High fluoroquinolone resistance proportions among multidrug-resistant tuberculosis driven by dominant L2 Mycobacterium tuberculosis clones in the Mumbai Metropolitan Region
Authors: Dreyer Viola , Mandal Ayan , Dev Prachi , Merker Matthias , Barilar Ivan , Utpatel Christian , Nilgiriwala Kayzad , Rodrigues Camilla , Crook Derrick W. ,   the CRyPTIC Consortium, Crook Derrick W. , Peto Timothy E. A. , Walker A. Sarah , Hoosdally Sarah J. , Gibertoni Cruz Ana L. , Carter Joshua , Earle Sarah , Kouchaki Samaneh , Yang Yang , Walker Timothy M. , Fowler Philip W. , Wilson Daniel , Clifton David A. , Iqbal Zamin , Hunt Martin , Knaggs Jeff , Cirillo Daniela M. , Borroni Emanuele , Battaglia Simone , Ghodousi Arash , Spitaleri Andrea , Cabibbe Andrea , Tahseen Sabira , Nilgiriwala Kayzad , Shah Sanchi , Rodrigues Camilla , Kambli Priti , Surve Utkarsha , Khot Rukhsar , Niemann Stefan , Kohl Thomas , Merker Matthias , Hoffmann Harald , Todt Katharina , Plesnik Sara , Ismail Nazir , Omar Shaheed Vally , Ngcamu Lavania Joseph Dumisani , Okozi Nana , Yao Shen Yuan , Thwaites Guy , Thuong Thuong Nguyen Thuy , Ngoc Nhung Hoang , Srinivasan Vijay , Moore David , Coronel Jorge , Solano Walter , Gao George F. , He Guangxue , Zhao Yanlin , Ma Aijing , Liu Chunfa , Zhu Baoli , Laurenson Ian , Claxton Pauline , Wilkinson Robert J. , Koch Anastasia , Lalvani Ajit , Posey James , Gardy Jennifer , Werngren Jim , Paton Nicholas , Jou Ruwen , Wu Mei-Hua , Xiao Yu-Xin , Ferrazoli Lucilaine , de Oliveira Rosangela Siqueira , Millard James , Warren Rob , Van Rie Annelies , Lapierre Simon Grandjean , Rabodoarivelo Marie-Sylvianne , Rakotosamimanana Niaina , Nimmo Camus , Musser Kimberlee , Escuyer Vincent , Cohen Ted , Rasigade Jean-Philippe , Wirth Thierry , Mistry Nerges , Niemann Stefan 
Published: August 2022

Abstract
Background
Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains are a serious health problem in India, also contributing to one-fourth of the global MDR tuberculosis (TB) burden. About 36% of the MDR MTBC strains are reported fluoroquinolone (FQ) resistant leading to high pre-extensively drug-resistant (pre-XDR) and XDR-TB (further resistance against bedaquiline and/or linezolid) rates. Still, factors driving the MDR/pre-XDR epidemic in India are not well defined.

Methods
In a retrospective study, we analyzed 1852 consecutive MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai by whole genome sequencing (WGS). Univariate and multivariate statistics was used to investigate factors associated with pre-XDR. Core genome multi locus sequence typing, time scaled haplotypic density (THD) method and homoplasy analysis were used to analyze epidemiological success, and positive selection in different strain groups, respectively.

Results
In total, 1016 MTBC strains were MDR, out of which 703 (69.2%) were pre-XDR and 45 (4.4%) were XDR. Cluster rates were high among MDR (57.8%) and pre-XDR/XDR (79%) strains with three dominant L2 (Beijing) strain clusters (Cl 1–3) representing half of the pre-XDR and 40% of the XDR-TB cases. L2 strains were associated with pre-XDR/XDR-TB (P < 0.001) and, particularly Cl 1–3 strains, had high first-line and FQ resistance rates (81.6–90.6%). Epidemic success analysis using THD showed that L2 strains outperformed L1, L3, and L4 strains in short- and long-term time scales. More importantly, L2 MDR and MDR + strains had higher THD success indices than their not-MDR counterparts. Overall, compensatory mutation rates were highest in L2 strains and positive selection was detected in genes of L2 strains associated with drug tolerance (prpB and ppsA) and virulence (Rv2828c). Compensatory mutations in L2 strains were associated with a threefold increase of THD indices, suggesting improved transmissibility.

Conclusions
Our data indicate a drastic increase of FQ resistance, as well as emerging bedaquiline resistance which endangers the success of newly endorsed MDR-TB treatment regimens. Rapid changes in treatment and control strategies are required to contain transmission of highly successful pre-XDR L2 strains in the Mumbai Metropolitan region but presumably also India-wide.