Fluoroquinolone heteroresistance in Mycobacterium tuberculosis: detection by genotypic and phenotypic assays in experimentally mixed populations

• Published in: Scientific reports Vol. 9; no. 1; pp. 11760 - 8
• Main Authors: Rigouts, L., Miotto, P., Schats, M., Lempens, P., Cabibbe, A. M., Galbiati, S., Lampasona, V., de Rijk, P., Cirillo, D. M., de Jong, B. C.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 13.08.2019; Nature Publishing Group
• Subjects: 45; 45/23; 45/77; 631/326/22/1290; 631/326/2521; Antibiotics; Antitubercular Agents - pharmacology; Bacilli; Drug Resistance, Microbial - genetics; Fluoroquinolones - pharmacology; Gatifloxacin; Genotype; Genotypes; High-Throughput Nucleotide Sequencing; Humanities and Social Sciences; Humans; Microbial Sensitivity Tests; multidisciplinary; Mutation; Mycobacterium tuberculosis; Mycobacterium tuberculosis - drug effects; Phenotype; Resistant mutant; Science; Science (multidisciplinary); Tuberculosis
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-48289-9

Heteroresistance - the simultaneous presence of drug-susceptible and -resistant organisms - is common in Mycobacterium tuberculosis . In this study, we aimed to determine the limit of detection (LOD) of genotypic assays to detect gatifloxacin-resistant mutants in experimentally mixed populations. A fluoroquinolone-susceptible M. tuberculosis mother strain (S) and its in vitro selected resistant daughter strain harbouring the D94G mutation in gyrA (R) were mixed at different ratio’s. Minimum inhibitory concentrations (MICs) against gatifloxacin were determined, while PCR-based techniques included: line probe assays (Genotype MTBDR sl and GenoScholar-FQ + KM TB II), Sanger sequencing and targeted deep sequencing. Droplet digital PCR was used as molecular reference method. A breakpoint concentration of 0.25 mg/L allows the phenotypic detection of ≥1% resistant bacilli, whereas at 0.5 mg/L ≥ 5% resistant bacilli are detected. Line probe assays detected ≥5% mutants. Sanger sequencing required the presence of around 15% mutant bacilli to be detected as (hetero) resistant, while targeted deep sequencing detected ≤1% mutants. Deep sequencing and phenotypic testing are the most sensitive methods for detection of fluoroquinolone-resistant minority populations, followed by line probe assays (provided that the mutation is confirmed by a mutation band), while Sanger sequencing proved to be the least sensitive method.