Alix Pantel1, Stéphanie Petrella2, Nicolas Veziris3, Stéphanie Matrat1, Aurélie Bouige1, Hélène Ferrand1, Wladimir Sougakoff3, Claudine Mayer2, Alexandra Aubry4. 1. Sorbonne Universités, UPMC Univ Paris 06, Paris, France. 2. Unité de Microbiologie Structurale, Département de Biologie Structurale et Chimie, Institut Pasteur, Paris, France UMR 3528, CNRS, Paris, France Université Paris Diderot, Sorbonne Paris Cité, Paris, France. 3. Sorbonne Universités, UPMC Univ Paris 06, Paris, France INSERM U1135, Immunity and Infectious Diseases Centre (Cimi-Paris), Team 13, Paris, France National Reference Centre for Mycobacteria, Laboratory of Bacteriology, Pitié-Salpêtrière Hospital, APHP, Paris, France. 4. Sorbonne Universités, UPMC Univ Paris 06, Paris, France INSERM U1135, Immunity and Infectious Diseases Centre (Cimi-Paris), Team 13, Paris, France National Reference Centre for Mycobacteria, Laboratory of Bacteriology, Pitié-Salpêtrière Hospital, APHP, Paris, France alexandra.aubry@upmc.fr.
Abstract
OBJECTIVES: Resistance to fluoroquinolones (FQs) in Mycobacterium tuberculosis (Mtb) is mainly due to mutations in DNA gyrase (GyrA2B2), with the most common substitutions located at positions 90 and 94 in GyrA. Two clinical MDR Mtb (MDR-TB) strains harbouring an A90E or D94N substitution in GyrA were found to be surprisingly susceptible to FQs (ofloxacin MIC ≤2 mg/L). We studied the impact of the additional GyrA substitutions found in these strains (T80A and T80A + A90G, respectively) on FQ susceptibility. METHODS: Mutants of interest were generated by site-specific mutagenesis of GyrA alleles. WT and mutant TB DNA gyrase subunits were overexpressed in Escherichia coli and purified, and the in vitro susceptibility to FQs of their DNA supercoiling reaction was studied. RESULTS: IC50s of mutant gyrase complexes bearing GyrA D94N and A90E were 3- to 36-fold higher than WT IC50s, whereas IC50s of gyrase bearing T80A + A90G + D94N and T80A + A90E were close to the WT IC50s. CONCLUSIONS: We demonstrated that substitutions T80A and A90G restore FQ susceptibility when associated with a substitution implicated in high-level FQ resistance. Line probe assay misclassification of MDR-TB strains as pre-XDR or XDR can be corrected by sequence analysis of gyrA.
OBJECTIVES: Resistance to fluoroquinolones (FQs) in Mycobacterium tuberculosis (Mtb) is mainly due to mutations in DNA gyrase (GyrA2B2), with the most common substitutions located at positions 90 and 94 in GyrA. Two clinical MDR Mtb (MDR-TB) strains harbouring an A90E or D94N substitution in GyrA were found to be surprisingly susceptible to FQs (ofloxacin MIC ≤2 mg/L). We studied the impact of the additional GyrA substitutions found in these strains (T80A and T80A + A90G, respectively) on FQ susceptibility. METHODS: Mutants of interest were generated by site-specific mutagenesis of GyrA alleles. WT and mutant TB DNA gyrase subunits were overexpressed in Escherichia coli and purified, and the in vitro susceptibility to FQs of their DNA supercoiling reaction was studied. RESULTS: IC50s of mutant gyrase complexes bearing GyrAD94N and A90E were 3- to 36-fold higher than WT IC50s, whereas IC50s of gyrase bearing T80A + A90G + D94N and T80A + A90E were close to the WT IC50s. CONCLUSIONS: We demonstrated that substitutions T80A and A90G restore FQ susceptibility when associated with a substitution implicated in high-level FQ resistance. Line probe assay misclassification of MDR-TB strains as pre-XDR or XDR can be corrected by sequence analysis of gyrA.
Authors: Bidhan C Dhar; Adam J Reed; Suvra Mitra; Patricia Rodriguez Sanchez; Daria D Nedorezova; Ryan P Connelly; Kyle H Rohde; Yulia V Gerasimova Journal: Biosens Bioelectron Date: 2020-06-13 Impact factor: 10.618
Authors: Adebisi Ajileye; Nataly Alvarez; Matthias Merker; Timothy M Walker; Suriya Akter; Kerstin Brown; Danesh Moradigaravand; Thomas Schön; Sönke Andres; Viola Schleusener; Shaheed V Omar; Francesc Coll; Hairong Huang; Roland Diel; Nazir Ismail; Julian Parkhill; Bouke C de Jong; Tim E A Peto; Derrick W Crook; Stefan Niemann; Jaime Robledo; E Grace Smith; Sharon J Peacock; Claudio U Köser Journal: Antimicrob Agents Chemother Date: 2017-03-24 Impact factor: 5.191