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Phylogenetically related Mycobacterium tuberculosis isolates with wild type rpoB and rifampicin resistance levels around the critical concentration

P Lempens(1,2) A Mansy(1) S M Arega(1) S Akter(1) M Kaswa(3) C J Meehan(4) B C de Jong(1) L Rigouts(1,2)

1:Institute of Tropical Medicine Antwerp; 2:University of Antwerp; 3:Programme National de Lutte contre la Tuberculose, République Démocratique du Congo; 4:Nottingham Trent University

Rifampicin is the strongest anti-tuberculosis drug, resistance to which is mediated through mutations in the RNA polymerase (rpoB) gene. In addition to rpoB, other genes potentially associated with Mycobacterium tuberculosis rifampicin resistance have been described, although their significance is unclear.

Retrospective whole genome sequencing (WGS) analysis of a set of 324 phenotypically rifampicin-resistant isolates obtained from retreatment tuberculosis patients in Kinshasa, Democratic Republic of Congo, and stored at the Institute of Tropical Medicine Antwerp, showed that 50 (15.4%) isolates had a wild type rpoB gene. WGS analysis also revealed that a majority (33/50; 66.0%) of these isolates belonged to M. tuberculosis lineage 4.7, all having the same spoligotype (shared international type (SIT) 144). This association between discordant pheno-geno rifampicin results and specific genotypes suggested the evolution of (an) alternative, non-rpoB mediated resistance mechanism(s). In this study, further phenotypic and genotypic testing was done to characterise these isolates.

Several mutations in genes previously associated with rifampicin resistance potentially explained resistance in these isolates. Retesting the isolates on Löwenstein-Jensen medium yielded minimum inhibitory concentrations around the critical concentration of 40 mg/L, as did retesting of rifampicin-susceptible isolates of lineage 4.7. The majority of lineage 4.7 isolates tested rifampicin-susceptible in MGIT at the critical concentration of 0.5 mg/L. Future studies using RNA expression profiling could shed light on the relatively modest increase in rifampicin MICs observed in lineage 4.7 not linked to mutations in rpoB.

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