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Evolution and success of the multi-drug resistant M. tuberculosis SIT41 (LAM7-TUR) lineage

Periodic Reporting for period 1 - MDR Tuberculosis (Evolution and success of the multi-drug resistant M. tuberculosis SIT41 (LAM7-TUR) lineage)

Reporting period: 2015-10-01 to 2017-09-30

The proposed project “Evolution and success of the multidrug-resistant Mycobacterium tuberculosis SIT41 (LAM7-TUR) lineage” was carried out at the Infection Genetics Emerging Pathogen Evolution Team (IGEPE) under the supervision of Prof. Christophe Sola.
Mycobacterium tuberculosis (MTB) is the cause of tuberculosis (TB) in humans. Multidrug resistant tuberculosis (MDR TB) is a major concern for control TB programs. Identification of the MTB lineage is of importance to tuberculosis control as it has shown that strain type may play a role in disease outcome, variation in vaccine efficacy and emergence of drug resistance. MTB strains of Beijing and Latin-American-Mediterranean (LAM) lineages were associated with hyper-transmissibility and drug resistance. In 2013 the European Center for Diseases Prevention and Control (ECDC) effectively lunched molecular surveillance of MDR TB at EU level. Within this EU project the typing of M. tuberculosis strains in Bulgaria confirmed strong association between MDR and SIT41 (TUR) [SIT – Spoligo International Type] MTB lineage. The MDR TB cases with SIT41 strain genotype in Bulgaria increased from 40% to 60% in the period 2007-2013, while the drug sensitive genotype represented only 2%. Statistically 97% of the TB patients infected with drug sensitive SIT41 MTB strain develop MDR tuberculosis. SIT41 is marker genotype for MDR TB.
Moreover the project promoted the research career restart of the Fellow by obtaining during the course of the action a Doctor of Sciences degree.
The genotyping results were reported and extensively exploited by the Bulgarian TB program and the ECDC for epidemiological surveillance reports and the ECDC database of MDR-TB genotypes. Fifteen publications and congress reports were produced during the course of the Action. The poster presented at the annual congress of the European Society of Mycobacteriology (ESM) was awarded with third price for best congress poster.
The conclusions of the project are:
- The endemic SIT41 MDR genotype, spreading in Bulgaria, was not found in other countries, suggesting local evolution.
- The main characteristic of the Bulgarian SIT41 strains is the 11 VNTR copy number of QUB-26(4052) marker.
- Contact tracing analysis could not explain how patients living at a distance of >350 km have identical MIRU-VNTR and cgMLST profiles.
- Applying on-going WGS analysis we hope to date the start of this ongoing MDR endemy, to identify true transmissions across the country. The clonal expansion of SIT41 among the MDR-TB patients in Bulgaria remains to be further analyzed.
The Host laboratory offered outstanding quality, innovative research opportunities and work in a world-wide laboratory network of excellence (Brazil, South Africa, the Netherlands, Kazakhstan and others).
The work plan targeted working on two project objectives:

Objective 1: Application of the next generation sequencing data for genotyping of M. tuberculosis strains based on SNP data.
Description: Tracking the origins, evolution and success of the M. tuberculosis SIT41(LAM7-TUR) lineage by WGS of SIT41 genotype and other MTB strains (n=>1600); Identification of lineage specific SNPs and selection of SNPs for new typing scheme
Methodology: bioinformatics, application of new SNP typing scheme, spoligotyping, 24 loci MIRU-VNTR typing, whole genome sequencing
D1.1 Development of genotyping SNP based scheme for improved SIT41 identification and cluster subtyping
D1.2 Identification and evaluation of high and low confidence SNPs on the basis of known phenotypic drug sensitivity data of the sequenced MTB strains for prediction of drug resistance in MTB

Objective 2: Development of rapid screening strategy to identify the SIT41 (TUR) genotype including mutation analysis to first and second line tuberculostatics drug resistance.
Description: Validation of microbead-based multiplex test for identification of SIT41 genotype strains including mutation prediction of drug resistance to first and second line tuberculosis drugs for population health intervention. Development of high throughput screening scheme for SIT41 genotyping and drug resistance prediction on filter card formats.
D2.1 Validation of locally adapted multiplex test (specific for Bulgaria) for SIT41 typing and identification of MDR/XDR strains.
D2.2 Development of SIT41 screening strategy for simultaneous lineage and drug resistance genotyping of clinical samples on filter card formats

Research results:
More than 400 drug sensitive and 100 multi-drug resistant (MDR) M. tuberculosis strains have been genotyped applying an automated microbeads 'Luminex' based technique. The isolation of the strains was performed in collaboration with the National reference laboratory of Tuberculosis of Bulgaria. All MDR-TB strains were also genotpyed applying 24 loci MIRU-VNTR technique. Whole genome sequencing (WGS) analysis was performed successfully on 37 of the MDR-TB strains. WGS analysis was performed in collaboration with the San Rafaelle Scientific Center, Milano, Italy (Dr. Daniela Cirillo). With the applied three genotyping techniques the transmission evolution of the MDR SIT41 strains was well documented. Four main clones were identified. The biggest spoligo/MIRU-VNTR clone was composed by more than 65 TB strains (see publications section). We proved that the lineage and genotype identification by applying the MagPix system is highly efficient, reliable and fast compared to the traditional mambrane based genotyping.
A dataset of genotyping results including spoligo and 24 loci MIRU-VNTR genotypes was deposited to the EU Open Access database Zenodo (DOI: 10.5281/zenodo.1065289)
The evolutionary success of the Bulgarian SIT41 MDR-TB genotype was followed and confirmed. The genotype is highly specific and limited to Bulgaria. Comparative analysis with strains of SIT41 from Turkey (in collaboration with Prof. Riza Durmaz, Ankara) identified different MIRU-VNTR profiles. We confirmed that the Bulgarian MTB strains of SIT41/MIRU-VNTR genotype have independent evolution.
On going analysis aim to identify the number of the most informative SNPs responsible for drug resistance in MTB SIT41 genotype. The development of screening strategy for SIT41 genotype on DNA obtained from clinical samples fixed on filter cards, such as the FTA card system (Watman International Ltd.) or the GenoCard (HainLifesciences, Nehren, Germany) is on-going. Preliminary results with sputum samples inactivated in ethanol for biosafety and DNA fixation on filter cards were promising. DNA extraction will be standardised by using Chelex100 solution. Identification of genotype lineage and resistance mutations to first and second-line tuberculostatics will be tested on the MagPix system. The developed screening strategy will have practical applications addressing the patients treatment.
The research project results will support the EU Health Strategy, ECDC, WHO TB END, and the Global Fund to Fight TB initiatives. The action promoted partnership by developing of new knowledge and skills within EU member states and thus building extensive collaboration. In the long term, the action results will improve the epidemiological surveillance of the MDR TB cases in Bulgaria and EU.
Participation at the Microbiology and Infection Congress 2017, Germany