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.     

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.     

Genotyping of Yersinia pseudotuberculosis isolated in Siberia and Far East

Genotypic characteristics based three main factors of pathogenicity (presence of resident plasmids [pYV, pVM], gene of toxin-superantigen ypm and nine genes for high pathogenicity island [HPI]) of 212 strains of Y. pseudotuberculosis isolated in Siberia and Far East were studied. It was shown that strains of Y. pseudotuberculosis with one of two variants of plasmids 82:47 MDa and 47 MDa (60.8% and 31.6% respectively) are predominated. Gene ypmA was detected in 96.2% of isolated strains. Eight strains had none of the ymp gene variants. HPI were detected in 96.2% of isolated strains. Obtained characteristics of Y. pseudotuberculosis allowed to determine the dominating genogroup pWYV+, ypmA+, HPI- (95.8% of strains) that cause systemic infection.     

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.     

Impact of Yersinia pseudotuberculosis on the in vitro production of cytokines by whole blood cells of donors

The impact of two plasmid (47, 82 MD), single plasmid (47 MD) and non plasmid Y. pseudotuberculosis strains, Y. enterocolitica (47 MD) as well as Y. pseudotuberculosis superantigen (YPM) on the production of interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-alpha (IFN = alpha) and tumor necrosis factor alpha (TNF-alpha) by whole blood cells obtained from donors was studied. All Y. pseudotuberculosis and Y. enterocolitica strains stimulated the production of IFN-alpha, IL-1, IL-6 and TNF-alpha by whole blood cells, but considerably less than Y. pseudotuberculosis lipopolysaccharide and YPM. These data are indicative of the pathogenetic role played by 82 MD plasmid in manifestation of Y. pseudotuberculosis immunosuppressive properties. The maximum stimulation of the production of cytokines was observed under the action of YPM, which confirmed an important role played by this superantigen in the pathogenesis of Y. pseudotuberculosis.     

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.     

Plasmid profile of strains of various Legionella species

The plasmid profile of Legionella strains of different origin has been studied. 15 out of 32 Legionella cultures belonging to different strains have been found to contain plasmid DNA in an amount of 1 or 2 plasmids, with the exception of L. feelei having 6 plasmids. Only 1 out of 3 Legionella strains isolated in the USSR has been found to possess a plasmid with a molecular weight of about 80 MD. Plasmids with this molecular weight have been found in 13 Legionella strains under study, such plasmids in L. pneumophila strains of serogroup 1 (strains Flint 1 and Albuquerque 1) and serogroup 9 (strain No. 35282) having an exact molecular weight of 82.4 +/- 2.4 MD and being similar in molecular structure, which has been established as the result of their treatment with restrictases Pst 1 and Hind III.     

The isolation of Yersinia intermedia strains with a plasmid of 82-megadalton molecular weight in natural populations of Yersinia

For the first time Y. intermedia strains containing plasmids with a molecular weight of 82 MD have been detected in natural populations of urease-positive Yersinia. Such populations have been isolated from two species of birds and from the soil in the area where they have been killed (the Maritime Territory), as well as from washings from the surface of onions in a vegetable store (Chita Province). The strains, administered orally to white mice, proved to be nonpathogenic. Plasmids with a molecular weight of 82 MD are supposed to occur in natural populations of other Yersinia species.     

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.     

Virulence of Yersinia pseudotuberculosis isolated from pork and from the throats of swine

Yersinia pseudotuberculosis was isolated from retail pork and from healthy swine throats. These wild-type strains and their representative cured isogenic strains were tested for the presence of plasmids and several virulence factors, and these characteristics were compared with those of virulent strains from humans. Two pork isolates (serotype IVB) and four swine isolates (serotypes IIB, IIC, III, and IVB) harbored a 42- to 48-megadalton plasmid which had similar fragmentation patterns resulting from digestion with restriction endonuclease. These six strains were lethal for mice via oral challenge and were positive in autoagglutination and calcium dependency tests. They also invaded HeLa cells and induced cytotoxicity. Histopathological examination and indirect fluorescent-antibody staining provided definite evidence of the pathogenicity of these strains when tissue sections from orally infected mice were used. The virulence factors of wild-type pork and swine isolates with the 42- to 48-megadalton plasmid were identical to those of two human isolates (serotypes IVB and VB). Hence, these pork and swine isolates should be considered potentially pathogenic for humans. The finding suggests that retail pork and swine may play an important role in the epidemiology of human infections caused by Y. pseudotuberculosis.     

Virulence of Yersinia pseudotuberculosis isolated from pork and from the throats of swine.

Yersinia pseudotuberculosis was isolated from retail pork and from healthy swine throats. These wild-type strains and their representative cured isogenic strains were tested for the presence of plasmids and several virulence factors, and these characteristics were compared with those of virulent strains from humans. Two pork isolates (serotype IVB) and four swine isolates (serotypes IIB, IIC, III, and IVB) harbored a 42- to 48-megadalton plasmid which had similar fragmentation patterns resulting from digestion with restriction endonuclease. These six strains were lethal for mice via oral challenge and were positive in autoagglutination and calcium dependency tests. They also invaded HeLa cells and induced cytotoxicity. Histopathological examination and indirect fluorescent-antibody staining provided definite evidence of the pathogenicity of these strains when tissue sections from orally infected mice were used. The virulence factors of wild-type pork and swine isolates with the 42- to 48-megadalton plasmid were identical to those of two human isolates (serotypes IVB and VB). Hence, these pork and swine isolates should be considered potentially pathogenic for humans. The finding suggests that retail pork and swine may play an important role in the epidemiology of human infections caused by Y. pseudotuberculosis.     

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.     

Pseudotuberculosis in the Krasnodar Territory. The clinico-epidemiological characteristics of its morbidity and the biological properties of its strains]

Group cases of pseudotuberculosis, first registered at health-restoring institutions of the health resort of Gelendzhik (the Krasnodar Territory), are described. The etiology of these cases of the disease was established on the basis of clinico-epidemiological data, specific seroconversion in 66% of the examined patients and the isolation of Yersinia pseudotuberculosis strains from the feces of four patients. The combination of signs observed in the isolated strains, viz. the presence of plasmids with molecular weights of 75 and 45 MD, resistance to bactericidal factors of normal human serum and autoagglutination in cell culture medium, made it possible to consider these strains to be virulent. The cases of infection were probably caused by the use of fresh cabbage salad. The study of 3,128 rodents caught in the Krasnodar Territory, including those caught in the area of the health resort, resulted in the isolation of 105 Y. enterocolitica strains and strains of 5 other Yersinia species, but not Y. pseudotuberculosis strains. The concept on the saprozoonotic nature of pseudotuberculosis was substantiated.     

A comparative analysis of the Salmonella typhi strains isolated from patients and bacterial carriers

The comparative analysis of 133 S. typhi clinical strains isolated from patients and carriers in Dnepropetrovsk Province in 1978-1987 was carried out. As shown by this analysis, 10 Vi phage types were represented in the set of strains under study, phage types A and F1 being the most numerous ones. Phage type F1 occurred less frequently among the strains isolated from carriers. 31.1% of the strains were found to contain plasmids with different molecular weight ranging from 96 to 0.5 MD. The occurrence of plasmid-containing strains remained at the same level during the whole period under study. Low-molecular plasmids occurred more frequently in the strains isolated from carriers. The minimal suppressive concentrations of a number of antibiotics, such as penicillin, ampicillin, monomycin, chloramphenicol, tetracycline, rifampicin and streptomycin, were determined. 7% of the strains were resistant to penicillin, 9% to monomycin, 15%--to tetracycline and 2.6% to chloramphenicol. The correlation between penicillin and monomycin resistance of the strains and the presence of the plasmid with a molecular weight of 60 MD in these strains was established. All strains were shown to be highly variable in the degree of their virulence: from 10(2) to 10(8). The strains isolated from patients possessed greater virulence.     

Plasmid analysis of cadmium- and penicillin-resistant strains of staphylococci isolated in maternity hospitals]

The plasmid analysis of a collection of the staphylococci isolated was made in the obstetric hospitals of Nizhny Novgorod and Arzamas in 1986-1989. It revealed the presence of large complex plasmids with a molecular weight of 22 MD carrying the determinants of resistance to penicillins and cadmium and mercury ions in polyresistant strains. Conjugation transfer of the plasmid even between the staphylococci of different species was performed under experimental conditions. It was suggested that occurrence of the strains carrying the complex cadmium-penicillinase plasmid was due to violation of the sanitary or ecological requirements.     

The complete genome sequence of Yersinia pseudotuberculosis IP31758, the causative agent of Far East scarlet-like fever

The first reported Far East scarlet-like fever (FESLF) epidemic swept the Pacific coastal region of Russia in the late 1950s. Symptoms of the severe infection included erythematous skin rash and desquamation, exanthema, hyperhemic tongue, and a toxic shock syndrome. The term FESLF was coined for the infection because it shares clinical presentations with scarlet fever caused by group A streptococci. The causative agent was later identified as Yersinia pseudotuberculosis, although the range of morbidities was vastly different from classical pseudotuberculosis symptoms. To understand the origin and emergence of the peculiar clinical features of FESLF, we have sequenced the genome of the FESLF-causing strain Y. pseudotuberculosis IP31758 and compared it with that of another Y. pseudotuberculosis strain, IP32953, which causes classical gastrointestinal symptoms. The unique gene pool of Y pseudotuberculosis IP31758 accounts for more than 260 strain-specific genes and introduces individual physiological capabilities and virulence determinants, with a significant proportion horizontally acquired that likely originated from Enterobacteriaceae and other soil-dwelling bacteria that persist in the same ecological niche. The mobile genome pool includes two novel plasmids phylogenetically unrelated to all currently reported Yersinia plasmids. An icm/dot type IVB secretion system, shared only with the intracellular persisting pathogens of the order Legionellales, was found on the larger plasmid and could contribute to scarlatinoid fever symptoms in patients due to the introduction of immunomodulatory and immunosuppressive capabilities. We determined the common and unique traits resulting from genome evolution and speciation within the genus Yersinia and drew a more accurate species border between Y. pseudotuberculosis and Y. pestis. In contrast to the lack of genetic diversity observed in the evolutionary young descending Y. pestis lineage, the population genetics of Y. pseudotuberculosis is more heterogenous. Both Y. pseudotuberculosis strains IP31758 and the previously sequenced Y. pseudotuberculosis strain IP32953 have evolved by the acquisition of specific plasmids and by the horizontal acquisition and incorporation of different genetic information into the chromosome, which all together or independently seems to potentially impact the phenotypic adaptation of these two strains.     

Influence of culture conditions and virulence plasmids on expression of immunoglobulin-binding proteins of Yersinia pseudotuberculosis

The influence of culture conditions and plasmids on immunoglobulin (Ig)-binding activity of two isogenic strains of Yersinia pseudotuberculosis (plasmid-free strain 48(-)82(-) and strain 48(+)82(+) bearing plasmids pYV48 and pVM82) was studied. The highest activity was observed in the bacteria grown on glucose-containing liquid medium in the stationary growth phase. The Ig-binding activity of the bacteria cultured on the liquid medium at pH 6.0 was about 1.5-fold higher than that of the bacteria grown at pH 7.2. Expression of the Ig-binding proteins (IBPs) was most influenced by temperature of cultivation. The IBP biosynthesis was activated in the bacteria grown at 4 degrees C and markedly decreased in those grown at 37 degrees C. The Ig-binding activity of lysates from the bacteria was caused by proteins with molecular weights of 7-20 kD. The activities of the plasmid-free and plasmid-bearing Y. pseudotuberculosis strains (48(-)82(-) and 48(+)82(+), respectively) were analyzed, and the plasmids were shown to have no effect on the IBP expression and biosynthesis, which seemed to be determined by chromosomal genes.     

Effects of growth temperature and pVM82 plasmid on fatty acids of lipid A from Yersinia pseudotuberculosis

Effects of cultivation temperature (8 or 37 degrees C) and plasmid profile on the lipid A fatty acids of three isogenic Yersinia pseudotuberculosis strains (plasmidless (82-) and strains containing pVM82 (82+) or p57 (57+) plasmids) obtained by alkaline hydrolysis of the whole bacterial cells and differentiated from fatty acids of other membrane lipids were investigated. On the basis of the analysis, it is concluded that lipids A of all studied samples contain 3-hydroxytetradecanoic and dodecanoic acids, a part of which exists as the 3-dodecanoyloxytetradecanoic derivative. The effect of temperature appears in the higher contents of ester- and amide-linked 3-acyloxyalkanoic residues in lipid A from the "cold" variants of the bacteria and is determined by chromosomal genes. The plasmid effect is seen as various responses of the isogenic derivatives to change of growth temperature: in cells of strains 82+ and 82- grown in the cold, the share of lipid A fatty acids in the total population of cellular fatty acids is reduced, while in strains with plasmid p57 it is increased. The temperature variants of the 57+ strain differ by the low contents of amide-linked 3-acyloxyalkanoic acids. Finally, lack of plasmid pVM82 in the "warm" variants of the bacteria results in accumulation of glycolipid molecules deprived of dodecanoic acid. Correlation between growth temperature and plasmid profiles, on one hand, and lipid A fatty acid composition and potential pathogenic properties of the Y. pseudotuberculosis, on the other hand, and also possible mechanisms of thermal adaptation of this organism are discussed.     

Invasiveness and cytotoxicity as criteria in assessing Yersinia attenuation

The electron microscopic study of cells HEp-2 and the complex microbiological and morphological study of tissues and organs of guinea pigs and mice infected with the isogenic pairs of Yersinia strains (Y. pseudotuberculosis I and Y. enterocolitica 09) differing in the presence of the calcium-dependence plasmid, as well as Y. pseudotuberculosis mutants resistant to rifampicin, nalidixic acid or crystal violet without this plasmid, have revealed that the invasiveness and cytotoxicity of the infective agents are not directly related to the presence of the above-mentioned plasmid in these bacteria. The use of the quantitative characteristics of virulence, such as penetration ability, intracellular multiplication, dissemination and the formation of degenerative changes, has made it possible to find out that the mutants of Y. pseudotuberculosis I, yielding the negative result in the keratoconjunctivitis test and resistant to the above-mentioned antimicrobial substances, can be arranged in the following order according to the degree of attenuation: rifr mutants--nalr mutants--kvlr mutants. In contrast to Y. pseudotuberculosis I, the loss of the calcium-dependence plasmid by Y. enterocolitica 09 is accompanied by an essential decrease in their invasive and cytotoxic properties, but this relationship is indirect and unstable. The proposed criteria intended for use in the evaluation of the degree of the manifestation of the invasive and cytotoxic properties of bacteria can be useful for the selection of optimally attenuated Yersinia strains showing promise as vaccine strains.