Laboratory evolution of Mycobacterium on agar plates for analysis of resistance acquisition and drug sensitivity profiles

Abstract

Drug-resistant tuberculosis (TB) is a growing public health problem. There is an urgent need for information regarding cross-resistance and collateral sensitivity relationships among drugs and the genetic determinants of anti-TB drug resistance for developing strategies to suppress the emergence of drug-resistant pathogens. To identify mutations that confer resistance to anti-TB drugs in Mycobacterium species, we performed the laboratory evolution of nonpathogenic Mycobacterium smegmatis, which is closely related to Mycobacterium tuberculosis, against ten anti-TB drugs. Next, we performed whole-genome sequencing and quantified the resistance profiles of each drug-resistant strain against 24 drugs. We identified the genes with novel meropenem (MP) and linezolid (LZD) resistance-conferring mutation, which also have orthologs, in M. tuberculosis H37Rv. Among the 240 possible drug combinations, we identified 24 pairs that confer cross-resistance and 18 pairs that confer collateral sensitivity. The acquisition of bedaquiline or linezolid resistance resulted in collateral sensitivity to several drugs, while the acquisition of MP resistance led to multidrug resistance. The MP-evolved strains showed cross-resistance to rifampicin and clarithromycin owing to the acquisition of a mutation in the intergenic region of the Rv2864c ortholog, which encodes a penicillin-binding protein, at an early stage. These results provide a new insight to tackle drug-resistant TB.

Identification of drivers of mycobacterial resistance to peptidoglycan synthesis inhibitors

“…In conclusion, our study outputs imply that mycobacterial resistance to beta-lactams may not be as simplistic as once thought. Confirmation of mutations in Rv2864c and upstream MSMEG_6319 in mutants isolated with meropenem, corroborated previous descriptions of the involvement of the Msm gene in resistance to this carbapenem ( Maeda et al, 2021 ). This is also in agreement with our previous description that Mtb clinical strains with a mutation in LpqK, another lipoprotein with similarity to PBPs and D,D-carboxypeptidases, show reduced meropenem MICs ( Olivença et al, 2022 ).…”

“…Rv2864c is a putative high-molecular-mass PBP with a lipoprotein motif, recently shown to be relatively resistant to all beta-lactam subclasses when compared to other well-known PBPs ( Kumar et al, 2022 ). The mutation in the putative promoter region of MSMEG_6319 (AT701_RS30910) has been previously described in meropenem-resistant Msm mutants ( Maeda et al, 2021 ). The detection of a mutation in Rv2864c in the meropenem-resistant Mtb isolates in our study is in accordance with these observations and further supported by reports of increased cefotaxime sensitivity in a Mtb mutant generated through Rv2864c disruption and by CRISPR interference studies on Mycobacterium abscessus orthologue gene MAB_3167c ( Wivagg et al, 2016 ; Akusobi et al, 2022 ).…”

“…The detection of a mutation in Rv2864c in the meropenem-resistant Mtb isolates in our study is in accordance with these observations and further supported by reports of increased cefotaxime sensitivity in a Mtb mutant generated through Rv2864c disruption and by CRISPR interference studies on Mycobacterium abscessus orthologue gene MAB_3167c ( Wivagg et al, 2016 ; Akusobi et al, 2022 ). Maeda et al (2021) additionally report in their meropenem-selected mutants a non-synonymous mutation in the coding sequence of MSMEG_6319 and an intergenic variant upstream MSMEG_6406 (AT701_RS31385), orthologue of Rv3811 and which encodes a putative N-acetylmuramoyl-l-alanine amidase. We did not observe the two latter features, but two of our meropenem-exposed mutants alternatively displayed a mutation 11 bp upstream the TSS of MSMEG_6317 , adjacent to MSMEG_6319 .…”

mbtD and celA1 association with ethambutol resistance in Mycobacterium tuberculosis: A multiomics analysis

“…In contrast to the method of agar-based selection for laboratory evolution against antibiotics ( Maeda et al., 2021 ) or induced by high-concentration drugs ( Zhao et al., 2014 ), the strategy of a continuous drug concentration gradient can better simulate the accumulation of drugs in the human body. Determination of the MIC value of 14 anti-TB drugs showed that laboratory-induced mono-EMB drug-resistant strains were successfully obtained ( Supplementary Table S2 ).…”

Identificação dos fatores de virulência da Leptospira interrogans através de análises proteômicas comparativas

“…BrkB também deve atuar nos mecanismos de aderência e invasão desta bactéria nas células hospedeiras. Este domínio também pode ser encontrado em outras bactérias como Escherichia coli (Ezraty et al, 2005) e Mycobacterium tuberculosis (Maeda et al, 2021). Foi possível identificar apenas uma proteína com domínio Virul_fac_BrkB na L. interrogans e L. biflexa, sendo caracterizadas como proteínas ortólogas e, posteriormente, classificadas como proteínas pouco conservadas.…”