Whole-Genome Sequencing of Drug-Resistant Mycobacterium tuberculosis Strains, Tunisia, 2012–2016

To investigate transmission of drug-resistant strains of Mycobacterium tuberculosis in Tunisia, we performed whole-genome sequencing on 46 multidrug-resistant strains isolated during 2012–2016. Core-genome multilocus sequence typing grouped 30 strains (65.2%) into 3 clusters, indicating extensive recent transmission and Haarlem clone predominance. Whole-genome sequencing might help public health services undertake appropriate control actions.

T he emergence of drug-resistant strains of Mycobacterium tuberculosis is hampering the control of tuberculosis (TB) worldwide. In Tunisia in 2017, the estimated percentage of TB patients with multidrug-resistant (MDR)/rifampinresistant TB was 1.1% among those with new infection and 13% among those with previously treated infection (1).
Effective and rapid tools are needed to characterize and track the transmission chains of MDR/rifampin-resistant TB. Whole-genome sequencing (WGS) has shown higher discriminatory power for epidemiologic investigations than have other conventional genotyping methods (e.g., spoligotyping, IS6110 restriction fragment length polymorphism, and mycobacterial interspersed repetitive unit-variable-number tandem repeat). Indeed, WGS has enabled investigators to rule out false transmission events (2)(3)(4)(5)(6)(7). Furthermore, WGS enables simultaneous determination of polymorphisms and insertions/deletions linked to resistance to first-line and second-line drugs (8).
In this study, we used WGS to investigate transmission of MDR and extensively drug resistant (XDR) TB strains isolated in Tunisia over a 4-year period by applying the core-genome multilocus sequence typing (cgMLST) scheme and identifying the drug-resistance marker for first-line and second-line drug resistance. This study was approved by the ethics committee of A. Mami Pneumology Hospital, Ariana, Tunisia.

The Study
We retrospectively studied 46 MDR M. tuberculosis isolates collected from 46 HIV-negative patients in Tunisia during June 2012-June 2016, which represented 57 (80.7%) cases of MDR TB. Of the 46 isolates, 6 represented all (100%) XDR TB cases recorded in the country during that period. We performed drug-susceptibility testing for resistance to first-line drugs (except pyrazinamide) by using the proportion method on Lowenstein-Jensen medium. For pyrazinamide and second-line drugs, we performed drug-susceptibility testing on a Bactec MGIT 960 system (Becton, Dickinson and Company, http://www.bd.com). WGS was performed on the MiniSeq platform (Illumina Inc., https:// www.illumina.com) targeting a minimum average reads coverage of 50-fold. To analyze the mutations involved in drug resistance and related to lineage determination, we used PhyResSe and TGS TB (9,10). We performed the cgMLST scheme version 2.1, considering 2,891 core genes, by using Ridom SeqSphere+ version 5.0.0 software (Ridom GmbH, https://www.cgmlst.org) (7). To define a strain as a part of a recent transmission chain, we fixed a threshold of <6 allele variants. For statistical analyses, we calculated p values by using OpenEpi version 3 (https://www.openepi. com) and considered p<0.05 to be significant.
The cluster analysis ( Figure 2) showed that 30 (65.2%) of 46 isolates were grouped within 3 clusters, and most (90.0%) clustered MDR TB cases belonged to the Haarlem family ( Figure 1). Of note, a big cluster of 24 MDR strains linked to Haarlem (cluster 1) was detected over

Whole-Genome Sequencing of Drug-Resistant
Mycobacterium tuberculosis Strains, Tunisia, 2012-2016 the entire study period. We found no significant associations between this cluster and patient sex, age (<35 years, >35 years), and resistance to second-line drugs (XDR TB) (p>0.05); however, we found a significant association with this cluster and pyrazinamide resistance (p = 0.001) and with Bizerte (p = 0.014). Despite the association with the Bizerte region, patients from various regions were part of this cluster ( Figure 2). We also found a significant association between cluster 2 (Haarlem) and Beja (p<0.001) and between cluster 3 (Latin-American-Mediterranean) and Ben Arous (p<0.001). Epidemiologic links were confirmed for patients in these 2 transmission chains ( Table 1). All cluster 1 strains shared mutations in drug-resistance genes (e.g., rpoB, Ser450Leu, the compensatory  mutation rpoBVal534Met (11), katG Ser315Thr, embB Met306Ile, and gidBArg47Trp). However, diversity was noticed in pncA mutations conferring resistance to pyrazinamide. On the basis of pncA variation, this cluster was split into 6 groups (Table 1).
Four XDR isolates belonged to cluster 1. Three XDR isolates from 2 brothers (patients 10942 and 14666) and their neighbor (patient 8626) shared mutations conferring resistance to second-line drugs and to pyrazinamide (Tables 1, 2), supporting evidence of direct transmission of this XDR strain. The fourth XDR TB case (patient/strain 13792) differed in mutations conferring resistance to pyrazinamide and fluoroquinolones. This case probably shared an ancestral MDR strain with cluster 1, which had evolved differently because of poor adherence of the patient to drug therapy ( Table 2; Appendix). Five nonclustered MDR isolates linked to Haarlem (4 patients from Bizerte) were distant by 6-11 alleles from cluster 1 showing the same polymorphisms in rpoB, katG, embB, and gidB (Figure 2; Appendix). Of note, cluster 2 presented the compensatory mutation rpoB Val534Met, but no variation in embB, gidB, and pncA was detected (Appendix). This Haarlem cluster showed limited transmission compared with cluster 1, which was distant by 97 alleles (Figure 2).
Regarding second-line drugs, all 13 fluoroquinoloneresistant strains had a mutation in the quinolone resistancedetermining region of gyrA, gyrB, or both. For secondline injectable drugs, 4 XDR strains had the mutation rrs A1401G and 2 had the mutation eis C-14T and a frame shift in tlyA (Table 2).   (13,14). As indicated by statistical association, we conclude that this cluster is still spreading in the Bizerte area. However, diffusion in different regions of the country is alarming and requires intensified efforts to control and diagnose drug-resistant TB. Only 2 strains, belonging to cluster 1, did not have any mutations in pncA. The wild-type pncA isolates might represent the genotype of the first strains that emerged in Bizerte and evolved since 2001 by acquiring single-nucleotide polymorphisms in pncA and the other genes, including genes involved in resistance to second-line drugs. It has been reported that the mutation rate during a transmission chain or TB latency is not completely stable and is estimated at 0.3-0.5 single-nucleotide polymorphisms/ genome/year (4,5,15), which leads to increased numbers of allele variants for some isolates and might explain the results found for 5 MDR strains distant from cluster 1 by 6-11 alleles.
The main limitation of this study is the incomplete number of MDR TB cases (≈19% missing). Epidemiologic information to confirm all clustered cases is lacking.
In summary, cgMLST-based WGS showed extensive transmission of MDR/XDR TB in Tunisia over 4 recent years, thereby indicating that MDR TB is not fully controlled. Use of this molecular approach for surveillance purposes might enable the public health service to undertake appropriate control actions, particularly in specific settings of this country.