Gemifloxacin resistance in Mycobacterium tuberculosis without QRDR mutations in gyrA or gyrB : evidence for non-canonical resistance mechanisms

Tuberculosis (TB) remains a global health threat, with multidrug-resistant TB (MDR-TB) presenting major therapeutic challenges. Fluoroquinolones (FQs) are key second-line drugs. Resistance is typically linked to mutations within quinolone resistance-determining regions (QRDRs), though non-canonical mechanisms are increasingly recognized. To evaluate phenotypic susceptibility to gemifloxacin in clinical Mycobacterium tuberculosis isolates and determine the association with mutations in gyrA and gyrB QRDRs. Sixty clinical isolates (40 drug-susceptible [DS], 20 MDR) were tested using the resazurin microtiter assay (REMA). Minimum inhibitory concentrations (MICs) ranged from 0.25 to 128 µg/mL. Resistance was defined using an epidemiological cutoff value (ECOFF) of ≥8 µg/mL. Sanger sequencing of QRDR hotspots in gyrA (codons 90, 91, 94) and gyrB (codons 500–540) was performed on all six resistant isolates and two susceptible controls. MDR isolates exhibited significantly higher median MICs compared to DS isolates (4 µg/mL vs 2 µg/mL, P = 0.04). Six isolates (10%) were phenotypically resistant, including two DS and four MDR strains. Given our modest sample size ( n = 60), no mutations were detected within the sequenced QRDR segments of gyrA and gyrB in any resistant isolate or control. In this hypothesis-generating study, gemifloxacin resistance was observed in M. tuberculosis without mutations in the sequenced QRDR regions of gyrA / gyrB , implying alternative mechanisms, such as efflux pump activity or altered permeability. While limited by sample size, these preliminary findings challenge the reliance on molecular diagnostics targeting only QRDRs and underscore the importance of phenotypic testing for accurate detection of resistance. Larger multicenter studies are warranted to validate these observations and elucidate the underlying mechanisms. IMPORTANCE This study provides the first evidence that Mycobacterium tuberculosis can develop phenotypic resistance to gemifloxacin—despite having no mutations in the canonical gyrA/gyrB quinolone resistance-determining regions (QRDRs). Among 60 clinical isolates, 10% of gemifloxacin-resistant strains were QRDR-negative. Although gemifloxacin is no longer used clinically due to toxicity, its unique structure reveals hidden, non-target-based resistance mechanisms (e.g., efflux, permeability changes) that likely also compromise current fluoroquinolones like levofloxacin and moxifloxacin. This exposes a critical blind spot: current molecular diagnostics (e.g., Xpert MTB/XDR, GenoType) may falsely label resistant strains as susceptible, risking treatment failure. We argue that phenotypic drug susceptibility testing (DST) must be mandatory—not optional—in multidrug-resistant tuberculosis (MDR-TB) programs to safeguard against this silent, undetected resistance.