Phenotypic instability of Mycobacterium tuberculosis strains harbouring clinically prevalent drug-resistant mutations

Whole-genome sequencing (WGS) has shown great potential in the rapid diagnosis of drug-resistant tuberculosis;1Finci I Albertini A Merker M et al.Investigating resistance in clinical Mycobacterium tuberculosis complex isolates with genomic and phenotypic antimicrobial susceptibility testing: a multicentre observational study.Lancet Microbe. 2022; 3: e672-e682Summary Full Text Full Text PDF PubMed Scopus (1) Google Scholar, 2Li J Feng S Chen Y Yu M Wei W Tian GB Antimicrobial susceptibility testing in clinical Mycobacterium tuberculosis isolates.Lancet Microbe. 2022; (published online Oct 27.)https://doi.org/10.1016/S2666-5247(22)00299-3Summary Full Text Full Text PDF Google Scholar however, the discrepancies between WGS-based drug susceptibility testing (DST) and phenotypic DST have not been well characterised due to a scarcity of high-quality phenotypic DST data. Recently, the largest, globally sourced dataset of Mycobacterium tuberculosis strains with WGS data and phenotypic DST data was generated by WHO,3Walker TM Miotto P Koser CU et al.The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis.Lancet Microbe. 2022; 3: e265-e273Summary Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 4WHOCatalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance.https://www.who.int/publications/i/item/9789240028173Date: June 25, 2021Date accessed: November 17, 2022Google Scholar providing a valuable and comprehensive resource for studying discrepancies between genotypic and phenotypic DST in detail. We characterised the phenotypic instability of genotypically resistant M tuberculosis strains on the basis of the dataset generated by WHO. High-quality genotypic and phenotypic DST data were available for 38 215 M tuberculosis isolates collected from 45 countries across six continents. The discrepant results were placed into two categories: (1) genotypic DST predicted drug-susceptibility while phenotypic DST found drug-resistance; and (2) genotypic DST predicted drug-resistance while phenotypic DST found drug-susceptibility. The first category potentially reflects the need to identify additional drug-resistant mutations, particularly for new and repurposed drugs (appendix), for which knowledge of resistance mechanisms is limited. However, although about 91% of isolates resistant to isoniazid and rifampicin harboured known drug-resistant mutations, the remaining proportion (around 9%) still represents a large number of patients whose resistance would not be detected by genotypic DST (appendix). We found the second scenario (ie, M tuberculosis strains with known drug-resistant mutations that have a drug-susceptible phenotype) especially interesting. When we analysed M tuberculosis strains with drug-resistant mutations identified by genotypic DST, we found the highest proportion of these strains to have phenotypic susceptibility to the antibiotic moxifloxacin (36%), and the lowest proportions to isoniazid (3%) and rifampicin (3%; appendix). We further found that for most antibiotics, strains that were phenotypically susceptible while having genotypic resistance had a variety of drug-resistant mutations, but were much more frequent with rare drug-resistant mutations, such as rpoB L430P (appendix). However, for moxifloxacin and ethambutol, we identified a high frequency of strains with this phenotypic instability with some of the clinically prevalent mutations (gyrA A90V, 52% strains; embB M306I, 39%). Although phenotypic instability was low with the most common drug-resistant mutations, such as rpoB S450L (1% strains) and katG S315T (1%), because of their high frequency in clinical strains they accounted for a large proportion of the phenotypically susceptible but genotypically resistant strains (rpoB S450L, 27% for rifampicin; katG S315T, 31% for isoniazid; appendix). In conclusion, we found that phenotypic instability of genotypically resistant strains is a characteristic that cannot be ignored in clinical M tuberculosis isolates. Future research is required to identify the molecular mechanisms underlying this phenotypic instability and develop corresponding solutions. We declare no competing interests. This study was supported by the National Natural Science Foundation of China (grant number 82272376 to QG) and the Shenzhen High-level Hospital Construction Fund (grant number G2022157 to QG). Download .pdf (.36 MB) Help with pdf files Supplementary appendix