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27261264
A novel resistance mutation in eccC5 of the ESX-5 secretion system confers ofloxacin resistance in Mycobacterium tuberculosis.

J Antimicrob Chemother, 2016/09;71(9):2419-27.

Eilertson B[1], Maruri F[2], Blackman A[2], Guo Y[3], Herrera M[4], van der Heijden Y[2], Shyr Y[3], Sterling TR[5]

Affiliations

PMID: 27261264DOI: 10.1093/jac/dkw168

Impact factor: 5.758

Abstract
background: Fluoroquinolone resistance in Mycobacterium tuberculosis is often conferred by DNA gyrase mutations. However, a substantial proportion of fluoroquinolone-resistant M. tuberculosis isolates do not have such mutations.
methods: Ofloxacin-resistant and lineage-matched ofloxacin-susceptible M. tuberculosis isolates underwent WGS. Novel candidate resistance mutations were confirmed by Sanger sequencing and conferral of resistance was assessed via site-directed mutagenesis and allelic exchange. Ofloxacin MIC was determined by resazurin microtitre assay (REMA) and the effects on MICs of efflux pump inhibitors (CCCP, reserpine and verapamil) were determined.
results: Of 26 ofloxacin-resistant isolates, 8 (31%) did not have resistance-conferring DNA gyrase mutations. The V762G mutation in Rv1783 (eccC5, encoding a protein in the ESX-5 membrane complex secretion system) was present on WGS in 8/26 (31%) resistant isolates and 0/11 susceptible isolates (P = 0.005). The mutation was identified in five isolates without DNA gyrase mutations and three isolates with such mutations; it was identified in both European-American and East Asian M. tuberculosis lineages. The ofloxacin MIC increased from 1 to 32 mg/L after introduction of the V762G mutation into M. tuberculosis H37Rv. In this strain with the V762G mutation, ofloxacin MIC did not change in the presence of efflux pump inhibitors.
conclusions: A novel V762G mutation in Rv1783 conferred ofloxacin resistance in M. tuberculosis by a mechanism other than drug efflux. This occurred in a substantial proportion of resistant isolates, particularly those without DNA gyrase mutations.
MeSH terms
Antitubercular Agents; Bacterial Proteins; Bacterial Secretion Systems; Case-Control Studies; DNA Mutational Analysis; Drug Resistance, Bacterial; Genome, Bacterial; Humans; Microbial Sensitivity Tests; Mutagenesis, Site-Directed; Mutation, Missense; Mycobacterium tuberculosis; Ofloxacin; Sequence Analysis, DNA; Tennessee; Tuberculosis; Virulence Factors
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