Correlation of different phenotypic drug susceptibility testing methods for four fluoroquinolones in Mycobacterium tuberculosis

Abstract

Background: Molecular resistance testing fails to explain all fluoroquinolone resistance, with a continued need for a suitable rapid phenotypic drug susceptibility testing method.

Objective: To evaluate the optimal method for phenotypic fluoroquinolone susceptibility testing.

Methods: Using Löwenstein-Jensen medium, Middlebrook 7H11 agar, BACTEC-MGIT 960 and the resazurin microtitre plate assay, we determined susceptibility to fluoroquinolones in Mycobacterium tuberculosis and investigated cross-resistance between ofloxacin, levofloxacin, moxifloxacin and gatifloxacin. We compared MICs of all four fluoroquinolones for 91 strains on Löwenstein-Jensen (as the gold standard) with their MICs in resazurin plates, and with ofloxacin susceptibility at a single concentration in MGIT and on 7H11 agar, in addition to sequencing of the gyrAB genes.

Results and conclusions: Applying a cut-off of 2 mg/L ofloxacin, 1 mg/L levofloxacin and 0.5 mg/L moxifloxacin and gatifloxacin in all methods, some discordance between solid medium and MGIT methods was observed, yet this tended to be explained by MICs around the cut-off. The high discordance between Löwenstein-Jensen (LJ) and resazurin plates suggests that the currently applied cut-offs for all fluoroquinolones in the resazurin method should decrease and minor changes in colour (from blue to purple) be considered as meaningful. High-level resistance in all assays to all drugs correlated well with the presence of gyrA mutations, in support of recent findings that fluoroquinolone resistance should be tested at different concentrations, as patients with lower levels of resistance may continue to benefit from high-dose fluoroquinolone-based therapy.

Figure 1.

Overview of the M. tuberculosis strains included in the study. OFX, ofloxacin; KAN, kanamycin; CAP, capreomycin; LVX, levofloxacin; MXF, moxifloxacin; GAT, gatifloxacin; S, susceptible; R, resistant; PM, proportion method; REMA, resazurin microtitre assay; LJ, Löwenstein–Jensen.

Figure 2.

MIC distribution of ofloxacin on Löwenstein–Jensen medium stratified to the historical resistance profile on Middlebrook 7H11 agar as well as gyrAB mutations. OFX, ofloxacin; S, susceptible; R, resistant; LJ, Löwenstein–Jensen.

Figure 3.

(Dis)agreement between Löwenstein–Jensen medium, Middlebrook 7H11 agar, REMA and MGIT 960 regarding ofloxacin susceptibility (cut-off 2 mg/L) considering Löwenstein–Jensen as gold standard: (a) all 91 M. tuberculosis strains and (b) excluding strains with an MIC around the cut-off (2 or 4 mg/L). Numbers inside the Venn diagram indicate ofloxacin-resistant strains in one or more methods. Black, concordantly resistant in all methods; light grey, ofloxacin resistant on Löwenstein–Jensen and resistant by one or more other methods; and dark grey, ofloxacin resistant by one or more methods, yet susceptible on Löwenstein–Jensen. W, gyrAB WT; M, gyrAB mutant; OFX, ofloxacin.

Figure 4.

Cumulative percentage of WT and QRDR mutant strains against respective MICs of ofloxacin, levofloxacin, moxifloxacin and gatifloxacin in REMA and on Löwenstein–Jensen medium. LJ, Löwenstein–Jensen; OFX, ofloxacin; LVX, levofloxacin; MXF, moxifloxacin; GAT, gatifloxacin. Dark/light curves represent strains without/with QRDR mutation detected and the vertical line indicates maximum discrimination between WT and QRDR mutant strains.