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.     

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.     

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.     

Plasmid composition of Yersinia pestis strains from different natural foci]

The study of the plasmid composition of 246 Y. pestis strains from different natural foci in the USSR and other countries revealed that 173 strains (70%) carried three known plasmids with a molecular weight of about 6, 45-50 and 60 megadaltons (MD) respectively. In 20 strains (8%) obtained from different sources additional cryptic plasmids were detected. In some cases the absence of one or two typical plasmids was observed. Replicon pPst was shown to have quite constant molecular weight (6 MD), whereas plasmids pCad and especially pFra exhibited certain variations of their molecular weight (45-49 MD and 60-149 MD respectively) in strains of different origin.     

Analysis of the plasmid composition of Yersinia pseudotuberculosis strains and its use for typing pseudotuberculosis pathogens

Electrophoresis in agarose gel has been used to study the plasmid spectra of 854 Yersinia pseudotuberculosis strains isolated from different sources. The plasmids found in the microbial strains are represented by the elements with molecular masses 82; 57; 45; 5.5; 4.4; 3.5; 2.7; 2.4; 2.3 Md. The variable spectra of plasmids is peculiar only for serovar I of Yersinia pseudotuberculosis. Plasmids p45 and p82 are classified as the main, while other plasmids as auxiliary ones. In accord with the classification all plasmid containing strains are divided into 8 plasmid strains. Using the proposed method for intraspecific typing of Yersinia pseudotuberculosis permits one to perfect the epidemiological analysis of pseudotuberculosis infection and make concrete the direction of prophylactic and antiepidemic measures.     

Analysis of insect toxin complex gene variability of Yersinia pestis and Yersinia pseudotuberculosis strains

The nucleotide sequences of the Tc's insect toxin complex genes have been analyzed in 18 natural strains of the main and non-main subspecies of Yersinia pestis isolated in different natural foci in the Russian Federation, as well as neighboring and more remote countries, as compared to the data on Y. pestis and Y. pseudotuberculosis strains stored in the NCBI GenBank database. The nucleotide sequences of these genes in plague agent strains have been found to be highly conserved, in contrast to those of the pseudotuberculosis agent. The sequences of two genes, tcaC and tccC2, have been found to be almost identical in Y. pestis strains, whereas other three genes (tcaA, tcaB, and tccC1) contain a few mutations, which, however, are not common for all strains of the plague agent. Exceptions are only strains of the Y. pestis biovar orientalis, whose tcaB gene is in a nonfunctional state due to a nucleotide deletion. The results suggest that the formation of the species Y. pestis as an agent of a natural focal infection with a transmissive mechanism has not resulted in degradation of the Tc's complex genes. Instead, these genes are likely to have been altered as the plague agent have been adapting to the new environment.     

小肠结肠炎耶尔耶尔森氏菌血清群的研究：Ⅱ.我国小肠...

Since 1980, we have collected 1120 strains of Yersinia enterocolitica, from the different parts of China. These strains have been obtained from various sources in man, animals and natural environment accompanied by their clinical or ecological information of Yersinia enterocolitica. The results of our tests have shown that the 747 strains have exhibited the clinical morphological and biochemical characteristics of Yersinia enterocolitica. Through comparing under the same conditions, out of the 747 strains 335 have been selected out with better antigenicity and have been produced antisera from their representative strains. This set of antisera is very satisfactory for its potency and specificity. This set of antisera is ready to supply and have good efficacy and application facilitated for control strains on identifying strains and their epidemiologic observation.     

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.     

Characterization of the Antigenic Subunits of the Envelope Protein of Yersinia pestis

Chemical, physical, and immunological properties of the envelope antigen of Yersinia pestis strains have been investigated. The antigen consists of two components with isoelectric points (pI) of 4.6 and 4.8. One component (pI 4.6) is a protein bound to a small carbohydrate moiety identified as an oligomeric galactan; the other component (pI 4.8) is a simple protein. These two components are antigenically identical. In buffered solution, the antigen exists as aggregates of molecular weights larger than 300,000. The aggregates dissociate into a variety of smaller molecular weight forms depending on the nature of the treatment for dissociation. Each aggregate can be further dissociated into a single antigenic subunit fraction containing protein and glycoprotein species with molecular weights in the range from 15,000 to 17,000. The subunits can be obtained by a dissociation treatment with 0.1% mercaptoethanol in 0.25% sodium dodecyl sulfate at 95 C for 5 min. The subunits will readily reaggregate into a variety of larger molecular weight forms on the removal of dodecyl sulfate.     

DNA probe for detecting Yersinia pestis and serovariant I of Yersinia pseudotuberculosis by detecting specific DNA repeating sequences]

In order to construct a DNA probe for the plague pathogen detection, we have obtained the recombinant plasmid pRD100 carrying an EcoRI-flanked 140 bp fragment from the genetically silent region of Yersinia pestis species-specific plasmid pYP1. When used as a DNA probe for hybridization of DNA from various strains of 25 bacterial species, this DNA fragment was shown to have the complementary sequences in all investigated Yersinia pestis strains (200), including the plasmid pYP1 lacking ones, and in all the studied Yersinia pseudotuberculosis serotype I strains (80). The search for the probe target in these species has led us to conclusion that it is a specific repeated DNA sequence present in more copies in Yersinia pestis than in Yersinia pseudotuberculosis serotype I. The hybridization of these sequences with the radioactive probe and 24 hours autography makes possible the detection of 1.3 x 10(5) cells of Yersinia pestis and 3 x 10(6) cells of Yersinia pseudotuberculosis serotype I immobilized on the nitrocellulose membranes. Use of the probe for analysis of the nitrocellulose membrane fixed spleen smears from animals that died of experimental plague made possible the detection of Yersinia pestis cells within 48 h.     

Screening of plasmids in museum strains of Yersinia pestis isolated from various natural foci

92 strains of Yersinia pestis isolated from different natural foci and stored for 3-40 years in the museum of live cultures have been studied. The strains having three typical plasmids, their different combinations, plasmidless strains or the strains carrying nontypical plasmids with the molecular masses 9, 15, 55, 80, 90 and 150 Md were found. The old museum strains are proposed to be used as a source of plasmids for the genetical research. The current control of plasmid contents in the museum strains is suggested by the plasmid changes in course of storage.     

Plasmid heterogeneity in populations of Yersinia pestis strains]

Yersinia pestis strains with the typical plasmid patterns were shown to have the heterogenic populations. Heterogeneity is increased by cultivation passages in artificial nutrient media and is manifested in plasmid elimination within several clones, plasmid integration into the chromosome, appearance of auxiliary plasmids or the ones with increased molecular masses. Passages of strains in experimental animals result in populations homogeneity with the typical plasmid patterns within all clones tested. The clones having changed the plasmid content and selected from heterogenic populations pertain their properties when cultivated in nutrient media and passaged in experimental animals.     

Genotyping Yersinia pestis strains from various natural foci

Seven genetic variants of Yersinia pestis were detected by finger-printing of 85 strains of this bacterium from natural foci by means of a BX probe. Variants of Y. pestis strains correlate with certain species of carriers.     

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.     

Alternative localization of the determinant of pathogenicity coded by the Yersinia pseudotuberculosis pVM82 plasmid]

The chromosomal DNA regions in Yersinia pseudotuberculosis strains occur that are homologous to 25 Md DNA segment of the plasmid pVM82 encoding the bacterial capability of immunosuppression. The character of the chromosomal DNA regions dispersion reacting with the 25 Md segment probes is different in epidemiologically hazardous and nonvirulent strains of Yersinia pseudotuberculosis. The specific DNA regions occur as well as identical ones. The suppression of antibody formation to a number of main Yersinia pseudotuberculosis antigens by epidemiologically hazardous strain is demonstrated. The suppression is analogous to the one previously described for Yersinia pseudotuberculosis strains harbouring the plasmid pVM82.     

New plasmidovars of Yersinia pestis isolated in Mongolia]

The plasmid spectres of 122 strains of Yersinia pestis isolated in Mongolia from patients, wild mammals and arthropods were studied. The populations of three plasmidovars of Yersinia pestis were found to be circulating in the natural foci of plague in Mongolia. The first plasmidovar harbours three plasmids with mol masses 6, 47, 65 Md. The second and third plasmidovars contain the plasmids with mol masses 6, 16, 47, 65 Md and 8, 47, 75-80 Md.     

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.     

Population structure of the Yersinia pseudotuberculosis complex according to multilocus sequence typing

Multilocus sequence analysis of 417 strains of Yersinia pseudotuberculosis revealed that it is a complex of four populations, three of which have been previously assigned species status [Y.?pseudotuberculosis sensu stricto (s.s.), Yersinia pestis and Yersinia similis] and a fourth population, which we refer to as the Korean group, which may be in the process of speciation. We detected clear signs of recombination within Y.?pseudotuberculosis s.s. as well as imports from Y.?similis and the Korean group. The sources of genetic diversification within Y.?pseudotuberculosis s.s. were approximately equally divided between recombination and mutation, whereas recombination has not yet been demonstrated in Y.?pestis, which is also much more genetically monomorphic than is Y.?pseudotuberculosis s.s. Most Y.?pseudotuberculosis s.s. belong to a diffuse group of sequence types lacking clear population structure, although this species contains a melibiose-negative clade that is present globally in domesticated animals. Yersinia similis corresponds to the previously identified Y.?pseudotuberculosis genetic type G4, which is probably not pathogenic because it lacks the virulence factors that are typical for Y.?pseudotuberculosis s.s. In contrast, Y.?pseudotuberculosis s.s., the Korean group and Y.?pestis can all cause disease in humans.     

Biochemical characteristics of Yersinia enterocolitica strains isolated in the Maritime Territory

The detailed study of 179 strains, considered to be typical and atypical representatives of Y. enterocolitica upon their isolation, has been carried out. Of these, 129 strains have been found to belong to Y. enterocolitica with their typical biochemical properties and 50 strains, to new Yersinia species. The ecological sources of all the isolated strains are indicated. The necessity of the thorough epidemiological, clinical and laboratory study of the etiological role of Yersinia in acute intestinal diseases in humans is pointed out.     

Detection and characterization of the plasmids of the plague microbe which determine the synthesis of pesticin I, fraction I antigen and "mouse" toxin exotoxin

Plasmid DNA was isolated from Yersinia pestis strains containing pesticin I or fraction I antigen and "mouse" toxin determinants. Specificity of DNA preparations was studied by using them for transformation of plague agent strains carrying no plasmids. pPstI plasmid (molecular weight 7,0-7,8 MD) encoded pesticin I, fibrinolysin and plasmacoagulase synthesis. Fraction I antigen and "mouse" toxin production determinants were borne on pFraI/Tox plasmid (molecular weight about 50 MD). The observation that some Y. pestis cultures, having lost the ability to synthesize one of pFraI/Tox products, still retained this plasmid in their cells, is regarded as an evidence for a complicated regulation of pFraI/Tox function.