title: Non-pncAGene-Mutated but Pyrazinamide-Resistant Mycobacterium tuberculosis: Why Is That?
abstract: Pyrazinamide (PZA) is a key component for the effective treatment of drug-susceptible and PZA-susceptible multidrug-resistant (MDRPZA-S) tuberculosis (TB).pncAgene mutations are usually detected in a clear majority (>90%) of PZA-resistant strains but obviously not in all. Rapid and reliable PZA drug susceptibility testing (DST) is critical whenever PZA is to be used in a treatment regimen, not least for the treatment of MDRPZA-STB. In this study, we selected 26 PZA-resistant isolates reported to carry a wild-typepncAgene. To confirm resistance, susceptibility testing was repeated using 100 mg/liter and 200 mg/liter PZA for all the 26 isolates and Sanger sequencing was repeated on the 18 isolates that remained PZA resistant. Apart from the eight isolates initially misclassified as PZA resistant, the retests identified three factors responsible for the phenotype-genotype discrepancy:panDorrpsAmutations identified by whole-genome sequencing (WGS) (n= 7), heteroresistance (n= 8), and mixed populations withMycobacterium avium(n= 3). Additionally, we performed WGS on 400 PZA-susceptible isolates and 15 consecutive MDRPZA-Rclinical isolates. Of the 400 PZA-susceptible isolates, only 1 harbored a nonsynonymouspncAmutation (Thr87Met), whereas a nonsynonymousrpsAmutation was found in 17 isolates. None of these isolates carried a nonsynonymouspanDmutation, while all 15 of the MDRPZA-Risolates harbored a nonsynonymouspncAmutation. Our findings indicate that it is necessary to consider the occurrence ofpanDmutations in PZA-resistant isolates, as well as heteroresistance, for the development and evaluation of new molecular techniques to ensure high-quality DST performance. The identification of nonsynonymousrpsAmutations in both PZA-susceptible and PZA-resistant isolates also implies that further studies are needed in order to determine the role ofrpsAin PZA resistance.
authors.affiliation: Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden jim.werngren@folkhalsomyndigheten.se.
authors.forename: Jim
authors.initials: J
authors.lastname: Werngren
authors.affiliation: Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Alm
authors.affiliation: Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Mansjö
mesh_terms: D000581:Amidohydrolases / Q000235:genetics*; D000995:Antitubercular Agents / Q000494:pharmacology*; D060085:Coinfection / Q000382:microbiology; D024881:Drug Resistance, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009162:Mycobacterium avium / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D011718:Pyrazinamide / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D014376:Tuberculosis / Q000382:microbiology; D000073336:Whole Genome Sequencing
chemical_list: D000995:Antitubercular Agents; D011718:Pyrazinamide; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis
keywords: M. tuberculosis; drug susceptibility testing; heteroresistance; panD; pncA; pyrazinamide; pyrazinamide resistance; rpsA