Single Nucleotide Polymorphisms in hsp65 and MACPPE12 Genes of Mycobacterium avium subsp. hominissuis

Russian Journal of Genetics - Mycobacterium avium subsp. hominissuis (MAH) are typical inhabitants of the environment, which are known as opportunistic pathogens of animals and humans. The aim of...     

Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.     

At Baltic crossroads: a molecular snapshot of Mycobacterium tuberculosis population diversity in Kaliningrad, Russia

The Kaliningrad region is the westernmost part of the Russian Federation; it includes an enclave on the Baltic Sea inside the European Union separated from mainland Russia by Lithuania and Poland. The incidence of tuberculosis in Kaliningrad has shown a steady and dramatic increase from 83/100 000 in 2000 to 134/100 000 in 2006; the rate of multidrug-resistant tuberculosis (MDR-tuberculosis) in the Kaliningrad region was reported to be 30.5% among newly diagnosed tuberculosis patients. This study presents a first molecular snapshot of the population diversity of Mycobacterium tuberculosis in this region. A total of 90 drug-resistant and susceptible M. tuberculosis strains from Kaliningrad were subjected to spoligotyping, 12-locus MIRU typing and mutation analysis of the drug resistance genes rpoB and katG . A comparison with international databases showed that the M. tuberculosis population in this region shares a joint pool of strains with the European part of Russia, and also exhibits a certain affinity with those of its northern European neighbours, such as Poland and Germany. Comparison of the genotyping and drug resistance data emphasized that the high prevalence of the MDR Beijing genotype strains is a major cause of the adverse epidemiological situation of MDR-tuberculosis in the Kaliningrad region.     

Mycobacterium tuberculosis Latin American-Mediterranean Family and Its Sublineages in the Light of Robust Evolutionary Markers

Mycobacterium tuberculosis has a clonal population structure, and the Latin American-Mediterranean (LAM) family is one of the largest and most widespread within this species, showing evidence for remarkable pathobiology and a confusing phylogeny. Here, we applied robust phylogenetic markers to study the evolution of the LAM family and its major sublineages circulating in Russia and neighboring countries. A total of 250 M. tuberculosis isolates were confirmed to belong to the LAM family based on the analysis of the LAM-specific single-nucleotide polymorphisms (SNPs) in the Rv3062 and Rv0129c genes. At this stage, the family status was rectified for 121 isolates misleadingly assigned by CRISPR spoligotyping to non-LAM families (T1- or T5-RUS1). Consequently, the reestimated LAM prevalence rate increased 2-fold in Russia and Kazakhstan and 4-fold in Belarus. The majority (91.8 to 98.7%) of the LAM isolates from all three countries belonged to the LAM-RUS sublineage. In contrast, the Ibero-American LAM RD-Rio sublineage was identified in only 7 Russian isolates. Taken together, our findings and further analyses suggest a monophyletic origin of LAM-RUS: at a historically distant time, in Russia, in a small founding bacterial/human population. Its dissemination pattern and high prevalence rate in Northern Eurasia may indicate a long-term coexistence of the LAM-RUS sublineage and local human populations hypothetically leading to coadaptation and reduced pathogenicity of the relatively more ancient clones, such as spoligotype international type 254 (SIT254), compared to the more recent SIT252 and SIT266 clones. In contrast, rare LAM RD-Rio isolates were likely brought to Russia through occasional human contact. The spread of RD-Rio strains is not as global as commonly claimed and is determined largely by human migration flows (rather than by pathobiological properties of these strains). Consequently, a host population factor appears to play a major role in shaping the in situ dissemination pattern of the imported strains in an autochthonous population.     

Corynebacterium diphtheriae spoligotyping based on combined use of two CRISPR loci

A large diphtheria epidemic in the 1990s in Russia and neighboring countries underlined the importance of permanent surveillance of the circulating and emerging clones of Corynebacterium diphtheriae, and hence there is a need for highly discriminatory, simple and portable typing methods. In the complete genome sequence of C. diphtheriae strain NCTC13129, we previously identified in silico two clustered, regularly interspaced short palindromic repeat (CRISPR) loci, and developed a macroarray-based method to study polymorphism in the larger DRB locus. We named this method spoligotyping (spacer oligonucleotide typing), analogously to a similar method of Mycobacterium tuberculosis genotyping. Here, we included in the analysis novel spacers of the other CRISPR locus in C. diphtheriae (DRA); both loci were simultaneously co-amplified and co-hybridized against the membrane with 27 different immobilized spacer-probes. The use of additional DRA spacers improved strain differentiation and discriminated within large DRB clusters. The 156 Russian strains of the epidemic clone were subdivided into 45 combined spoligotypes compared to 35 DRB-spoligotypes and only two ribotypes ('Sankt-Peterburg' and 'Rossija'). The spoligotyping method allows digital presentation of profiles and therefore it is perfectly suitable for interlaboratory comparison and database management; it may become a powerful tool for epidemiological monitoring and phylogenetic analysis of C. diphtheriae.     

Mycobacterium tuberculosis Population in Northwestern Russia: An Update from Russian-EU/Latvian Border Region

This study aimed to characterize the population structure of Mycobacterium tuberculosis in Pskov oblast in northwestern Russia, to view it in the geographical context, to compare drug resistance properties across major genetic families. Ninety M. tuberculosis strains from tuberculosis (TB) patients, permanent residents in Pskov oblast were subjected to LAM-specific IS6110-PCR and spoligotyping, followed by comparison with SITVITWEB and MIRU-VNTRplus databases. The Beijing genotype (n = 40) was found the most prevalent followed by LAM (n = 18), T (n = 13), Haarlem (n = 10), Ural (n = 5), and Manu2 (n = 1); the family status remained unknown for 3 isolates. The high rate of Beijing genotype and prevalence of LAM family are similar to those in the other Russian settings. A feature specific for M. tuberculosis population in Pskov is a relatively higher rate of Haarlem and T types. Beijing strains were further typed with 12-MIRU (followed by comparison with proprietary global database) and 3 hypervariable loci QUB-3232, VNTR-3820, VNTR-4120. The 12-MIRU typing differentiated 40 Beijing strains into 14 types (HGI = 0.82) while two largest types were M2 (223325153533) prevalent throughout former USSR and M11 (223325173533) prevalent in Russia and East Asia. The use of 3 hypervariable loci increased a discrimination of the Beijing strains (18 profiles, HGI = 0.89). Both major families Beijing and LAM had similar rate of MDR strains (62.5 and 55.6%, respectively) that was significantly higher than in other strains (21.9%; P = 0.001 and 0.03, respectively). The rpoB531 mutations were more frequently found in Beijing strains while LAM drug resistant strains mainly harbored rpoB516 and inhA −15 mutations. Taken together with a high rate of multidrug resistance among Beijing strains from new TB cases (79.3% versus 44.4% in LAM), these findings suggest the critical impact of the Beijing genotype on the current situation with MDR-TB in the Pskov region in northwestern Russia.     

Biological properties of some nitrogen-fixing associative enterobacteria

Enterobacteria isolates from potato tubers were able to fix nitrogen, to protect plants against phytopathogens and to produce phytohormones thus increasing the plant yield. These isolates were previously phenotypically identified as Erwinia carotovora; however, they differed from typical E. carotovora in a number of biological characteristics and were found to be nonphytopathogenic (avirulent) due to the lack of pectate lyase activity. A data matrix, containing 31 strains and 105 biological characteristics was used for computer cluster analysis. The avirulent strains formed a separate cluster more closely related to Klebsiella spp. strains (with a 0.67 level of similarity) than to typical phytopathogenic bacteria of the E. carotovora group (with a 0.48 level of similarity). A phylogenetic analysis based on restriction polymorphisms of an amplified ribosomal DNA spacer region revealed that the avirulent strains studied here were different from all Erwinia, Klebsiella and other enterobacteria species strains. The AP PCR/hybridization technique showed cross homology of amplified DNA of these avirulent strains and a lack of such homology with the DNA from strains of other species. Numerical taxonomy data, rDNA analysis and AP PCR/hybridization assays confirmed that these avirulent bacteria may be regarded as an independent group of enterobacteria.     

Genotypic characteristics of Mycobacterium avium subsp. hominissuis strains

Mycobacterium avium are typical environmental, non-tuberculosis microorganisms that occasionally cause mycobacteriosis, an infectious disease in wild and domestic animals, birds, and humans. Here, we report the results of the first study on the genetic diversity of the Russian population of M. avium. A total of 85 M. avium subsp. hominissuis (MAH) clinical strains were isolated from patients (including 30 HIV-positive individuals) with mycobacteriosis in St. Petersburg, 2008–2011. The identification of the microorganisms was carried out using biochemical tests and the PCR detection of the mobile elements IS901 and IS900, as well as of the polymorphism of restriction fragments of the hsp65 gene. The genetic diversity of the isolates was evaluated by VNTR typing based on eight variable-number tandem repeats (VNTRs) (292, X3, 25, 47, 3, 7, 10, and 32 [Thibault et al., 2007]). The MAH population studied was characterized by 15 VNTR types, including nine unique patterns and six clusters of isolates with identical eight-digit profiles. The largest clusters (22221128 and 24221128) included 45 (59.2%) and 15 (19.7%) isolates, respectively; the others contained 2–7 strains. The strains of the cluster 2533112’8 possessed a truncated TR10 locus (allele 2′). Taking into account the absence of the epidemiological links between the patients and the fact that the infection was presumably delivered from the environment, the high rate of clustering of MAH isolates can be explained by the low discriminatory power of the eight-locus VNTR-typing scheme (HGDI 0–0.61).     

Emerging multidrug resistant Mycobacterium tuberculosis strains of the Beijing genotype circulating in Russia express a pattern of biological properties associated with enhanced virulence

The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.