[MOLECULAR CHARACTERISTICS OF THE MULTIDRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS IN THE NORTHWEST RUSSIA].

The goal of this work was to study the genotypic characteristics of the multidrug-resistant (MDR, i.e., resistant to at least rifampicine and isoniazid) Mycobacterium tuberculosis strains isolated in 2011-2012 from tuberculosis (TB) patients in the Northwest Russia. Spoligotyping of 195 M. tuberculosis isolates identified 14 different spoligotypes and assigned isolates to the genetic families Beijing (n = 162, 83%), LAM (n = 15), H3/URAL (n = 14), as well as T, Haarlem and X. Spoligotypes SIT1 (Beijing), SIT42 (LAM) and SIT262 (H3/URAL) were the most prevalent. Irrespective to the genotype, all the isolates were resistant to streptomycin. The multidrug resistance was accompanied by the resistance to ethionamide (56%), amikacin (31%), kanamycin (40%), and capreomycin (33%). The ethambutol resistance was found in 71% (n = 115) and 42% (n = 14) of the Beijing and non-Beijing strains, respectively (p < 0.05). In conclusion, the multidrug resistant M. tuberculosis population circulating in the Northwest Russia continues to be dominated by the Beijing family strains.

Novel macroarray-based method of Corynebacterium diphtheriae genotyping: evaluation in a field study in Belarus.

In this study, we evaluated a novel macroarray-based spoligotyping method for Corynebacterium diphtheriae strain typing. A total of 20 C. diphtheriae biotype gravis toxigenic isolates collected in Belarus from suspected foci of diphtheria infection (diphtheria cases, carriers, or contacts) were subjected to DNA fingerprinting. All strains had an identical ribotyping profile that was identified as ribotype 'Rossija' by comparison with the international ribotype database at the Institut Pasteur of Paris. A spoligotyping method based on simultaneous reverse-hybridization analysis of two CRISPR (clustered, regularly interspaced short palindromic repeats) loci differentiated these strains into three spoligotypes. Comparison of the spoligotyping results with the epidemiological linkage network helped us to resolve suspected links in the chains of transmission. To conclude, the C. diphtheriae spoligotyping method demonstrated its utility in the field study, in particular, underlining the importance of the use of both CRISPR loci. The generated discrete data can be presented in digital binary format and be easily exchanged between laboratories and stored in local and global databases.

Early ancient sublineages of Mycobacterium tuberculosis Beijing genotype: unexpected clues from phylogenomics of the pathogen and human history.

OBJECTIVE: The Mycobacterium tuberculosis Beijing family is an epidemiologically important lineage subdivided into large-scale phylogenetic sublineages: ancient, endemic in East Asia, and global modern. Here, we analysed ancient sublineages of the Beijing genotype in the Omsk region of southwestern Siberia, an intriguing area at the intersection of European Russia, Siberia, and Central Asia. METHODS: The study included 423 M. tuberculosis strains isolated in 2013-2017 and subjected to drug susceptibility testing, genotyping, and whole genome sequencing. RESULTS: The Beijing genotype constituted 280 out of 423 strains. Forty Beijing strains belonged to the early ancient sublineage (wild type mutT4-48). Of these, 11 belonged to the 14717-15 MIRU-VNTR cluster and had intact RD181, 29 belonged to the 1071-32 cluster and had the RD181 deletion. Thirty-nine ancient strains were multidrug-resistant (MDR) and 20 pre-extensively drug resistant (XDR)/XDR. Comparison with global data demonstrated that these clones circulate mainly in Asian Russia with certain phylogenetic affinity to strains from Japan, Korea, and northeastern China. The genome-wide analysis revealed 29-37 single nucleotide polymorphism distances between isolates from different Russian regions within these two clusters. CONCLUSIONS: Based on phylogenetic, phylogeographic, genomic, and historical data, we hypothesize that these two clones or their direct ancestors were probably brought to Russia ∼70 years ago after the Second World War with Japanese prisoners of war and, until recently, were mainly circulating in Siberia and the Far East. Their elevated prevalence in Omsk along with the extremely strong association with not only MDR but also pre-XDR/XDR also observed in other locations highlight their epidemic potential and the need for monitoring and attention from health authorities.

PCR-based methodology for detecting multidrug-resistant strains of Mycobacterium tuberculosis Beijing family circulating in Russia.

The Beijing genotype of Mycobacterium tuberculosis has been identified in 40-50% of the clinical isolates studied in Russia during the last decade. This genotype has been reported to be associated with multiple drug resistance and possesses some significant pathogenic properties. Therefore, early identification of such strains is of extreme importance in the timely detection of drug resistance. The present study was performed on 354 strains isolated in Russia from 1996 to 2002 and previously characterised by IS 6110-restriction fragment length polymorphism (RFLP) typing and spoligotyping. These strains included 198 Beijing family strains and 156 strains of other genotypes (IS 6110-RFLP profiles). A subsequent polymerase chain reaction (PCR) analysis with IS 6110-derived outwardly oriented primers (IS 6110-PCR) easily discriminated the Beijing strains from non-Beijing strains. The multiplex allele-specific (MAS)-PCR assays were further used to detect mutations in katG315 and rpoB531, associated with resistance to isoniazid and rifampin, respectively. The katG315 and rpoB531 mutations were found to be more prevalent among Beijing (96.8% and 77.3%) than among non-Beijing strains (85.7% and 28%). Consequently, we propose a two-step methodology based on routine PCR and simple agarose gel electrophoresis in order to detect (i) a Beijing family strain using IS 6110-PCR, and, (ii) its possible resistance to the major anti-tuberculosis drugs using specific MAS-PCR assays.