Abstract
In order to correlate the mutations inside the entire gyrA and gyrB genes with the level of resistance to ofloxacin (OFX) and moxifloxacin (MFX) in isolates of multidrug-resistant Mycobacterium tuberculosis (MDR-TB), a total of 111 isolates were categorized into OFX-susceptible (MIC, ≤2 μg/ml) and low-level (MIC, 4 to 8 μg/ml) and high-level (MIC, ≥16 μg/ml) OFX-resistant isolates and MFX-susceptible (MIC, ≤0.5 μg/ml) and low-level (MIC, 1 to 2 μg/ml) and high-level (MIC, ≥4 μg/ml) MFX-resistant isolates. Resistance-associated mutations inside the gyrA gene were found in 30.2% of OFX-susceptible and 72.5% and 72.2% of low-level and high-level OFX-resistant isolates and in 28.6% of MFX-susceptible and 58.1% and 83.9% of low-level and high-level MFX-resistant isolates. Compared with OFX-susceptible isolates, low-level and high-level OFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (17.0%, 65.0%, and 72.2%, respectively; P < 0.001) and a higher prevalence of the gyrB G512R mutation (0.0%, 2.5%, and 16.7%, respectively; P = 0.006). Similarly, compared with MFX-susceptible isolates, low-level and high-level MFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (14.3%, 51.6%, and 80.6%, respectively; P < 0.001) as well as a higher prevalence of the gyrB G512R mutation (0.0%, 0.0%, and 12.9%, respectively; P = 0.011). D94G and D94N mutations in gyrA and the G512R mutation in gyrB were correlated with high-level MFX resistance, while the D94A mutation was associated with low-level MFX resistance. The prevalence of mutations at gyrA codons 88 to 94 and the gyrB G512R mutation were higher among fluoroquinolone (FQ)-susceptible East Asian (Beijing) and Indo-Oceanic strains than they were among Euro-American strains, implying that molecular techniques to detect FQ resistance may be less specific in areas with a high prevalence of East Asian (Beijing) and Indo-Oceanic strains.
INTRODUCTION
Fluoroquinolones (FQs) are used as second-line drugs for the treatment of tuberculosis (TB). These broad-spectrum antibacterial agents with bactericidal activity against Mycobacterium tuberculosis exert their bactericidal effects by inhibiting mycobacterial DNA gyrase activity, which prevents bacterial DNA from unwinding and replicating (1–3).
Moxifloxacin (MFX), a “fourth-generation” FQ, has been shown to have better activity against M. tuberculosis than ofloxacin (OFX) and is recommended by the World Health Organization (WHO) for the treatment of multidrug-resistant TB (MDR-TB; defined as resistant to at least two of the most effective antituberculosis drugs, isoniazid and rifampin) (2, 3). Unfortunately, the widespread use of FQs to treat bacterial infections has led to the emergence of FQ-resistant MDR-TB and extensively drug-resistant TB (XDR-TB; defined as MDR-TB resistant to any FQ and a second-line injectable drug) (4, 5), thereby complicating patient care.
Mutations in genes encoding DNA gyrase subunits gyrA and gyrB are the most common mechanism conveying FQ resistance in TB (6, 7). The most frequent resistance-associated mutations occur in a conserved region of the gyrA gene (codons 74 to 113) and, less frequently, the gyrB gene (codons 461 to 499), known as the quinolone resistance-determining region (QRDR) (2, 7, 8). However, many previous studies found that up to 60% of FQ-resistant M. tuberculosis isolates lack known mutations in the QRDR of gyrA or gyrB or both (7, 9), which compromises the sensitivity and specificity of molecular testing methods (7).
In MDR-TB, it is unclear whether resistance-associated mutations outside the QRDR of gyrA and gyrB are associated with FQ resistance (7). In this study, we evaluated the possible association between the level of resistance to OFX and MFX and mutations in and outside the QRDR of the gyrA and gyrB genes in M. tuberculosis isolates.
MATERIALS AND METHODS
Selection of isolates.
This study was conducted at the Chest Hospital, a tertiary TB referral hospital in Taiwan. M. tuberculosis was identified by a combination of morphology, the growth rate of the colonies, and biochemical tests (10). Drug susceptibility to first-line anti-TB drugs, including isoniazid (0.2 μg/ml), rifampin (1.0 μg/ml), and ethambutol (5.0 μg/ml), and second-line anti-TB drugs, including streptomycin (2.0 μg/ml), kanamycin (6.0 μg/ml), and ofloxacin (2.0 μg/ml), was tested using modified proportional disk elution methods as described previously (5, 11). This study was approved by the Institutional Review Board of Chest Hospital (B-ER-102-164).
From 2006 to 2011, a total of 55 consecutive OFX-resistant MDR isolates were collected, and we selected one out of every two OFX-susceptible MDR isolates, leading to 56 OFX-susceptible MDR isolates collected. M. tuberculosis isolates were preserved at −80°C and were subcultured onto Lowenstein-Jensen (LJ) medium prior to determining MIC values. MICs were measured by the broth microdilution method using Sensititre microtiter trays (Trek Diagnostic Systems, Cleveland, OH, USA). The range of concentrations tested was 0.25 to 32 μg/ml for OFX and 0.06 to 8 μg/ml for MFX. The results were read at 21 days via the mirror method according to the instructions supplied by the manufacturer. The isolates were categorized into OFX-susceptible (MIC, ≤2 μg/ml) and low-level (MIC, 4 to 8 μg/ml) and high-level (MIC, ≥16 μg/ml) OFX-resistant isolates and MFX-susceptible (MIC, ≤0.5 μg/ml) and low-level (MIC, 1 to 2 μg/ml) and high-level (MIC, ≥4 μg/ml) MFX-resistant isolates. Genotyping (spoligotyping and 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat [MIRU-VNTR] genotyping) was performed as previously described (12, 13), and major lineages were determined.
DNA sequencing.
The entire gyrA and gyrB genes were amplified with 8 primer sets, GYRA1 (−43 to 802 bp; forward 5′-GAT GTC TAA CGC AAC CCT GCG TTC GAT and reverse 5′-AGG TAC GAC CGC GGG AAT CCT CTT CTA), GYRA2 (593 to 1,376 bp; forward 5′-CCG ACG CGG TGT TCT GG and reverse 5′-TCG ATT TTG GCC AGG TCG TC), GYRA3 (1,230 to 2,049 bp; forward 5′-GTC GGA GAC CGT CGA TA and reverse 5′-GAA CCT GAT GGA CTG CCC), and GYRA4 (1,883 to 2,678 bp; forward 5′-GCA ACG GGC TGG TGA AAA AG and reverse 5′-GAC CAA GCC ATC CGC ATT C) for the gyrA gene, and GYRB1 (−365 to 446 bp; forward 5′-ATC GCC GCA GCG GTT G and reverse 5′-ACC TGA GAC CAC TCG TAC CC), GYRB2 (287 to 1,089 bp; forward 5′-TCG ACG TGG TGA TGA CAC AA and reverse 5′-GCT GAC CTT CAC CGA GAT CA), GYRB3 (939 to 1,744 bp; forward 5′-CGG CAC CCA CGA AGA GG and reverse 5′-ATG CGA ATT CCG GGT CAC TG), and GYRB4 (1,546 to 2,203 bp; forward 5′-GAG TTC GAT ATC GGC AAG CTG and reverse 5′-CCC GTC GCG CAC CTC) for the gyrB gene. The sequences were assembled by Sanger sequencing and were compared to the reference strain sequence (H37Rv; GenBank accession number NC_000962.3) using BioEdit software.
Statistical analysis.
The sequences from isolates were compared to the reference strain sequence, and the proportions of resistant and susceptible isolates harboring specific mutations were compared using the chi-square test or Fisher's exact test as appropriate. A P value of <0.05 was considered to indicate statistical significance; all tests were two tailed. All statistical analyses were performed with the statistical package Stata for Windows (version 11; College Station, Texas).
RESULTS
Of the 111 MDR isolates, 38 (34.2%), 50 (45.0%), and 24 (21.6%) were also resistant to ethambutol, streptomycin, and kanamycin, respectively. Of these, 54 isolates (48.6%) belonged to the East Asian (Beijing) strain, 25 (22.5%) were Euro-American strains, 30 (27.0%) were Indo-Oceanic strains, and the remaining 2 isolates (1.8%) had unique patterns. Of the 111 isolates, 53 (47.7%) were OFX-susceptible isolates, 40 (36.0%) were low-level OFX-resistant isolates, and 18 (16.2%) were high-level OFX-resistant isolates. In addition, 49 (44.1%) were MFX-susceptible isolates, 31 (27.9%) were low-level MFX-resistant isolates, and 31 (27.9%) were high-level MFX-resistant isolates (Table 1).
TABLE 1.
MICs of ofloxacin and moxifloxacin established for multidrug-resistant Mycobacterium tuberculosis with resistance-associated mutations in the gyrA and gyrB genes
| Gene mutations (codon/nucleotide) | Total no. of isolates | No. of isolates |
||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC (μg/ml) of ofloxacin |
MIC (μg/ml) of moxifloxacin |
|||||||||||||||||||
| 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | >32 | 0.06 | 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | >8 | |||
| gyrA | gyrB | |||||||||||||||||||
| G88C (Ggc/Tgc) | G512R (Ggg/Agg) | 1 | 1 | 1 | ||||||||||||||||
| A90V (gCg/gTg) | 4 | 1 | 1 | 2 | 1 | 1 | 1 | 1 | ||||||||||||
| A90V (gCg/gTg) | A403S (Gcg/Tcg) | 1 | 1 | 1 | ||||||||||||||||
| A90V (gCg/gTg) & A384V (gCa/gTa) | M291I (atG/atC) | 2 | 1 | 1 | 1 | 1 | ||||||||||||||
| S91P (Tcg/Ccg) | 2 | 1 | 1 | 2 | ||||||||||||||||
| D94G (GAc/GGc) | 13 | 1 | 1 | 1 | 7 | 2 | 1 | 1 | 1 | 3 | 6 | 2 | ||||||||
| D94G (GAc/GGc) | G512R (Ggg/Agg) | 1 | 1 | 1 | ||||||||||||||||
| D94G (GAc/GGc) & P190L (cCg/cTg) | 1 | 1 | 1 | |||||||||||||||||
| D94G (GAc/GGc) & A384V (gCa/gTa) | M291I (atG/atC) | 2 | 1 | 1 | 2 | |||||||||||||||
| D94N (GAc/AAc) | 6 | 3 | 3 | 1 | 3 | 1 | 1 | |||||||||||||
| D94N (GAc/AAc) | H244N (Cac/Aac) | 1 | 1 | 1 | ||||||||||||||||
| D94N (GAc/AAc) & A132S (Gcc/Tcc) | H244N (Cac/Aac) | 1 | 1 | 1 | ||||||||||||||||
| D94N (GAc/AAc) & A132S (Gcc/Tcc) | G512R (Ggg/Agg) | 1 | 1 | 1 | ||||||||||||||||
| D94A (GAc/GCc) | 1 | 1 | 1 | |||||||||||||||||
| D94A (GAc/GCc) | A403S (Gcg/Tcg) | 2 | 1 | 1 | 1 | 1 | ||||||||||||||
| D94A (GAc/GCc) | N499T (AAc/ACc) | 1 | 1 | 1 | ||||||||||||||||
| D94A (GAc/GCc) & A384V (gCa/gTa) | 1 | 1 | 1 | |||||||||||||||||
| D94A (GAc/GCc) & A384V (gCa/gTa) | M291I (atG/atC) & A504T (Gcg/Acg) | 1 | 1 | 1 | ||||||||||||||||
| D94V (GAc/GTc) | 2 | 1 | 1 | 1 | 1 | |||||||||||||||
| D94H (GAc/CAc) | 3 | 1 | 2 | 1 | 1 | 1 | ||||||||||||||
| D94Y (GAc/TAc) | 1 | 1 | 1 | |||||||||||||||||
| A288D (gCc/gAc) | 1 | 1 | 1 | |||||||||||||||||
| T335A (Acc/Gcc) | 1 | 1 | 1 | |||||||||||||||||
| A384V (gCa/gTa) | M291I (atG/atC) | 8 | 1 | 1 | 4 | 2 | 1 | 3 | 1 | 2 | 1 | |||||||||
| D461N (Gac/Aac) | 1 | 1 | 1 | |||||||||||||||||
| I485V (Atc/Gtc) | 1 | 1 | 1 | |||||||||||||||||
| N499D (AAc/GAc) | 3 | 1 | 1 | 1 | 1 | 1 | 1 | |||||||||||||
| A508S (Gcg/Tcg) | 1 | 1 | 1 | |||||||||||||||||
| G512R (Ggg/Agg) | 1 | 1 | 1 | |||||||||||||||||
| 46 | 5 | 10 | 13 | 6 | 4 | 5 | 1 | 2 | 2 | 14 | 9 | 8 | 6 | 4 | 1 | 2 | ||||
| Total | 111 | 10 | 13 | 15 | 15 | 14 | 26 | 11 | 3 | 4 | 2 | 20 | 17 | 10 | 15 | 16 | 17 | 9 | 5 | |
The sequencing results of the entire gyrA and gyrB genes and the MIC distributions of OFX and MFX are shown in Table 1. A total of 111 (100%) and 108 (97.3%) isolates had gyrA E21Q and S95T natural polymorphisms, respectively. Resistance-associated mutations in gyrA were observed in 16/53 (30.2%) OFX-susceptible and 29/40 (72.5%) low-level and 13/18 (72.2%; P < 0.001) high-level OFX-resistant strains as well as in 14/49 (28.6%) MFX-susceptible and 18/31 (58.1%) low-level and 26/31 (83.9%; P < 0.001) high-level MFX-resistant isolates. In addition, a total of 11 (20.8%) OFX-susceptible and 10 (25.0%) low-level and 8 (44.4%; P = 0.149) high-level OFX-resistant strains and 8 (16.3%) MFX-susceptible and 10 (32.3%) low-level and 11 (35.5%; P = 0.103) high-level MFX-resistant strains carried mutations in gyrB. Among 29 isolates with gyrB mutations, 22 (75.9%) also had mutations in gyrA.
Of the 111 isolates, nine isolates (8.1%) had double mutations in the gyrA gene, one (0.9%) isolate had double mutations in the gyrB gene, and 24 (21.6%) isolates had 2 to 3 mutations in both genes. No mutations in gyrA or gyrB were found in the remaining 34 (64.2%) OFX-susceptible isolates or in the 9 (22.5%) low-level and 3 (16.7%; P < 0.001) high-level OFX-resistant isolates or in the remaining 33 (67.3%) MFX-susceptible isolates or in the 10 (32.3%) low-level and 3 (9.7%; P < 0.001) high-level MFX-resistant isolates.
As shown in Table 2, the most prevalent mutations were at codons 88 to 94 of the gyrA gene, with mutation at codon 94 being the most prevalent. Compared with OFX-susceptible isolates, low-level and high-level OFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (9/53 [17.0%] versus 26/40 [65.0%] and 13/18 [72.2%], respectively; P < 0.001) and a higher prevalence of the gyrB G512R mutation (0/53 [0.0%] versus 1/40 [2.5%] and 3/18 [16.7%], respectively; P = 0.006). Similarly, compared with MFX-susceptible isolates, low-level and high-level MFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (7/49 [14.3%] versus 16/31 [51.6%] and 25/31 [80.6%], respectively; P < 0.001) and at gyrA codon 94 (4/49 [8.2%] versus 10/31 [32.3%] and 24/31 [77.4%]; P < 0.001) and a higher prevalence of the gyrB G512R mutation (0/49 [0.0%] versus 0/31 [0.0%] and 4/31 [12.9%], respectively; P = 0.011). Three (5.7%) OFX-susceptible isolates, four (6.9%) OFX-resistant isolates, two (4.1%) MFX-susceptible isolates, and five (8.1%) MFX-resistant isolates carried mutations only in gyrB, and they were not associated with resistance to OFX or MFX.
TABLE 2.
Mutations in the gyrA and gyrB genes and associated levels of ofloxacin and moxifloxacin resistance in multidrug-resistant Mycobacterium tuberculosis
| Gene mutations (codon/nucleotide) | Ofloxacin |
Moxifloxacin |
||||||
|---|---|---|---|---|---|---|---|---|
| No. susceptible (n = 53) | No. resistant |
P value | No. susceptible (n = 49) | No. resistant |
P value | |||
| Low-level (n = 40) | High-level (n = 18) | Low-level (n = 31) | High-level (n = 31) | |||||
| gyrA | ||||||||
| Codon 88–94 | 9 | 26 | 13 | <0.001 | 7 | 16 | 25 | <0.001 |
| G88C (Ggc/Tgc) | 0 | 0 | 1 | 0.074 | 0 | 0 | 1 | 0.559 |
| A90V (gCg/gTg) | 2 | 5 | 0 | 0.112 | 3 | 4 | 0 | 0.106 |
| S91P (Tcg/Ccg) | 1 | 1 | 0 | 0.801 | 0 | 2 | 0 | 0.152 |
| Codon 94 | 6 | 20 | 12 | <0.001 | 4 | 10 | 24 | <0.001 |
| D94G (GAc/GGc) | 2 | 11 | 4 | 0.002 | 2 | 3 | 12 | <0.001 |
| D94N (GAc/AAc) | 0 | 4 | 5 | 0.001 | 0 | 2 | 7 | 0.001 |
| D94A (GAc/GCc) | 1 | 3 | 2 | 0.170 | 0 | 4 | 2 | 0.032 |
| D94H (GAc/CAc) | 1 | 2 | 0 | 0.752 | 1 | 1 | 1 | 1.000 |
| D94V (GAc/GTc) | 2 | 0 | 0 | 0.653 | 1 | 0 | 1 | 1.000 |
| D94Y (GAc/TAc) | 0 | 0 | 1 | 0.162 | 0 | 0 | 1 | 0.559 |
| A132S (Gcc/Tcc) | 0 | 1 | 1 | 0.143 | 0 | 0 | 2 | 0.152 |
| P190L (cCg/cTg) | 0 | 1 | 0 | 0.523 | 0 | 0 | 1 | 0.559 |
| A288D (gCc/gAc) | 0 | 1 | 0 | 0.523 | 1 | 0 | 0 | 1.000 |
| T335A (Acc/Gcc) | 1 | 0 | 0 | 1.000 | 1 | 0 | 0 | 1.000 |
| A384V (gCa/gTa) | 7 | 5 | 2 | 1.000 | 6 | 4 | 4 | 1.000 |
| gyrB | ||||||||
| A227D (gCc/gAc) | 1 | 0 | 0 | 1.000 | 1 | 0 | 0 | 1.000 |
| H244N (Cac/Aac) | 0 | 1 | 1 | 0.143 | 0 | 1 | 1 | 0.310 |
| M291I (atG/atC) | 7 | 4 | 2 | 0.923 | 6 | 3 | 4 | 1.000 |
| A403S (Gcg/Tcg) | 1 | 2 | 0 | 0.752 | 0 | 3 | 0 | 0.041 |
| D461N (Gac/Aac) | 1 | 0 | 0 | 1.000 | 1 | 0 | 0 | 1.000 |
| I485V (Atc/Gtc) | 0 | 1 | 0 | 0.523 | 0 | 1 | 0 | 0.559 |
| Any codon 499 | 1 | 1 | 2 | 1.000 | 1 | 1 | 2 | 0.811 |
| N499D (AAc/GAc) | 1 | 1 | 1 | 0.565 | 1 | 1 | 1 | 1.000 |
| N499T (AAc/ACc) | 0 | 0 | 1 | 0.162 | 0 | 0 | 1 | 0.564 |
| A504T (Gcg/Acg) | 0 | 0 | 1 | 0.162 | 0 | 0 | 1 | 0.564 |
| A508S (Gcg/Tcg) | 1 | 0 | 0 | 1.000 | 0 | 1 | 0 | 0.559 |
| G512R (Ggg/Agg) | 0 | 1 | 3 | 0.006 | 0 | 0 | 4 | 0.011 |
| Wild type | 34 | 9 | 3 | <0.001 | 33 | 10 | 3 | <0.001 |
As shown in Table 2, the D94G mutation, the most common mutation in the gyrA gene, was associated with OFX resistance (15/17, 88.2%; P = 0.001) and high-level MFX resistance (12/17, 70.6%; P < 0.001). Also, we found that the D94N mutation was significantly associated with high-level OFX (5/9, 55.6%; P = 0.001) and with high-level MFX resistance (7/9, 77.8%; P = 0.001) and that the D94A mutation was associated with low-level MFX resistance (4/6, 66.7%; P = 0.032). The mutation at G512R in gyrB was associated with high-level OFX (3/4, 72.5%; P = 0.006) and MFX resistance (4/4, 100%; P = 0.011). Although the gyrB mutation G512R was associated with high-level OFX and MFX resistance, 75% (3/4) of the isolates had resistance-associated mutations in gyrA (one with a G88C mutation, one with a D94G mutation, and one with a D94N mutation). The remaining one isolate that only had the gyrB mutation G512R also had high-level OFX and MFX resistances.
Mutations at codons 88 to 94 of the gyrA gene were associated with OFX and MFX resistance in East Asian (Beijing), Euro-American, and Indo-Oceanic strains (Table 3). However, compared with Euro-American strains, OFX- and MFX-susceptible East Asian (Beijing) and Indo-Oceanic strains were more likely to have mutations at codons 88 to 94 of the gyrA gene.
TABLE 3.
Mutations in the gyrA and gyrB genes and ofloxacin and moxifloxacin resistance in different strains of multidrug-resistant Mycobacterium tuberculosis
| Gene mutations (codon/nucleotide) | East Asian (Beijing) strain |
Euro-American strain |
Indo-Oceanic strain |
|||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Ofloxacin-resistant |
Moxifloxacin-resistant |
Ofloxacin-resistant |
Moxifloxacin-resistant |
Ofloxacin-resistant |
Moxifloxacin-resistant |
|||||||||||||
| No. no (n = 29) | No. yes (n = 25) | P value | No. no (n = 25) | No. yes (n = 29) | P value | No. no (n = 9) | No. yes (n = 16) | P value | No. no (n = 10) | No. yes (n = 15) | P value | No. no (n = 15) | No. yes (n = 15) | P value | No. no (n = 14) | No. yes (n = 16) | P value | |
| gyrA | ||||||||||||||||||
| Codon 88–94 | 5 | 17 | <0.001 | 4 | 18 | 0.001 | 0 | 10 | 0.003 | 0 | 10 | 0.001 | 4 | 10 | 0.066 | 3 | 11 | 0.014 |
| G88C (Ggc/Tgc) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1.000 | 0 | 1 | 1.000 | ||||
| A90V (gCg/gTg) | 0 | 1 | 0.463 | 1 | 0 | 0.463 | 0 | 3 | 0.280 | 0 | 3 | 0.250 | 2 | 1 | 1.000 | 2 | 1 | 0.586 |
| S91P (Tcg/Ccg) | 1 | 1 | 1.000 | 0 | 2 | 0.493 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| Any codon 94 | 4 | 15 | 0.001 | 3 | 16 | 0.001 | 0 | 7 | 0.027 | 0 | 7 | 0.020 | 2 | 8 | 0.05 | 1 | 9 | 0.007 |
| D94G (GAc/GGc) | 2 | 8 | 0.032 | 2 | 8 | 0.086 | 0 | 4 | 0.260 | 0 | 4 | 0.125 | 0 | 3 | 0.224 | 0 | 3 | 0.228 |
| D94N (GAc/AAc) | 0 | 1 | 0.463 | 0 | 1 | 1.000 | 0 | 2 | 0.520 | 0 | 2 | 0.500 | 0 | 4 | 1.000 | 0 | 4 | 0.103 |
| D94A (GAc/GCc) | 0 | 3 | 0.093 | 0 | 3 | 0.240 | 0 | 1 | 1.000 | 0 | 1 | 1.000 | 1 | 1 | 1.000 | 0 | 2 | 0.485 |
| D94H (GAc/CAc) | 1 | 2 | 0.591 | 1 | 2 | 1.000 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| D94V (GAc/GTc) | 1 | 0 | 1.000 | 0 | 1 | 1.000 | 0 | 0 | 0 | 0 | 1 | 0 | 1.000 | 1 | 0 | 0.467 | ||
| D94Y (GAc/TAc) | 0 | 1 | 0.463 | 0 | 1 | 1.000 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| A132S (Gcc/Tcc) | 0 | 1 | 0.463 | 0 | 1 | 1.000 | 0 | 1 | 1.000 | 0 | 1 | 1.000 | 0 | 0 | 0 | 0 | ||
| P190L (cCg/cTg) | 0 | 1 | 0.463 | 0 | 1 | 1.000 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| A288D (gCc/gAc) | 0 | 0 | 0 | 0 | 0 | 1 | 1.000 | 1 | 0 | 0.4 | 0 | 0 | 0 | 0 | ||||
| T335A (Acc/Gcc) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1.000 | 1 | 0 | 0.467 | ||||
| A384V (gCa/gTa) | 1 | 3 | 0.326 | 1 | 3 | 0.615 | 1 | 1 | 1.000 | 1 | 1 | 1.000 | 5 | 3 | 0.682 | 4 | 4 | 1.000 |
| gyrB | ||||||||||||||||||
| A227D (gCc/gAc) | 0 | 0 | 0 | 0 | 1 | 0 | 0.360 | 1 | 0 | 0 | 0 | 0 | 0 | |||||
| H244N (Cac/Aac) | 0 | 0 | 0 | 0 | 0 | 1 | 1.000 | 0 | 1 | 1.000 | 0 | 1 | 1.000 | 0 | 1 | 1.000 | ||
| M291I (atG/atC) | 1 | 2 | 0.591 | 1 | 2 | 1.000 | 1 | 1 | 1.000 | 1 | 1 | 1.000 | 5 | 3 | 0.682 | 4 | 4 | 1.000 |
| A403S (Gcg/Tcg) | 0 | 0 | 0 | 0 | 0 | 1 | 1.000 | 0 | 1 | 1.000 | 1 | 1 | 1.000 | 0 | 2 | 0.485 | ||
| D461N (Gac/Aac) | 1 | 0 | 1.000 | 1 | 0 | 0.463 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| I485V (Atc/Gtc) | 0 | 1 | 0.463 | 0 | 1 | 1.000 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| N499D (AAc/GAc) | 0 | 0 | 0 | 0 | 1 | 2 | 1.000 | 1 | 2 | 1.000 | 0 | 0 | 0 | 0 | ||||
| N499T (AAc/ACc) | 0 | 0 | 0 | 0 | 0 | 1 | 1.000 | 0 | 1 | 1.000 | 0 | 0 | 0 | 0 | ||||
| A504T (Gcg/Acg) | 0 | 1 | 0.463 | 0 | 1 | 1.000 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
| A508S (Gcg/Tcg) | 1 | 0 | 0 | 1 | 1.000 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |||||
| G512R (Ggg/Agg) | 0 | 3 | 0.093 | 0 | 3 | 0.240 | 0 | 0 | 0 | 0 | 0 | 1 | 1.000 | 0 | 1 | 1.000 | ||
Table 4 shows the sensitivity, specificity, positive likelihood ratio (LR+), negative likelihood ratio (LR−), and accuracy of using gyrA codon 88 to 94 mutations and the gyrB G512R mutation for identifying resistance to OFX and MFX. For OFX, the sensitivity was 69.0% (95% confidence interval [CI], 55.5% to 80.5%), the specificity was 83.0% (70.2% to 91.9%), the LR+ was 4.1 (2.2 to 7.5), the LR− was 0.4 (0.3 to 0.6), and the accuracy rate was 75.7% (66.6% to 83.3%). For MFX, the corresponding values were 67.7% (54.7% to 79.1%), 85.7% (72.8% to 94.1%), 4.7 (2.3 to 9.6), 0.4 (0.3 to 0.5), and 75.7% (66.6% to 83.3%), respectively. The specificity and LR+ of predicting OFX and MFX resistance in East Asian (Beijing) and Indo-Oceanic strains were slightly lower than those in Euro-American strains.
TABLE 4.
Performance of mutation detection (gyrA codon 88 to 94 or gyrB G512R) for identification of ofloxacin and moxifloxacin resistance (low-level and high-level) in different strains of multidrug-resistant Mycobacterium tuberculosis
| Agent | Strain | No. of isolates |
Performance, % (95% confidence interval) |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Resistant |
Susceptible |
|||||||||
| Mutation positive | Mutation negative | Mutation positive | Mutation negative | Sensitivity | Specificity | LR+ | LR− | Accuracy | ||
| Ofloxacin | All | 40 | 18 | 9 | 44 | 69.0 (55.5–80.5) | 83.0 (70.2–91.9) | 4.1 (2.2–7.5) | 0.4 (0.3–0.6) | 75.7 (66.6–83.3) |
| East Asian (Beijing) | 18 | 7 | 5 | 24 | 72.0 (50.6–87.9) | 82.8 (64.2–94.2) | 4.2 (1.8–9.6) | 0.3 (0.2–0.6) | 77.8 (64.4–88.0) | |
| Euro-American | 10 | 6 | 0 | 9 | 62.5 (35.4–84.8) | 100.0 (66.4–100.0) | >10.0 | 0.4 (0.2–0.7) | 76.0 (54.9–90.6) | |
| Indo-Oceanic | 10 | 5 | 4 | 11 | 66.7 (38.4–88.2) | 73.3 (44.9–92.2) | 2.5 (1.0–6.2) | 0.5 (0.2–1.0) | 70.0 (50.6–85.3) | |
| Moxifloxacin | All | 42 | 20 | 7 | 42 | 67.7 (54.7–79.1) | 85.7 (72.8–94.1) | 4.7 (2.3–9.6) | 0.4 (0.3–0.5) | 75.7 (66.6–83.3) |
| East Asian (Beijing) | 19 | 10 | 4 | 21 | 65.5 (45.7–82.1) | 84.0 (63.9–95.5) | 4.1 (1.6–10.4) | 0.4 (0.2–0.7) | 74.1 (60.3–85.0) | |
| Euro-American | 10 | 5 | 0 | 10 | 66.7 (38.4–88.2) | 100.0 (69.2–100.0) | >10.0 | 0.3 (0.2–0.7) | 80.0 (59.3–93.2) | |
| Indo-Oceanic | 10 | 5 | 4 | 11 | 66.7 (38.4–88.2) | 73.3 (44.9–92.2) | 2.5 (1.0–6.2) | 0.5 (0.2–1.0) | 70.0 (50.6–85.3) | |
DISCUSSION
Our study showed that 72.4% of OFX-resistant and 71.0% of MFX-resistant MDR-TB isolates had mutations in the gyrA gene, but those mutations were also found in 30.2% of OFX-susceptible and 28.6% of MFX-susceptible isolates. Mutations in the gyrB gene were less commonly found in OFX-resistant (31.0%) and MFX-resistant (33.9%) isolates, but they were found in 20.8% of OFX-susceptible and 16.3% of MFX-susceptible isolates. Mutations at codons 88 to 94 of gyrA and the gyrB G512R mutation were significantly associated with OFX and MFX resistance; however, among East Asian (Beijing) and Indo-Oceanic strains, OFX- or MFX-susceptible isolates tended to harbor more of those mutations than Euro-American strains.
The most common mutations in the gyrA gene that confer resistance in M. tuberculosis to fluoroquinolones are the A90V, S91P, and D94 (G, A, H, N, or Y) mutations (2, 7, 8, 14, 15). In this study, we found a rare mutation at codon 94 (D94V) in two isolates. The two isolates were susceptible to OFX, but one showed high-level resistance to MFX. Although some previous studies have shown that the levels of resistance to FQs are not associated with the type of mutation present in the gyrA and gyrB genes (16), we found that the D94G and D94N mutations in gyrA and the G512R mutation in gyrB were correlated with high-level MFX resistance, while the D94A mutation was associated with low-level MFX resistance. Similar findings were reported by Nosova et al. and by Kam et al. (15, 17). However, this association is not absolute since the MICs of FQs for isolates with the same mutation vary widely.
Mutations in the gyrB gene are less commonly associated with FQ resistance in M. tuberculosis (7, 8). We found that 20.8% (11/53) of OFX-susceptible isolates, 16.3% (8/49) of MFX-susceptible isolates, 25.0% (10/40) and 32.2% (10/31) of low-level OFX- and MFX-resistant isolates, respectively, and 44.4% (8/18) and 35.5% (11/31) of high-level OFX- and MFX-resistant isolates, respectively, had mutations in gyrB. However, among 29 isolates with gyrB mutations, 22 (75.9%) also had mutations in gyrA, and 18 (62.1%) isolates concomitantly had mutations at codon 94 in gyrA. Among 7 isolates having only gyrB mutations, the sole isolate with a gyrB G512R mutation had high-level OFX and MFX resistance. However, due to the small numbers of isolates with isolated gyrB mutations, no such mutation was significantly independently associated with high-level OFX or MFX resistance.
We found that 24 isolates (21.6%) had 2 to 3 mutations in the gyrA gene or the gyrB gene or in both genes. Of 24 isolates with multiple mutations, 6 (25.0%) had high-level OFX resistance and 10 (41.7%) had high-level MFX resistance. We also found that the rate of high-level resistance to OFX (25.0%) or MFX (41.7%) among isolates with multiple mutations was similar to that among isolates with a single mutation (22.0% for OFX and 43.9% for MFX). Similar findings were reported by Yin and Yu and Matrat et al. (16, 18).
A substantial proportion of FQ-resistant isolates did not harbor mutations in gyrA or gyrB. In mycobacteria, FQ resistance can also be conferred by increased FQ efflux or by DNA mimicry in laboratory strains (19, 20). We found that isolates with low-level MFX resistance had fewer mutations in the gyrA and gyrB genes than isolates with high levels of resistance to MFX. This may indicate that alternative mechanisms of FQ resistance may play a more significant role in conferring low-level MFX resistance (7). This suggests that molecular diagnostic tests that focus on these codons will have lower sensitivity in isolates with low-level FQ resistance.
In this study, mutations at codons 88 to 94 of gyrA were found in 17.0% (9/53) of OFX-susceptible isolates and in 14.3% (7/49) of MFX-susceptible isolates. Surprisingly, those isolates carrying resistance-conferring mutations were susceptible to FQs. Among those susceptible isolates harboring mutations at codons 88 to 94, none were Euro-American strains, but 5 (55.6%) and 4 (44.4%) OFX-susceptible isolates and 4 (57.1%) and 3 (42.9%) MFX-susceptible isolates were East Asian (Beijing) and Indo-Oceanic strains. In addition, there was a limitation because the number of isolates in this study was small, which limited the power to investigate the effect of specific mutations among different lineage types on MICs.
The recommended epidemiological cutoff (ECOFF), which differentiates wild-type and non-wild-type strains, for MFX in Middlebrook 7H10 is 0.5 μg/ml. However, in view of enhanced moxifloxacin activity, two critical concentrations, low level (0.5 μg/ml) and high level (2.0 μg/ml), have been suggested by the World Health Organization since 2012 (21). Previous studies by Sirgel et al. (2) and Zhang et al. (22) have found that mutations at codon 94 in gyrA were linked to high-level fluoroquinolone resistance. A recent report by Kambli et al. (23) also supported the view that the majority of isolates with mutations at codon 94 in gyrA were associated with a high-level resistance to MFX with MICs of 2.5 mg/ml in 76% (32/42) of isolates with a D94G mutation. In the present study, we found that the D94G and D94N mutations in gyrA and the G512R mutation in gyrB were correlated with high-level MFX resistance and that high-level MFX resistance was seen in 70.6% (12/17), 77.8% (7/9), and 100% (4/4) of isolates with a D94G mutation of gyrA, a D94N mutation of gyrA, and a G512R mutation of gyrB, respectively. However, inconsistent with the report by Li et al. (24), the D94A mutation was only associated with low-level MFX resistance. Using detection of the presence of gyrA D94G and D94N and the gyrB G512R mutations, the performance of prediction of high-level MFX-resistance can improve; the sensitivity was 67.7% (48.5% to 82.7%), the specificity was 91.3% (82.3% to 96.1%), the LR+ was 7.7 (3.7 to 16.4), the LR− was 0.2 (0.2 to 0.6), and the accuracy rate was 83.8% (75.6% to 90.1%).
In conclusion, mutations at codons 88 to 94 of gyrA and the G512R mutation in the gyrB gene were associated with OFX and MFX resistance. The level of resistance was not equally affected by mutations in the two genes. However, some OFX- or MFX-susceptible East Asian (Beijing) and Indo-Oceanic strains also harbored those particular mutations, implying that molecular techniques to detect FQ resistance may be less specific in areas with a high prevalence of those strains.
ACKNOWLEDGMENT
We declare no conflict of interest.
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