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



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.



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.



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.
Division of Infectious Diseases, Department of Medicine, State University of New York Downstate, New York, NY, USA Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA.
Brandon
B
Eilertson
Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA Vanderbilt Tuberculosis Center, Vanderbilt University School of Medicine, Nashville, TN, USA.
Fernanda
F
Maruri
Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA Vanderbilt Tuberculosis Center, Vanderbilt University School of Medicine, Nashville, TN, USA.
Amondrea
A
Blackman
Vanderbilt Center for Quantitative Sciences, Vanderbilt University School of Medicine, Nashville, TN, USA.
Yan
Y
Guo
Vanderbilt Technologies for Advance Genomics, Vanderbilt University Medical Center, Nashville, TN, USA.
Miguel
M
Herrera
Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA Vanderbilt Tuberculosis Center, Vanderbilt University School of Medicine, Nashville, TN, USA.
Yuri
Y
van der Heijden
Vanderbilt Center for Quantitative Sciences, Vanderbilt University School of Medicine, Nashville, TN, USA.
Yu
Y
Shyr
Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA Vanderbilt Tuberculosis Center, Vanderbilt University School of Medicine, Nashville, TN, USA timothy.sterling@vanderbilt.edu.
Timothy R
TR
Sterling
D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D058947:Bacterial Secretion Systems / Q000235:genetics; D016022:Case-Control Studies; D004252:DNA Mutational Analysis; D024881:Drug Resistance, Bacterial; D016680:Genome, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D015242:Ofloxacin / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D013714:Tennessee; D014376:Tuberculosis / Q000382:microbiology; D037521:Virulence Factors / Q000235:genetics*
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; C000621160:ESX-5 protein, Mycobacterium tuberculosis; D037521:Virulence Factors; D015242:Ofloxacin