salmonella typhimurium, a Shigella flexneri, and a Klebsiella pneumoniae. The 4G1 antibody reacted with only the proteins of the two Y. enterocolitica strains. In conclusion, the equivalent of the heat-modifiable protein was present in Y. enterocolitica and Y. pseudotuberculosis. Moreover, this protein also carried a species-specific antigenic determinant.     

Characteristics of Yersinia pseudotuberculosis isolated from animals in Brazil

Strains (105) of Yersinia pseudotuberculosis isolated in Brazil between 1982 and 1990 were bio-serotyped. They were also studied for plasmid profile, autoagglutination and calcium dependence at 37 °C, Congo red uptake, pyrazinamidase activity, esculin hydrolysis, salicin fermentation and drug sensitivity: 95·24% were biotype 2, serogroup O:3; 2·86% were biotype 1, serogroup O:1; and 1·90% were biotype 2, non-agglutinable. Plasmids were found in 77·14% of the strains (one in each strain). There was total correlation between the presence of the virulence plasmid and autoagglutination, calcium dependence at 37 °C and Congo red uptake. The esculin, salicin and pyrazinamidase tests were not efficient in differentiating pathogenic from non-pathogenic Y. pseudotuberculosis isolates. All strains were highly sensitive to the drugs used. These results indicate that Y. pseudotuberculosis is a potential pathogen for humans in Brazil, especially because the bio-serogroups detected among animals are those most frequently associated with human diseases.     

Occurrence of Yersinia enterocolitica in wild animals.

Yersinia species were isolated from 16 of 495 small wild animals and from 1 of 38 foxes. The animals were trapped in seven regions of Hokkaido, Japan. Of the 17 strains isolated, 9 were Yersinia enterocolitica O6; 2 were Y. enterocolitica O5A; 1 was Y. enterocolitica, O4; 1 was Y. enterocolitica O9; 1 was Yersinia pseudotuberculosis IVB; and 3 were sucrose-negative strains. Yersinia pestis was not isolated. The O6 organism was most prevalent in large red-back mice (Clethrionomys rufocanus bedfordiae) and showed significant differences in its mode of distribution according to region. Incidence of the O6 organism in the ileum of the animal was threefold that in the cecum, and the organism was recovered at approximately 10(5) cells per g of cecal contents per c. rufocanus bedfordiae animal.