Analysis of genomic polymorphism of typical and atypical strains of the plague pathogen using polymerase chain reaction with universal primers

An analysis of genome polymorphism of the Y. pestis strains by using the method of polymerase chain reaction (PCR) for the tandem repeats of bacteriophage M13 DNA revealed a species similarity of both typical and atypical (according to diagnostic signs) plague-microbe strains. Strain Y. pestis A-1726 with the atypical differential-and-diagnostic properties, without the amplicon specified for Y. pestis and sized 1000 b.p., was identified among 27 analyzed Y. pestis strains. The amplicon profiles of the basic Y. pestis subtype were found to be different from such profiles of other Y. pestis subtypes.     

Numerical analysis of the phenotypic properties and total genomic characteristics of strains of Yersinia pestis related to different subspecies

The numerical analysis of the phenotypical properties of Y. pestis strains, classified with 5 subspecies by 60 signs, was carried out. In comparing the properties of strains belonging to the main (nomenclature) subspecies with strains of other subspecies, the similarity index varied within the range 82-95%. A high degree of genetic affinity between 21 Y. pestis strains of five subspecies was demonstrated by the method of molecular DNA-DNA hybridization. The level of DNA homology with respect to the alpha-CTP.[3H] reference mark of Y. pestis P-1300 in strains belonging to different subspecies was found to be 84-97%. The plasmid spectrum of 25 examined strains of these three subspecies proved to be identical and consisted of plasmids similar in their electrophoretic motility to marker plasmids from Y. pestis strains EV from the Research Institute of Epidemiology and Hygiene and Otten.     

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 population variability of Yersinia pestis in soil samples from the natural focus of plague

Three Y. pestis strains were found to exist in the experimental soil ecosystem at a temperature of 4 degrees - 8 degrees C for a longer period (10 months, the term of observation) than at room temperature (3.5 months). Y. pestis population structure was characterized by relative stability in strains of the subspecies altaica and heterogeneity in the strain of the main subspecies, manifested by the loss of the pgm locus by vegetative cells and the preservation of pgm+ variants in the latent (uncultivable) form (LF). In the populations of all strains uniformity in calcium dependence, the tendency towards a decrease in the synthesis of factor 1, nutritional requirements in amino acids was observed. An important factor of the preservation of Y. pestis in the soil was LF formation. At room temperature this process quickly resulted in the death of the population. At 4 degrees - 8 degrees C A. pestis altaica avirulent strain could be inoculated onto solid nutrient media for a two-fold longer period (for 4 month) than the strain with selective virulence and for 5.5 months longer than Y. pestis pestis highly virulent strain.     

Genetic variations in the pgm locus among natural isolates of Yersinia pestis

A PCR-based screening method was used to study the genetic variations of the pgm locus among natural isolates of Yersinia pestis from China. Our results indicate that genetic variations in the pgm locus are well correlated with biovars of Y. pestis and plague foci, suggesting that the pgm locus plays a role in Y. pestis adaptation to its environment. The gene encoding two-component regulatory system sensor kinase became a pseudogene in all strains of biovar Orientalis due to a thymidine deletion, while it is intact in all the strains of the other biovars. Only strains from Foci H and L are the same as Yersinia pseudotuberculosis in that they have an intact transmembrane helix in the sensor kinase protein, which is lost in all the other strains because of the 18 bp in-frame deletion. The IS100 element that flanks the 39 terminus of the pgm locus was inserted into the chromosome during the within-species microevolution of Y. pestis, which is absent in strains from Foci G, H and L and also in Y. pseudotuberculosis. This fact indicates that the strains from these three foci are of an older lineage of Chinese Y. pestis. It is this IS100 element's absence that maintained high stability of the pgm locus in the Y. pestis strains from these three foci. The IS285 element insertion in the pigmentation segment and the IS100 element insertion in the downstream flanking region of the pgm locus are only present in strains from Foci H and L. The flanking region outside the 59 terminus of the upstream IS100 element is identical in the strains from these two foci, which is different in the other strains. All of these unique characteristics suggest that they are of a special lineage of Chinese Y. pestis.     

PCR快速鉴定鼠疫耶尔森氏菌

建立一种简便、快速、特异的PCR检测方法,用于鼠疫耶尔森氏菌的快速鉴定。针对鼠疫耶尔森氏菌特异的一段染色体序列3a设计引物,扩增-276bp片段的鼠疫标识序列。应用该PCR反应体系,对我国17个生态型及1个待定的生态型共计275株鼠疫耶尔森氏菌及48株相关菌株的PCR扩增结果表明,实验菌株均扩增出预期的276bp片段产物带,48株相关菌株均阴性,其检测灵敏度为100pg DNA。说明该方法用于鼠疫耶尔森氏菌的检测鉴定简便、快捷,具有很高的特异性和敏感性。     

Detection of viable Yersinia pestis by fluorescence in situ hybridization using peptide nucleic acid probes

A successful method has been developed for the detection of live Yersinia pestis, the plague bacillus, which incorporates nascent RNA synthesis. A fluorescent in situ hybridization (FISH) assay using peptide nucleic acid (PNA) probes was developed specifically to differentiate Y. pestis strains from closely related bacteria. PNA probes were chosen to target high copy mRNA of the Y. pestis caf1 gene, encoding the Fraction 1 (F1) antigen, and 16S ribosomal RNA. Among Yersinia strains tested, PNA probes Yp-16S-426 and Yp-F1-55 exhibited binding specificities of 100% and 98%, respectively. Y. pestis grown in the presence of competing bacteria, as might be encountered when recovering Y. pestis from environmental surfaces in a post-release bioterrorism event, was recognized by PNA probes and neither hybridization nor fluorescence was inhibited by competing bacterial strains which exhibited faster growth rates. Using fluorescence microscopy, individual Y. pestis bacteria were clearly differentiated from competing bacteria with an average detection sensitivity of 7.9x10(3) cells by fluorescence microscopy. In the current system, this would require an average of 2.56x10(5) viable Y. pestis organisms be recovered from a post-release environmental sample in order to achieve the minimum threshold for detection. The PNA-FISH assays described in this study allow for the sensitive and specific detection of viable Y. pestis bacteria in a timely manner.     

Genotyping of Yersinia pestis strains on the basis of variation of ribosomal rRNA biosynthesis genes

Analysis of restriction fragment length polymorphism of rRNA genes of Yersinia pestis and Y. pseudotuberculosis strains, circulating in Russian Federation and abroad revealed the effectiveness of ribotyping for differentiation between these microorganisms, as well as for differentiation between different Y. pestis biovars and main and nonmain subspecies of this agent. Use of this method was shown to be promising as a component for the complex molecular typing system of Y. pestis. Variant ribotypes of main and non-main subspecies of Y. pestis strains are presented.     

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.     

Function of large plasmids of the plague pathogen

Function of the largest plasmid of Yersinia pestis was studied. Assay of four strains of highly virulent Y. pestis isolated from different natural sources demonstrated that the characters of endotoxin production, sugar and alcohol fermentation, nitrate reduction as well as requirement in nutrients are mediated by no Y. pestis plasmids. On the contrary, FI and FII antigens are determined by the largest plasmid in some strains and are chromosome-mediated in others. It may well be that the genes for these antigens are comprised within the transposon-like DNA fragment.     

Genetic analysis of biochemical differences of Yersinia pestis strains

Literature data and results of our experimental studies on genetic base of biochemical differentiation of Yersinia pestis strains of various subspecies and biovars are summarized in the review. Data on variability of genes coding biochemical features (sugar and alcohol fermentation, nitrate reduction), the differential development of which are the base of existing phenotypic schemes of Y. pestis strains classification, are presented. Variability of these genes was shown to have possible use for the development of genetic classification of Y. pestis strains of various subspecies and biovars.     

环介导等温扩增技术检测鼠疫耶尔森菌的敏感性和特异性研究

为观察环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术能否适用于我国不同疫源地鼠疫耶尔森菌所有基因组型的检测,本研究建立了一种基于3a靶序列设计特异性引物快速检测鼠疫耶尔森菌的LAMP方法.选择分离自我国11个鼠疫自然疫源地的65株野生代表性鼠疫耶尔森菌株,同...     

Growth of Male-Specific Bacteriophage in Pasteurella Harboring F-Genotes Derived from Escherichia coli

When either the F' lac or the F'Cm plasmid was transferred from Escherichia coli into Pasteurella pseudotuberculosis, the P. pseudotuberculosis (F') strains isolated formed plaques with both ribonucleic acid (RNA)-containing and deoxyribonucleic acid-containing male-specific phages. In contrast, strains of P. pestis harboring E. coli (F') plasmids did not form plaques with male-specific phages, although such strains permitted limited multiplication of phage MS2. The adsorption and burst size of MS2 were approximately the same in both species of Pasteurella, but the per cent of adsorbed MS2 that produced infective centers was much lower in P. pestis than it was in P. pseudotuberculosis. By use of a sib-selection technique of P. pestis (F') cells, we isolated a single clone that could form MS2 plaques. (32)P-labeled MS2 adsorbed equally to and its RNA penetrated equally into both the typical MS2-nonpermissive P. pestis cells and the MS2-permissive P. pestis cells. No host modification occurred after growth of MS2 in Pasteurella. Our data suggest that typical strains of P. pestis inhibit the intracellular development of phage MS2.     

Yersinia pestis pFra shows biovar-specific differences and recent common ancestry with a Salmonella enterica serovar Typhi plasmid

Population genetic studies suggest that Yersinia pestis, the cause of plague, is a clonal pathogen that has recently emerged from Yersinia pseudotuberculosis. Plasmid acquisition is likely to have been a key element in this evolutionary leap from an enteric to a flea-transmitted systemic pathogen. However, the origin of Y. pestis-specific plasmids remains obscure. We demonstrate specific plasmid rearrangements in different Y. pestis strains which distinguish Y. pestis bv. Orientalis strains from other biovars. We also present evidence for plasmid-associated DNA exchange between Y. pestis and the exclusively human pathogen Salmonella enterica serovar Typhi.     

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.     

Comparative study of the structure of some genetic probes used for typing Yersinia pestis strains

A nucleotide sequence common for genetic probes used for detection and investigation of Y. pestis strains MK, IS100, and HRSIII was identified on the basis of restriction, hybridization, and computer analysis. This region of chromosomal DNA is a part of low-molecular BX-probe (about 170 bp) we have developed. The results of genomic fingerprinting of Y. pestis strains by the BX-probe are promising as regards its future utilization for typing the strains isolated in various endemic areas.     

Resistance to levomycetin and activity of several enzymes in Escherichia coli and the agent of plague]

The authors compared the activity of acetyl-CoA-synthetase and of the enzymes belonging to the group of asparaginic acid in levomycetin sensitive and resistant strains of Y. pestis and E. coli. There were revealed marked differences in the activity of aspartase, fumarase, synthetase and desamidase of L-asparagin, and also of the enzyme activated by acetate in the E. coli strains with plasmide resistance. Transmission of R-factor to the pestis was accompanied by decomposition of L-asparadein, formation of AC-CoA. At the same time transformation of L-asparaginic acid catalyzed by aspartase remained on the same low level in the sensitive pestis cultures and their variants with the R-factor. When the resistance was controlled by chromosomal resistance markers, the activity of the enzymes providing formation of L-asparagic acid, its amide and L-malic acid showed no significant change. In chromosomal type of resistance in the mutants of pestis and E. coli the acetyl-CoA-synthetase reaction was as a rule somewhat increased.     

Results of screening of plasmids of Yersinia pestis strains from various regions of endemic central-asian plague focus

The results of the plasmid screening of Yersinia pestis strains isolated from four autonomous focuses on the northern border of the Central Asian zone of plague natural focality are presented. The plasmid profile of Yersinia pestis strains from the focuses is characterized as stable and independent of the source and time of strain isolation. The peculiar characteristic of the strains isolated in Tuva is the presence of an "additional" 15-16 Md plasmid in those strains.     

A study of the nucleotide sequence variability of rha locus genes of Yersinia pestis main and non-main subspecies

A study of the structural and regulatory genes, determining rhamnose fermentation, that are located in the rha locus of the chromosome of Yersinia pestis main and non-main subspecies and of Yersinia pseudotuberculosis of serogroups I-III was performed. The nucleotide sequence of Y. pestis main subspecies differs substantially from those of non-main subspecies and Y. pseudotuberculosis by the presence of a nucleotide substitution in 671 bp location of rhaS gene resulting presumably in the Y. pestis non-main subsp inability to utilize rhamnose. This results in incapability of Y. pestis non-main subspecies to utilize rhamnose. Other nucleotide substitutions found in Y. pestis non-main subspecies strains influence only upon expression efficiency of this diagnostic criterion.