Pathogenetic significance of the psychrophilia of Yersinia pseudotuberculosis

The work presents the data indicating that the temperature of Y. pseudotuberculosis cultivation is very important in regulating the activity of pathogenicity factors, necessary for the initiation of the pathogenic process in the cells of the macroorganism. Low temperature (8-10 degrees C), necessary for the growth of Y. pseudotuberculosis, facilitates the activation of invasive and toxic pathogenicity factors. At a growth temperature of 37 degrees C the inhibition of such necessary attributes of virulence as adhesion and invasion into epithelial cells occurs in Y. pseudotuberculosis, which decreases the capacity of these bacteria for inducing the infectious process. The virulence of Y. pseudotuberculosis population, lost as the result of its cultivation in synthetic culture media at a temperature of 37 degrees C, has been found to be restored at low temperature.     

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.     

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.     

Yersinia enterocolitica 03 findings on porcine tongues in comparison with yersiniosis incidence in man in Czechoslovakia

Positive isolations of Yersinia obtained in repeated bacteriological examinations of porcine tongues at three slaughter-houses in Prague and a single examination at the slaughter-house at Kladno were compared with notified yersiniosis morbidity. The incidence of illnesses caused by Y. enterocolitica 03 does not exceed values of 4.5/100,000 and 3.5/100,000 population in the Czech and Slovak Socialist Republics, respectively, and is equal to a sixtieth part of the notified shigellosis and salmonellosis morbidity. Cultivation of 334 pooled samples consisting of 1142 porcine tongues yielded 12 strains (1.05%) of Y. enterocolitica 03, five strains (0.44%) of Y. pseudotuberculosis and 55 strains (4.82%) of other Yersinia organisms (indole-positive serotypes). Because of the low isolation rates obtained for the individual Yersinia species, Y. enterocolitica 03 in particular, the isolation efficiency of different cultivation techniques and culture media was statistically evaluated for all Yersinia organisms jointly. Primary cultivation on deoxycholate-citrate medium yielded five of the 12 Y. enterocolitica 03 strains isolated. The other Yersinia strains grew only after preliminary propagation. Yersinia pseudotuberculosis grew almost exclusively (4 out of 5 strains) on McConkey's agar.     

PCR detection of virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and investigation of virulence gene distribution

PCR-based assays were developed for the detection of plasmid- and chromosome-borne virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis, to investigate the distribution of these genes in isolates from various sources. The results of PCR genotyping, based on 5 virulence-associated genes of 140 strains of Y. enterocolitica, were compared to phenotypic tests, such as biotyping and serotyping, and to virulence plasmid-associated properties such as calcium-dependent growth at 37 degrees C and Congo red uptake. The specificity of the PCR results was validated by hybridization. Genotyping data correlated well with biotype data, and most biotypes resulted in (nearly) homogeneous genotypes for the chromosomal virulence genes (ystA, ystB, and ail); however, plasmid-borne genes (yadA and virF) were detected with variable efficiency, due to heterogeneity within the bacterial population for the presence of the virulence plasmid. Of the virulence genes, only ystB was present in biotype 1A; however, within this biotype, pathogenic and apathogenic isolates could not be distinguished based on the detection of virulence genes. Forty Y. pseudotuberculosis isolates were tested by PCR for the presence of inv, yadA, and lcrF. All isolates were inv positive, and 88% of the isolates contained the virulence plasmid genes yadA and lcrF. In conclusion, this study shows that genotyping of Yersinia spp., based on both chromosome- and plasmid-borne virulence genes, is feasible and informative and can provide a rapid and reliable genotypic characterization of field isolates.     

DNA, RNA, and protein biosynthesis in cells of Yersinia pseudotuberculosis at various cultivation temperatures

Y. pseudotuberculosis cells cultivated at temperatures of 37 degrees C and 8 degrees C were found to be capable of incorporating exogenic precursors into DNA, RNA and protein. The linear growth of thymidine incorporation occurred during 8 hours of cultivation at 37 degrees C, then the amount of the incorporated label decreased. At 8 degrees C the level of thymidine incorporation into DNA gradually increased for 80 hours and longer, but not reaching the level of incorporation observed at 37 degrees C. The incorporation of uridine into RNA of Y. pseudotuberculosis cells reached its maximum after 4 hours of cultivation at 37 degrees C, at a lower temperature of cultivation the incorporation of uridine into bacterial cells was almost linear, though slower, and lasted for 20 hours. The content of radioactive alanine in Y. pseudotuberculosis protein increased during 16 hours of cultivation at a high temperature, while at 8 degrees C the growth of the incorporation level lasted for at least 40 hours. For all precursors under study the incorporation rate into the cell biopolymers at the initial stages of cultivation was higher at 37 degrees C, than at a lower temperature.     

Evaluation of a Single Procedure Allowing the Isolation of Enteropathogenic Yersinia along with Other Bacterial Enteropathogens from Human Stools

Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most Gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other Gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools.     

The temperature-induced early and late development of resistance to the bactericidal action of blood serum in Yersinia pseudotuberculosis]

Y. pseudotuberculosis strains were grown at 6 degrees-8 degrees C and then incubated at 37 degrees C. 3-6 hours later serum resistance appeared in the strains having plasmid virulence and producing outer membrane polypeptide with a molecular weight of 120 kD, known as P1. 10-12 hours later serum resistance appeared in the strain having the virulence plasmid, but not producing P1, as well as in strains in which the plasmid was eliminated.     

Variation in lipid A structure in the pathogenic yersiniae

Important pathogens in the genus Yersinia include the plague bacillus Yersinia pestis and two enteropathogenic species, Yersinia pseudotuberculosis and Yersinia enterocolitica. A shift in growth temperature induced changes in the number and type of acyl groups on the lipid A of all three species. After growth at 37 degrees C, Y. pestis lipopolysaccharide (LPS) contained the tetra-acylated lipid IV(A) and smaller amounts of lipid IV(A) modified with C10 or C12 acyl groups, Y. pseudotuberculosis contained the same forms as part of a more heterogeneous population in which lipid IV(A) modified with C16:0 predominated, and Y. enterocolitica produced a unique tetra-acylated lipid A. When grown at 21 degrees C, however, the three yersiniae synthesized LPS containing predominantly hexa-acylated lipid A. This more complex lipid A stimulated human monocytes to secrete tumour necrosis factor-alpha, whereas the lipid A synthesized by the three species at 37 degrees C did not. The Y. pestis phoP gene was required for aminoarabinose modification of lipid A, but not for the temperature-dependent acylation changes. The results suggest that the production of a less immunostimulatory form of LPS upon entry into the mammalian host is a conserved pathogenesis mechanism in the genus Yersinia, and that species-specific lipid A forms may be important for life cycle and pathogenicity differences.     

Chromosomal-encoded siderophores are required for mouse virulence of enteropathogenic Yersinia species

Abstract Yersinia enterocolitica and Y. pseudotuberculosis are enteropathogenic for humans. Essential virulence functions of these pathogens are determined by a 40-mDa plasmid. Plasmid-bearing Y. pseudotuberculosis strains and Y. enterocolitica strains of serotypes 0 : 8, 0 : 13, 0 : 20 and 0 : 40 are lethal for mice. In contrast, human pathogenic Y. enterocolitica strains of serotype 0 : 3, 0 : 9 and 0 : 5.27 are not mouse-lethal. Using a sensitive siderophore-indicator CAS-agar, we were able to detect siderophore production in all mouse-lethal Y. enterocolitica and Y. pseudotuberculosis strains mentioned above. By Tn5-transposon insertions into the chromosome of a serotype 0 : 8 strain we obtained two siderophore-deficient mutants. Introduction of the virulence plasmid did not render these mutants mouse-lethal, indicating that siderophore production is an essential virulence factor. The human nonpathogenic, aerobactin-producing strains of Y. intermedia, Y. kristensenii and Y. frederiksenii remained avirulent for mice after receiving the virulence plasmid of Y. enterocolitica . Obviously the siderophore aerobactin does not contribute to virulence in the genus Yersinia .     

Virulence plasmid-associated autoagglutination in Yersinia spp.

The autoagglutination of Yersinia enterocolitica was dependent on the presence of the virulence plasmid and on the active growth of bacteria in tissue culture media at 37 degrees C. Cultures with a high initial concentration of bacteria failed to autoagglutinate , indicating that synthesis of new virulence plasmid-associated surface factors was essential for autoagglutination. The synthesis of a plasmid-encoded polypeptide (molecular weight, 240,000), designated P1, that could be dissociated under strongly reducing conditions into subunits of 52,500 daltons was found to be correlated with autoagglutination. Further, a strain of Yersinia pseudotuberculosis [ YPIII ( PIB102 )], which has Tn5 inserted within the structural gene of P1 that prevents the synthesis of P1, failed to autoagglutinate , in contrast to the wild-type strain, strongly indicating that P1 is involved in this phenomenon. It was also found by immunoblotting that in addition to the common response to temperature, the P1 proteins of Y. enterocolitica and Y. pseudotuberculosis were immunologically related.     

The effect of temperature and selective agents on the growth of Yersinia enterocolitica serotype O:3 in pure culture

This study examined the individual and combined effects of the selective agents normally present in Yersinia-selective agar (i.e. cefsulodin, irgasan and novobiocin) on the growth kinetics of plasmid-bearing (P+) and plasmid-cured (P-) Yersinia enterocolitica serotype O:3 at 25 and 37 degrees C. Growth studies were carried out in pure culture, and the data obtained were subjected to linear regression analysis to determine lag phase duration(s) and growth rates of the examined strains. In general, the presence of selective agents increased the duration of the lag phase at 37 degrees C, with longer lag phases noted in all cases in which two or more selective agents were present. Growth rates in CIN broth base (CIN NA) and CIN NA plus commercial supplement (SR 109) (CIN) were faster at 37 than 25 degrees C, but in some cultures of incomplete CIN NA broth with less than three supplements added, growth tended to be faster at 25 than 37 degrees C. Generally, plasmid-bearing strains grew slower than plasmid-cured strains in most media at 37 degrees C due to virulence plasmid expression retarding growth. In some instances at 37 degrees C, it was observed that the growth rates of both plasmid-bearing and plasmid-cured strains were comparable, indicating the influence of added selective agent/s negating any effects associated with virulence plasmid expression. The effects of selective agents, incubation temperature and virulence plasmid carriage on the growth kinetics of Y. enterocolitica are discussed.     

Effect of a culturing method and growth phase on composition of lipopolysaccharides in Yersinia pseudotuberculosis

The effects of the culturing method (suspension cultures in a liquid nutrient broth or colonies on a solid agarized medium) and the growth phase on the lypopolysaccharide (LPS) composition of Yersinia pseudotuberculosis (O:Ib serovar, strain KS 3058) grown in cold (5 degrees C) were studied. The amount the LPS synthesized by cells depended on the bacteria growth phase for both media. The LPS acylation degree was constant, whereas the length of the O-specific polysaccharide chain varied with the culture age and achieved maximum in the stationary growth phase for both media. The bacteria culturing on the nutrient agar stimulated more intensive synthesis of LPS, which were extracted more easily, had longer polysaccharide O-chains, and were more toxic than LPS of the bacteria cultured in the liquid medium. It was proposed that the culturing of Yersinia pseudotuberculosis in cold as colonies on the agar surface causes an increase in the bacterial virulence.     

Chemotaxis of Yersinia pseudotuberculosis as a mechanism in its search for target tissues of the host organism

Y. pseudotuberculosis cells grown at biologically low temperature have been shown capable of chemotaxis with respect to carbohydrates and amino acids. During cultivation at 36-37 degrees C Y. pseudotuberculosis cells retain this property for 10-15 hours and then lose it. The mechanism of chemotaxis makes it possible for Y. pseudotuberculosis to "find" human and animal tissues and can facilitate the realization of the pathogenicity potential of these bacteria. When administered orally to mice motile bacteria, i. e. those grown at 6-8 degrees C, have been more virulent for the animals than nonmotile ones cultivated at 36-37 degrees C.     

Recognition of pathogenic Yersinia enterocolitica by crystal violet binding and polymerase chain reaction

Cefsulodin-Irgasan-Novobiocin (CIN) agar is used for the selective isolation and enumeration of Yersinia enterocolitica from clinical specimens and food. The medium contains crystal violet and about 1 mmol l^-1 calcium and can be used for the phenotypic characterization of strains that carry a virulence plasmid. At 32°C, irrespective of pathogenicity, colonies are translucent with a pale pink centre surrounded by a transparent border ('bullseye'), while at 37°C pathogenic strains grow as calcium-dependent microcolonies which, because of crystal violet binding, are intensely coloured. These results were confirmed by the polymerase chain reaction with primers directed at the vir F gene, which is present only in pathogenic strains of Y. enterocolitica. Pathogenic strains of Y. enterocolitica can be recognized by growth at 37°C on Yersinia selective agar.     

Lack of correlation between the presence of plasmids and fimbriae in Yersinia enterocolitica and Yersinia pseudotuberculosis

Thirty-nine strains of Yersinia enterocolitica and 10 strains of Yersinia pseudotuberculosis were studied for the presence of fimbriae, plasmids and two plasmid associated phenotypic expressions, autoagglutination and Ca2+ dependent growth at 37 degrees C. All of the strains studied became fimbriated, which was confirmed by electron microscopy and haemagglutination tests. Fimbriation was not correlated with the presence or absence of plasmids.     

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.     

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.     

Production of bacteriocin-like substances byYersinia frederiksenii,Y. kristensenii,andY. intermedia strains

The production of bacteriocin-like substances by strains of Yersinia frederiksenii, Y. kristensenii and Y. intermedia in broth culture was established. These substances showed a selective activity against Y. enterocolitica, Y. frederiksenii, Y. kristensenii and Y. intermedia strains. Electron micrographs revealed the presence of phage tails in culture media. The production of these substances was detected in cultures grown at 25 degrees C but not in those grown at 37 degrees C, while these bacteriocin-like substances were active at 25 and 37 degrees C. Y. enterocolitica serogroups 0:3 and 0:9 were more susceptible to these bacterin-like substances than strains of Yersinia isolated from environmental sources.