A highly rifampicin resistant Mycobacterium tuberculosis strain emerging in Southern Brazil
Introduction
Tuberculosis (TB) remains a major public health emergency worldwide and current efforts to control the disease have been threatened by the high rates of drug-resistant TB (DR-TB) cases [1]. The overall success rate for TB treatment is 82%, but multidrug resistance (MDR-TB) - resistance to isoniazid (INH) and rifampicin (RIF), the two main anti-TB drugs - is associated with worse treatment outcome, dropping the cure rates to 60% [1]. RIF is one of the most important drugs used in the anti-TB treatment, due to its high bactericidal effects. RIF mechanism action consists of binding at RNA polymerase (RNAp) beta-subunit, encoded by rpoB gene, resulting in the inhibition of the bacterial mRNA transcription [2,3]. The occurrence of RIF-resistance in Mycobacterium tuberculosis (M. tuberculosis) is generally associated to single nucleotide substitutions at rpoB gene, mainly at rifampicin resistance determining region (RRDR), an 81-base pair region comprising from codon 426 to 452 [4].
Previous studies conducted in Rio Grande do Sul, a high TB burden setting and largest state in Southern Brazil, described an uncommon insertion of 12 nucleotides (12 nt) at the codon 435 (codon 516 in Escherichia coli based rpoB numbering system) in the rpoB gene among MDR M. tuberculosis strains. The 12 nt insertion results in a duplication of four amino acids (QNNP) and was firstly described by Perizzolo et al. (2012) [5]. Following studies analyzing MDR strains from Rio Grande do Sul [[6], [7], [8], [9]] [[6], [7], [8], [9]] [[6], [7], [8], [9]] also reported strains carrying on the 12 nt insertion, and to our knowledge, this insertion was not described in any other region worldwide.
Hence, for an enhanced TB control management in Southern Brazil, is need the elucidation concerning molecular and phenotypic consequences of that 12 nt insertion, as well as, comprehension on this strain spreading into population. For this propose, we screened RIF-resistant M. tuberculosis strains from Rio Grande do Sul State using a WGS-based population study approach, in order to identify strains carrying on the 12 nt insertion. Afterward, we examine the phylogenetic relationship among these strains, the phenotypic consequences of the insertion into resistance level and biological cost, and also the in silico prediction to RIF binding effect.
Section snippets
Sample collection and drug susceptibility testing (DST)
In total 16 M. tuberculosis strains carrying on the 12 nt insertion at rpoB gene were identified on a WGS-based state-wide study that is being currently conducted in Rio Grande do Sul, Southern Brazil. The study included 174 RIF-resistant strains collected from 2011 to 2014, and 169 of them presented multidrug resistance. The M. tuberculosis clinical strains were from the State Central Laboratory (LACEN - Rio Grande do Sul), the reference laboratory in charge of performing drug susceptibility
Population and phenotypic drug resistance
Among the 174 RIF-resistant strains from Rio Grande do Sul, collected from 2011 to 2014, we identified 16 (9.19%) strains carrying on a 12 nt insertion at codon 435 of the rpoB gene, all of them MDR. Amid those 16 MDR strains, one was additionally resistant to EMB and other to STR, according to DST results. Regarding the clinical and epidemiological characteristics, 14 were male, 14 previously treated for TB and seven HIV-infected. Besides that, unfavorable outcome was seen for 13 patients, the
Discussion
Rio Grande do Sul is a high burden TB state in Southern Brazil with elevated rates of MDR-TB. The state has an estimated population of 11,3 million people [45] and in 2018 were accounted for 4541 new TB cases [46] and around 82 MDR diagnosed (data from LACEN-RS). Our group has been exploring M. tuberculosis genetic diversity and its transmission across Rio Grande do Sul State in the last two decades. In 2012, Perizzolo et al. [5] first described the presence of a 12 nt insertion at rpoB gene
Conclusion
Here we described the key molecular and phenotypic aspects from a M. tuberculosis strain emerging in Southern Brazil in recent years that carries an uncommon 12 nt insertion at rpoB gene leading high-level RIF-resistance. The phylogenetic approach demonstrated a potential transmission from the prison inmate population to community of the 12 nt insertion strain, demonstrating an need for enhanced surveillance on TB transmission amid the inmate population and the anti-TB treatment abandonment,
Funding
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES), (grantnumber: 001), and supported by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), (grant number: 17/1265–8 INCT-TB).
Declaration of competing interest
The authors declare no competing interests.
Acknowledgments
We are grateful to TGen, C-Path and ReSeqTB for supporting whole genome sequencing and to Brazilian Network of Tuberculosis Research (REDE-TB) for enabling this partnership. The authors would like to thank Centro de Desenvolvimento Científico e Tecnológico (CDCT)/Centro Estadual de Vigilância em Saúde/Secretaria Estadual da Saúde do Rio Grande do Sul for the support and infrastructure.
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MLR and PEAS contributed equally.