Thymine- and thymidine-dependence of Yersinia pestis mutants with altered thymidylate synthetase

Thymine- and thymidine-dependent mutants of Y. pestis strain EV-76 have been isolated and characterized. Obtaining Y. pestis thymine-dependent mutants in trimethoprim-containing media with full nutritional value in the presence of thymine and thymidine and the capacity of natural strains from the foci of infection in Transcaucasia and Mongolia to grow in such media indicate that Y. pestis has gene tpp controlling thymidine phosphorylase, but this enzyme is strongly suppressed under normal conditions. The capacity for its suppression under definite conditions and the degree of the activation of thymidine phosphorylase determine the realization of Thy and Thyd phenotypes in Y. pestis mutants under study, though both types of these mutants have a mutation damage of gene thy A coding the synthesis of thymidylate synthetase.     

The sensitivity of bacterial strains of Yersinia pestis isolated from voles to normal human serum]

The work deals with the results of the study of the viability of Y. pestis strains, isolated from voles in different natural foci of plague, in human normal serum (HNS) and its dependence on the assimilation of ions of exogenic iron. The cultures isolated at the Transcaucasian mountain focus of infection and having no small plasmid pYP were found highly sensitive to HNS. The introduction of the sources of iron, such as hemoglobin or ferritin, into the serum decreased its bactericidal effect.     

Increased invasiveness as one of the manifestations of phenotype variability of the plague microbe in fleas

Experimental studies conducted on genetically connected virulent subcultures of Y. pestis showed that the death of albino mice infected by flea bite occurred earlier than in the animals infected by a syringe subcutaneously. A high invasiveness of Y. pestis subcultures isolated from fleas (in comparison with the initial strains and subcultures from the animals) persisted for 2--3 passages in their cultivation on artificial nutrient media.     

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28 degrees C and 37 degrees C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Several proteins with molecular weights ranging from 34 kDa to 71 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could also be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discussed.     

Development of an improved selective agar medium for isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

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...     

The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague

Iron acquisition in Yersinia pestis is fundamental to the success of plague pathogenesis. We have previously identified an approximately 5.6 kb region (yfe) of Y. pestis genomic DNA, capable of restoring iron-deficient growth but not siderophore production to an Escherichia coli mutant (SAB11) incapable of synthesizing the siderophore, enterobactin. The yfe locus of Y. pestis, found in both pigmented (Pgm+) and nonpigmented (Pgm-) strains, comprises five genes arranged in two distinct operons (yfeA-D and yfeE ). The larger of these, yfeABCD, encodes an ABC transport system, whose expression is iron and Fur regulated and is repressed in cells grown in the presence of manganese. Cells from a Pgm-, Yfe- (DeltayfeAB ) mutant strain of Y. pestis exhibited reduced transport of both 55Fe and 54Mn. Furthermore, cells containing an intact yfe locus showed reduced 55Fe uptake when competing amounts of MnCl2 or ZnCl2 were present, whereas 54Mn uptake was inhibited by FeCl3 but not by ZnCl2. Similarly, yfe mutants of Y. pestis exhibited growth defects on media supplemented with the iron chelators 2,2'-dipyridyl or conalbumin. These growth defects were not relieved by supplementation with MnCl2. A ybt-, DeltayfeAB mutant of Y. pestis was completely avirulent in mice infected intravenously (LD50 > 1.7 x 107 cfu) compared with its parental ybt-, yfe+ strain, which had an LD50 of < 12. In addition, compared with its ybt+, yfe+ parent, a ybt+, DeltayfeAB mutant of Y. pestis had an approximately 100-fold increase in the LD50 from a subcutaneous route of infection. These data suggest that the Yfe and Ybt systems may function effectively to accumulate iron during different stages of the infectious process of bubonic plague.     

Y. pestis phage of a new serovar

Phage II, isolated from Y. pestis strain 2247 obtained from a desert focus in Central Asia, has been studied. The phage is classified with moderate phages and essentially differs from moderate phages of serovar 2. The sources of isolation, high specificity and the absence of common serological features with presently known Y. pestis phages of serovars 1 and 2 permit the classification of this phage with new serovar 3 of Y. pestis phages.     

Virulence, viability and antibiotic sensitivity of the causative agent of plague growing on nutrient media at 28 and 37 degrees C

A comparative study of virulence, viability and antibiotic sensitivity of Y. pestis strains grown at 28 degrees C and 37 degrees C in yeast-casein medium, yeast medium with Hottinger's meat digest and yeast medium with protein hydrolysate obtained from sunflower seed groats has been made. These media have been found to be suitable for the prolonged cultivation of Y. pestis at 28 degrees C and 37 degrees C, for the determination of its sensitivity to antibiotics, as well as for the preservation of Y. pestis cultures.     

Role of histamine receptors of mouse and guinea pig peritoneal leukocytes in the pathogenetic effect of Yersinia pestis adenylate cyclase]

The effect of purified preparation of adenylate cyclase isolated from Y. pestis on peritoneal leukocytes of white mice and guinea pigs was investigated. Y. pestis adenylate cyclase was shown to accomplish its pathogenic action via histamine-specific receptors on the surface of eukaryotic cells. The involvement of H1 and H2 histamine receptors on target cells in the adenylate cyclase action leading to development of plague infection is discussed.     

Yersinia pestis L-forms in rodents and ectoparasites

Y. pestis L-forms and bacterial forms persist in the body of great gerbils for 40 days. L-forms are poorly phagocytized and can persist in phagocytes for a long time. In guinea pigs immunized with vaccine EV, Y. pestis antigen could be detected till day 160. An unstable L-form was isolated from Ornithodoros mites 3 years after their experimental infection with Y. pestis. Bacterial forms persist in mites for 1-3 years. For 5 years Y. pestis antigen is regularly detected in a high percentage of mites.     

Proline-dependent wild strains and proline-dependent mutants of the plague microbe]

It is found that the growth of Yersinia pestis wild strains, isolated from Citellus musicus Menetrié in the Central Caucasus, depends on the presence of proline in the medium. Proline can not be substituted by glutamic acid, other amino acids or vitamins. 28 proline-requiring mutants were selected from Y. pestis marmot strain 20b. Three groups of proline-requiring Y. pestis mutants are established, similar to those of Escherichia coli. The requirement of proline does not affect the virulency, pigment formation and calcium dependence.     

Yersinia philomiragia sp. n., a New Member of the Pasteurella Group of Bacteria, Naturally Pathogenic for the Muskrat (Ondatra zibethica)

A bacterium experimentally pathogenic for muskrats (Ondatra zibethica), white mice, mountain voles (Microtus montanus), and deer mice (Peromyscus maniculatus) was isolated from the tissues of a sick muskrat captured on the Bear River Migratory Bird Refuge (Brigham City, Utah) and from four surface water samples collected within 15 miles of that point. In culture, the cells are chiefly coccoid, but in the tissues of muskrats and voles they resemble the bizarre forms of Yersinia pestis, except for their smaller size. The characteristics of the organism are described and the name Yersinia philomiragia sp. n. is proposed.     

Cold temperature-induced modifications to the composition and structure of the lipopolysaccharide of Yersinia pestis

Following a report of variations in the lipopolysaccharide (LPS) structure of Yersinia pestis at mammalian (37 degrees C) and flea (25 degrees C) temperatures, a number of changes to the LPS structure were observed when the bacterium was cultivated at a temperature of winter-hibernating rodents (6 degrees C). In addition to one of the known Y. pestis LPS types, LPS of a new type was isolated from Y. pestis KM218 grown at 6 degrees C. The core of the latter differs in: (i) replacement of terminal galactose with terminal d-glycero-d-manno-heptose; (ii) phosphorylation of terminal oct-2-ulosonic acid with phosphoethanolamine; (iii) a lower content of GlcNAc, and; (iv) the absence of glycine; lipid A differs in the lack of any 4-amino-4-deoxyarabinose and presumably partial (di)oxygenation of a fatty acid(s). The data obtained suggest that cold temperature switches on an alternative mechanism of control of the synthesis of Y. pestis LPS.     

Immunogenicity of Yersinia pestis grown on nutrient media at 28 and 37 degrees C

The immunogenicity of Y. pestis strain EV, grown in yeast-casein medium, yeast medium with Hottinger digest and yeast medium with sunflower-seed protein at 28 degrees C and 37 degrees C, for guinea pigs and white mice has been studied. As revealed in this study, these media ensure the formation of highly immunogenic populations of Y. pestis strain EV and, therefore, can be used for growing Y. pestis vaccine strains. Considerable fluctuations in the content of such highly protective antigen as fraction 1 do not affect the immunogenicity of live cultures of Y. pestis strain EV. This is due to the leveling of differences in the content of this antigen in the process of the multiplication of these bacteria in laboratory animals.     

Multiple antimicrobial resistance in plague: an emerging public health risk

Antimicrobial resistance in Yersinia pestis is rare, yet constitutes a significant international public health and biodefense threat. In 1995, the first multidrug resistant (MDR) isolate of Y. pestis (strain IP275) was identified, and was shown to contain a self-transmissible plasmid (pIP1202) that conferred resistance to many of the antimicrobials recommended for plague treatment and prophylaxis. Comparative analysis of the DNA sequence of Y. pestis plasmid pIP1202 revealed a near identical IncA/C plasmid backbone that is shared by MDR plasmids isolated from Salmonella enterica serotype Newport SL254 and the fish pathogen Yersinia ruckeri YR71. The high degree of sequence identity and gene synteny between the plasmid backbones suggests recent acquisition of these plasmids from a common ancestor. In addition, the Y. pestis pIP1202-like plasmid backbone was detected in numerous MDR enterobacterial pathogens isolated from retail meat samples collected between 2002 and 2005 in the United States. Plasmid-positive strains were isolated from beef, chicken, turkey and pork, and were found in samples from the following states: California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York and Oregon. Our studies reveal that this common plasmid backbone is broadly disseminated among MDR zoonotic pathogens associated with agriculture. This reservoir of mobile resistance determinants has the potential to disseminate to Y. pestis and other human and zoonotic bacterial pathogens and therefore represents a significant public health concern.     

Yersinia pestis kills Caenorhabditis elegans by a biofilm-independent process that involves novel virulence factors

It is known that Yersinia pestis kills Caenorhabditis elegans by a biofilm-dependent mechanism that is similar to the mechanism used by the pathogen to block food intake in the flea vector. Using Y. pestis KIM 5, which lacks the genes that are required for biofilm formation, we show that Y. pestis can kill C. elegans by a biofilm-independent mechanism that correlates with the accumulation of the pathogen in the intestine. We used this novel Y. pestis-C. elegans pathogenesis system to show that previously known and unknown virulence-related genes are required for full virulence in C. elegans. Six Y. pestis mutants with insertions in genes that are not related to virulence before were isolated using C. elegans. One of the six mutants carried an insertion in a novel virulence gene and showed significantly reduced virulence in a mouse model of Y. pestis pathogenesis. Our results indicate that the Y. pestis-C. elegans pathogenesis system that is described here can be used to identify and study previously uncharacterized Y. pestis gene products required for virulence in mammalian systems.     

cAMP-binding protein--a protein kinase of the plague pathogen]

In Y. pestis a cyclic AMP-binding protein was detected, isolated to a homogeneous state, and its physico-chemical properties were studied. The protein is a highly molecular compound with a molecular weight of 180 kD, capable of being released into the environment in the process of cell growth and having protein kinase activity, not depending on the presence of cyclic AMP. Y. pestis neuraminidase is one of the substrates appearing due to the action of protein kinase detected in this study. Y. pestis protein kinase may alter the spectrum of protein phosphorylation in the leukocytes of white mice. The direct participation of this protein in the development of infection is supposed.     

Experimentally induced plague infection in the northern grasshopper mouse (Onychomys leucogaster) acquired by consumption of infected prey

In this study, 20 laboratory reared Onychomys leucogaster from a parental population that is naturally exposed to plague were each fed a white mouse that had been inoculated with Yersinia pestis. Three of the 20 O. leucogaster died, four survived with antibody titers against Y. pestis and 13 survived with no titer against Y. pestis. In contrast, when 20 O. leucogaster from a plague naive parental population were fed infected prey, seven died and 13 survived with no antibody titer against Y. pestis. Our results suggest another means by which O. leucogaster from populations that are naturally exposed to plague may acquire the disease.     

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.