SUMMARY: Increased interest in the pathogenic potential ofYersinia pestishas emerged because of the potential threats from bioterrorism. Pathogenic potential is based on genetic factors present in a population of microbes, yet most studies evaluating the role of specific genes in virulence have used a limited number of strains. ForY. pestisthis issue is complicated by the fact that most strains available for study in the Americas are clonally derived and thus genetically restricted, emanating from a strain ofY. pestisintroduced into the United States in 1902 via marine shipping and subsequent spread of this strain throughout North and South America. In countries from the former Soviet Union (FSU), Mongolia, and China there are large areas of enzootic foci ofY. pestisinfection containing genetically diverse strains that have been intensely studied by scientists in these countries. However, the results of these investigations are not generally known outside of these countries. Here we describe the variety of methods used in the FSU to classifyY. pestisstrains based on genetic and phenotypic variation and show that there is a high level of diversity in these strains not reflected by ones obtained from sylvatic areas and patients in the Americas.

Yersinia pestis, the pathogen of plague, has greatly influenced human history on a global scale. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), an element participating in immunity against phages' invasion, is composed of short repeated sequences separated by unique spacers and provides the basis of the spoligotyping technology. In the present research, three CRISPR loci were analyzed in 125 strains of Y. pestis from 26 natural plague foci of China, the former Soviet Union and Mongolia were analyzed, for validating CRISPR-based genotyping method and better understanding adaptive microevolution of Y. pestis. Using PCR amplification, sequencing and online data processing, a high degree of genetic diversity was revealed in all three CRISPR elements. The distribution of spacers and their arrays in Y. pestis strains is strongly region and focus-specific, allowing the construction of a hypothetic evolutionary model of Y. pestis. This model suggests transmission route of microtus strains that encircled Takla Makan Desert and ZhunGer Basin. Starting from Tadjikistan, one branch passed through the Kunlun Mountains, and moved to the Qinghai-Tibet Plateau. Another branch went north via the Pamirs Plateau, the Tianshan Mountains, the Altai Mountains and the Inner Mongolian Plateau. Other Y. pestis lineages might be originated from certain areas along those routes. CRISPR can provide important information for genotyping and evolutionary research of bacteria, which will help to trace the source of outbreaks. The resulting data will make possible the development of very low cost and high-resolution assays for the systematic typing of any new isolate.

The species Yersinia pestis is commonly divided into three classical biovars, Antiqua, Medievalis, and Orientalis, belonging to subspecies pestis pathogenic for human and the (atypical) non-human pathogenic biovar Microtus (alias Pestoides) including several non-pestis subspecies. Recent progress in molecular typing methods enables large-scale investigations in the population structure of this species. It is now possible to test hypotheses about its evolution which were proposed decades ago. For instance the three classical biovars of different geographical distributions were suggested to originate from Central Asia. Most investigations so far have focused on the typical pestis subspecies representatives found outside of China, whereas the understanding of the emergence of this human pathogen requires the investigation of strains belonging to subspecies pestis from China and to the Microtus biovar. Multi-locus VNTR analysis (MLVA) with 25 loci was performed on a collection of Y. pestis isolates originating from the majority of the known foci worldwide and including typical rhamnose-negative subspecies pestis as well as rhamnose-positive subspecies pestis and biovar Microtus. More than 500 isolates from China, the Former Soviet Union (FSU), Mongolia and a number of other foci around the world were characterized and resolved into 350 different genotypes. The data revealed very close relationships existing between some isolates from widely separated foci as well as very high diversity which can conversely be observed between nearby foci. The results obtained are in full agreement with the view that the Y. pestis subsp. pestis pathogenic for humans emerged in the Central Asia region between China, Kazakhstan, Russia and Mongolia, only three clones of which spread out of Central Asia. The relationships among the strains in China, Central Asia and the rest of the world based on the MLVA25 assay provide an unprecedented view on the expansion and microevolution of Y. pestis.

φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis , the etiologic agent of plague, and used by the CDC in the identification of Y. pestis . φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc ( l - glycero - d - manno- heptose/ d -glucopyranose)-Kdo/Ko (3-deoxy- d - manno -oct-2-ulopyranosonic acid/ d - glycero - d - talo- oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis .

Plague still poses a significant threat to human health, and interest has been renewed recently in the possible use of Yersinia pestis as a biological weapon by terrorists. The septicaemic and pneumonic forms are always lethal if untreated. Attempts to treat this deadly disease date back to the era of global pandemics, when various methods were explored. The successful isolation of the plague pathogen led to the beginning of more scientific approaches to the treatment and cure of plague. This subsequently led to specific antibiotic prophylaxis and therapy for Y. pestis. The use of antibiotics such as tetracycline and streptomycin for the treatment of plague has been embraced by the World Health Organization Expert Committee on Plague as the ‘gold standard’ treatment. However, concerns regarding the development of antibiotic-resistant Y. pestis strains have led to the exploration of alternatives to antibiotics. Several investigators have looked into the use of alternatives, such as immunotherapy, non-pathogen-specific immunomodulatory therapy, phage therapy, bacteriocin therapy, and treatment with inhibitors of virulence factors. The alternative therapies reported in this review should be further investigated by comprehensive studies of their clinical application for the treatment of plague.