Vrste, filogenetske karakteristike i genetička osnova rezistencije multirezistentnih sojeva Mycobacterium tuberculosis kompleksa izolovanih u Srbiji

Abstract

Jedan od najvećih problema sa kojim se suočavaju programi kontrole tuberkuloze (TB) je širenje multirezistentnih (MDR) sojeva Mycobacterium tuberculosis kompleksa (MTK), odnosno sojeva rezistentnih najmanje na rifampicin i izoniazid, dva najvažnija antituberkulotika (ATL). Molekularno genetička karakterizacija MDR sojeva MTK prisutnih u datoj populaciji predstavlja važan korak u uspostavljanju adekvatnih mera kontrole MDR TB, ali ciljana istraživanja genoma MDR sojeva izolovanih u Srbiji do sada nisu rađena. Stoga su ovim istraživanjem obuhvaćeni svi MDR sojevi MTK izolovani u Srbiji tokom poslednjih šest godina i izvršena je molekularna identifikacija do nivoa vrste, genotipizacija izolata u cilju sagledavanja njihove filogenetske strukture i karakteristika transmisije, kao i detekcija mutacija odgovornih za rezistenciju na ATL. Za istraživanje su formirane dve kolekcije sojeva: svi MDR sojevi MTK izolovani u Srbiji u periodu 01.01.2008.-31.05.2014. godina (115 sojeva) i svi sojevi MTK izolovani od pacijenata obolelih od plućne TB u Srbiji tokom perioda 01.12.2010.-30.11.2011. godina (971 soj). Molekularna identifikacija ispitivanih sojeva MTK izvršena je primenom komercijalnog eseja zasnovanog na reakciji reverzne hibridizacije. Za genotipizaciju MDR sojeva MTK korišćene su konvencionalne tehnike spoligotipizacija i MIRU-VNTR tipizacija, a sekvenciranje genoma nove generacije (NGS) izvršeno je na Illumina MiSeq sekvenatoru, tehnikom sekvenciranja putem sinteze i reverzibilnih terminatora. Klasteri MDR sojeva MTK identifikovani primenom klasičnih tehnika genotipizacije definisani su kao grupe od najmanje dva soja sa identičnim genotipovima, a klasteri definisani na osnovu rezultata NGS kao grupe sojeva čiji su se genomi razlikovali u ≤12 polimorfizama pojedinačnih nukleotida (SNP). Identifikacija faktora rizika za transmisiju MDR TB u Srbiji izvedena je na osnovu poređenja demografskih, kliničkih, epidemioloških i laboratorijskih karakteristika pacijenata čiji su izolati bili svrstani u klastere i pacijenata čiji su MDR sojevi imali jedinstvene genotipove. Izvršena je sveobuhvatna analiza rezultata sekvenciranja gena koji se smatraju ključnim za rezistenciju na ATL prve i druge linije. Rezultati ovog istraživanja pokazali su da svi ispitivani MDR sojevi pripadaju vrsti M. tuberculosis i da ne postoji razlika u profilima vrsta MTK koje izazivaju TB i MDR TB u Srbiji...

The spread of multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains, defined as resistant to at least the two most important antituberculous drugs isoniazid and rifampicin, is a major threat for tuberculosis (TB) control programmes. Molecular genetic characterization of MDR MTBC strains circulating in a population is essential to guide establishment of efficient MDR TB control strategies, but no such analysis of MDR MTBC strains isolated in Serbia has been performed so far. Therefore, this research included a representative collection of MDR MTBC strains isolated in Serbia, and molecular identification to the species level, comprehensive genotyping, and detection of mutations in genes conferring resistance to the first- and second-line anti-TB drugs were performed. The research included two collections of MTBC strains: all MDR MTBC strains isolated in Serbia from January 1st 2008 to May 31st 2014 (115 strains), and all MTBC strains isolated from patients diagnosed with pulmonary TB in Serbia from December 1st 2010 to November 30th 2011 (971 strains). Molecular identification of all MTBC strains was performed by using a commercial assay based on reverse hybridization. Spoligotyping and MIRU-VNTR methods were used as classical genotyping techniques, while next generation sequencing (NGS) by synthesis and reversible terminators was performed using the Illumina MiSeq sequencer. In classical genotyping analyses, a cluster was defined as a minimum of two strains harbouring identical genotype pattern. Genomic clusters were defined where no more than 12 single-nucleotide polymorphisms (SNPs) separated a patient isolate from that of at least one other patient in the cluster. Risk factors for active transmission of MDR TB in Serbia were identified by comparison of demographic, clinical, epidemiologic, and laboratory characteristics of patients whose strains were grouped in clusters and patients whose strains exhibited unique genotypes. Comprehensive analysis of mutations detected in genes associated with resistance to anti-TB drugs was performed. All MTBC strains analysed in this research were identified as M. tuberculosis species, which clearly shows that causative agents of TB and MDR TB in Serbia have identical species profile. NGS analysis revealed a presence of six genotypes among MDR MTBC strains, namely TUR, Clade 1, Beijing, Haarlem, S, and Ural...