Title: Transcontinental spread and evolution of an MDR M. tuberculosis clade over five decades

Authors: Matthias Merker^1,2,3,*,Jean-Philippe Rasigade^4,5,7,*, Maxime Barbier^4,5,*,Helen Cox⁶, Silke Feuerriegel^1,2, Thomas A. Kohl^1,2, Egor Shitikov⁸, Kadri Klaos⁹, Cyril Gaudin¹⁰, Rudy Antoine¹¹, Roland Diel¹², Sonia Borrell^13,14, Sebastien Gagneux^13,14, Vladyslav Nikolayevskyy¹⁵, Sönke Andres¹⁶, Valeriu Crudu¹⁷, Ulrich Nübel^18,19, Philip Supply^11,#, Stefan Niemann^1,2,# Thierry Wirth^4,5,# * and # contributed equally

Affiliations: ¹Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany; ²German Center for Infection Research, Partner site Hamburg-Lübeck-Borstel-Riems, Germany; ³Evolution of the Resistome, Research Center Borstel, Borstel, Germany; ⁴Laboratoire Biologie Intégrative des Populations, Evolution Moléculaire,EPHE, PSL University, Paris, France; ⁵Institut de Systématique, Evolution, Biodiversité, ISYEB, Muséum national d’Histoire naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, Paris, France; ⁶Division of Medical Microbiology and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; ⁷CIRI INSERM U1111, University of Lyon, Lyon, France; ⁸Federal Research and Clinical Centre of Physical-Chemical Medicine, Moscow, Russian Federation; ⁹SA TUH United Laboratories, Mycobacteriology, Tartu, Estonia; ¹⁰Genoscreen, Lille, France; ¹¹Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 9017 - CIIL - Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France; ¹²Institute for Epidemiology, Schleswig-Holstein University Hospital, Kiel, Germany; ¹³Swiss Tropical and Public Health Institute, Basel, Switzerland; ¹⁴University of Basel, Basel, Switzerland; ¹⁵Imperial College London, London, United Kingdom; ¹⁶Division of Mycobacteriology (National Tuberculosis Reference Laboratory), Research Center Borstel, Borstel. Germany; ¹⁷National TB Reference Laboratory, Physiopneumology Institute, Chisinau, Republic of Moldova; ¹⁸Microbial Genome Research, Leibniz-Institut DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; ¹⁹German Center for Infection Research, Braunschweig site, Germany

Abstract: Transmission and evolution of multidrug resistant (MDR) Mycobacterium tuberculosis complex (Mtbc) strains are jeopardizing the long-term efficacy of new antibiotics and newly endorsed combination therapies. Especially certain MDR clades belonging to Mtbc lineage 2 (Beijing genotype) are extremely successful in multiple settings in Eurasia.

Here, we employed comparative genomics, and Bayesian statistics of 720 isolates from 23 countries to trace the epidemic spread over time, and to identify genetic factors of transmission success of the MDR W148 European/Russian clade.

The most recent common ancestor of this clade was dated around 1963 and the bacterial population underwent two successive epidemic expansion events in the late 1980s and late 1990s. These population expansions were accompanied by the evolution of resistance against up to 11 different anti-TB drugs. Notably, MDR strains acquired additional resistances against fluoroquinolones and second line injectable drugs, and thus an extensively drug resistant (XDR) genotype, on average within 20 years. Time-scaled haplotypic density analysis, as a surrogate marker for recent expansion events, revealed that widespread acquisition of compensatory mutations was associated with transmission success, particularly of the most resistant W148 strains. Virtually all W148 strains harbored a hypervirulence-associated ppe38 gene locus, and incipient recurrent emergence of prpR mutation-mediated drug tolerance was detected.

We show that the W148 European/Russian clade within Mtbc lineage 2 has evolved an outstanding genetic background which is an important factor for its’ long lasting, continent-wide epidemic success over five decades. The ongoing expansion of these strains with MDR/XDR genotypes is a challenge for phenotypic as well as genotypic antimicrobial susceptibility tests, and jeopardize the successful implementation of new MDR-TB antibiotics.