title: High proportion of heteroresistance in gyrA and gyrB in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates.
abstract: Heteroresistance is the coexistence of populations with differing nucleotides at a drug resistance locus within a sample of organisms. Although Sanger sequencing is the gold standard for sequencing, it may be less sensitive than deep sequencing for detecting fluoroquinolone heteroresistance in Mycobacterium tuberculosis. Twenty-seven fluoroquinolone monoresistant and 11 fluoroquinolone-susceptible M. tuberculosis isolates were analyzed by Sanger and Illumina deep sequencing. Individual sequencing reads were analyzed to detect heteroresistance in the gyrA and gyrB genes. Heteroresistance to fluoroquinolones was identified in 10/26 (38%) phenotypically fluoroquinolone-resistant samples and 0/11 (P = 0.02) fluoroquinolone-susceptible controls. One resistant sample was excluded because of contamination with the laboratory strain M. tuberculosis H37Rv. Sanger sequencing revealed resistance-conferring mutations in 15 isolates, while deep sequencing revealed mutations in 20 isolates. Isolates with fluoroquinolone resistance-conferring mutations by Sanger sequencing all had at least those same mutations identified by deep sequencing. By deep sequencing, 10 isolates had a single fixed (defined as >95% frequency) mutation, while 10 were heteroresistant, 5 of which had a single unfixed (defined as <95% frequency) mutation and 5 had multiple unfixed mutations. Illumina deep sequencing identified a higher proportion of fluoroquinolone-resistant M. tuberculosis isolates with heteroresistance than did Sanger sequencing. The heteroresistant isolates frequently demonstrated multiple mutations, but resistant isolates with fixed mutations each had only a single mutation.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA Vanderbilt Tuberculosis Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA brandon.o.eilertson@vanderbilt.edu.
authors.forename: Brandon
authors.initials: B
authors.lastname: Eilertson
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA Vanderbilt Tuberculosis Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Maruri
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA Vanderbilt Tuberculosis Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Amondrea
authors.initials: A
authors.lastname: Blackman
authors.affiliation: Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Miguel
authors.initials: M
authors.lastname: Herrera
authors.affiliation: Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Samuels
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA Vanderbilt Tuberculosis Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Sterling
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D024841:Fluoroquinolones / Q000494:pharmacology*; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D010641:Phenotype; D018088:Tuberculosis, Multidrug-Resistant / Q000235:genetics*; D014397:Tuberculosis, Pulmonary / Q000382:microbiology*
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D027081:DNA Gyrase