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Jann-Yuan Wang, Li-Na Lee, Hsin-Chih Lai, Shu-Kuan Wang, I-Shiow Jan, Chong-Jen Yu, Po-Ren Hsueh, Pan-Chyr Yang, Fluoroquinolone resistance in Mycobacterium tuberculosis isolates: associated genetic mutations and relationship to antimicrobial exposure, Journal of Antimicrobial Chemotherapy, Volume 59, Issue 5, May 2007, Pages 860–865, https://doi.org/10.1093/jac/dkm061
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Abstract
We assessed the fluoroquinolone (FQ) susceptibility of clinical isolates of Mycobacterium tuberculosis in an endemic area. The genetic mutations responsible for FQ resistance were also evaluated.
A total of 420 M. tuberculosis isolates during January 2004 to December 2005 were randomly selected. Data on the clinical characteristics of the patients were obtained from medical records. The MICs of ofloxacin, ciprofloxacin, levofloxacin and moxifloxacin were determined. Spoligotyping and sequencing of the gyrA and gyrB genes were performed for all isolates resistant to any tested FQ.
Of the 420 isolates, 52 (12.4%), 26 (6.2%), 26 (6.2%) and 30 (7.1%) were resistant to isoniazid, rifampicin, ethambutol and streptomycin, respectively. Multidrug resistance was found in 5.0% of isolates. For all tested FQs, the susceptibility rate was higher than 97%. Resistance to any first-line drug and isolation from a patient with prior anti-tuberculous treatment were correlated with FQ resistance. Multidrug resistance had the strongest correlation with FQ resistance (19% of isolates). Neither the previous use of FQs nor the duration of FQ exposure was correlated with the FQ susceptibility. Of the 14 FQ-resistant isolates, five (35.7%) had gyrA mutations (four D94G and one A90V) and another one (7.1%) had a gyrB mutation (N538D).
This study found FQ resistance in 3.3% of all clinical isolates of M. tuberculosis. FQ resistance was correlated with first-line drug resistance and prior anti-tuberculous treatment, suggesting the need for routine FQ susceptibility testing in patients with these characteristics.
Introduction
The fluoroquinolones (FQs) were introduced into clinical practice in Taiwan in 1986. FQs have broad-spectrum antimicrobial activity and so are widely used for the treatment of bacterial infections of the respiratory, gastrointestinal and urinary tracts, as well as sexually transmitted diseases and chronic osteomyelitis.1,2 In contrast to many other antibiotics used to treat bacterial infections, the FQs have excellent in vitro and in vivo activity against Mycobacterium tuberculosis.3–5 FQs are recommended for use as prophylactic treatment of patients exposed to multidrug-resistant tuberculosis (MDR-TB, defined as simultaneously resistant to at least isoniazid and rifampicin), for treatment of proven MDR-TB, for empirical treatment of TB disease in settings with high rates of MDR-TB and for patients with severe adverse reaction to first-line agents.6–8 Recent studies showed that previous FQ use and MDR-TB were associated with FQ resistance in M. tuberculosis isolates,9,10 therefore it is crucial to maintain information about the FQ susceptibility in different patient populations to guide selection of the most appropriate treatment. This is especially important for those patients with recurrent TB after treatment, those with MDR-TB as well as those who have previously received FQ therapy. This study was conducted to assess the FQ susceptibility of M. tuberculosis isolates from patients in a medical centre in Taiwan. In our hospital, isepamicin was a recently available aminoglycoside with a low level of toxicity to the kidney and inner ear,11 clarithromycin was the most commonly used macrolide in treating mycobacterial disease and linezolid was the only available oxazolidinone, and so these were also included in the survey.
Materials and methods
M. tuberculosis isolates
This study was conducted in northern Taiwan at a tertiary-care referral centre with 2000 beds. From January 2004 to December 2005, M. tuberculosis was isolated from 2984 clinical specimens, including 2780 respiratory and 204 non-respiratory specimens. Of them, a total of 2778 isolates were preserved, including 2592 (93.2%) from respiratory specimens and 186 (91.2%) from non-respiratory specimens. Preserved isolates were frozen at −80°C in Dubos liquid medium prior to being subcultured on Lowestein–Jensen medium and incubated at 37°C in an aerobic atmosphere with 5–10% CO2.12 Of the 2778 preserved isolates, 420, from 420 patients, were randomly selected. Susceptibility to isoniazid, rifampicin, ethambutol and streptomycin was tested using the modified proportion disc elution method on 7H10 agar (BBL, Becton Dickson, Franklin Lakes, NJ, USA) as described previously.12
Test for MIC
The range of concentrations tested for ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, clarithromycin, linezolid and isepamicin was 0.06–128 mg/L. Except for isepamicin, which was tested against 117 M. tuberculosis isolates, all drugs were tested against all 420 isolates. The MICs were determined by serial dilution on agar plates as described previously.13 Resistance was defined as an MIC of >2 mg/L for ciprofloxacin and ofloxacin, >1 mg/L for levofloxacin and >0.5 mg/L for moxifloxacin.9,14
Clinical evaluation of patients
The medical records (including pharmacy records and the interview records from TB case managers), as well as the registered information on the National Surveillance Network of Communicable Disease (Center for Disease Control, Taiwan) of the selected patients were reviewed to identify prior FQ exposure, prior history of TB and anti-tuberculous treatment prior to collection of the clinical specimens.
Spoligotyping and sequencing of the quinolone resistance-determining regions (QRDRs)
Isolates resistant to any tested FQ were selected. Isolation of mycobacterial DNA and spoligotyping were performed as previously described.15,16 Amplification of the QRDRs of the gyrA and gyrB genes of M. tuberculosis was carried out with primers GYRA-1 (5′-CAGCTACATCGACTATGCG) and GYRA-2 (5′-GGGCTTCGGTGTGTACCTCAT) for the gyrA gene,17 and with primers GYRB-1 (5′-CCACCGACATCGGTGGATT) and GYRB-2 (5′-CTGCCAClTGAGTTTlGTACA) for the gyrB gene.18 Briefly, the PCR mixture (100 μL) contained ∼1 ng of DNA template, final concentrations of 1.0 μM of each set of primers, 200 μM deoxynucleoside triphosphate (dATP, dCTP, dGTP and dTTP; Pharmacia, Uppsala, Sweden) and 10 μL Taq buffer and 5.0 U/μL Taq polymerase (Gibco-Bethesda Research Laboratories). Amplification was performed for 40 cycles (1 min at 94°C, 1 min at 52°C and 2 min at 72°C). The QRDRs of each isolate were amplified in two independent reactions. The PCR products were purified and sequenced in both directions by an automated sequencer.10
Statistical analysis
Analysis of the association of factors with FQ resistance was performed using the χ2 test.
Results
In 2003, the incidence and mortality of TB in Taiwan was 62.4 and 5.8 per 100 000, respectively. Among all TB patients, 7.5% were infected by MDR-TB.19 Of the 420 isolates, 52 (12.4%), 26 (6.2%), 26 (6.2%) and 30 (7.1%) were resistant to isoniazid, rifampicin, ethambutol and streptomycin, respectively. The overall rate of resistance to any one drug was 17.1%, and the MDR rate was 5.0%. The MIC distributions of the seven tested drugs are shown in Table 1. The mean age of the 420 TB patients was 58.0 years (range: 4 months–94 years). A total of 176 (41.9%) patients had underlying disease, the most common being diabetes mellitus (81 patients, 19.3%) and malignancy (71 patients, 16.9%). Serostatus of HIV was tested in 307 (73.1%) patients and was positive in 10 (2.4%). Of the 113 patients with unknown HIV serostatus, all were free of other AIDS-defining illnesses before 30 June 2006. Of the 22 patients receiving immunosuppressant treatment, 11 were post-transplant patients, 8 had autoimmune disease and the remaining 2 had interstitial lung disease. The clinical characteristics of the patients are summarized in Table 2.
Drug . | Total number of isolates . | MIC (mg/L) . | Cut-off MIC for resistance . | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
0.06 . | 0.12 . | 0.25 . | 0.5 . | 1 . | 2 . | 4 . | 8 . | 16 . | 32 . | 64 . | 128 . | |||
OFX | 420 | 2 | 2 | 10 | 116 | 243 | 40 | 3 | 2 | 2 | 0 | 0 | 0 | >2 |
CIP | 420 | 0 | 2 | 25 | 141 | 162 | 84 | 3 | 1 | 1 | 1 | 0 | 0 | >2 |
LVX | 420 | 3 | 17 | 139 | 216 | 39 | 3 | 1 | 2 | 0 | 0 | 0 | 0 | >1 |
MXF | 420 | 20 | 110 | 227 | 53 | 8 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | >0.5 |
CLR | 420 | 15 | 11 | 9 | 11 | 12 | 16 | 31 | 124 | 43 | 55 | 43 | 50 | >2 |
LZD | 420 | 7 | 28 | 125 | 39 | 204 | 16 | 1 | 0 | 0 | 0 | 0 | 0 | >1 |
ISP | 117 | 0 | 0 | 1 | 15 | 81 | 20 | 0 | 0 | 0 | 0 | 0 | 0 | >1 |
Drug . | Total number of isolates . | MIC (mg/L) . | Cut-off MIC for resistance . | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
0.06 . | 0.12 . | 0.25 . | 0.5 . | 1 . | 2 . | 4 . | 8 . | 16 . | 32 . | 64 . | 128 . | |||
OFX | 420 | 2 | 2 | 10 | 116 | 243 | 40 | 3 | 2 | 2 | 0 | 0 | 0 | >2 |
CIP | 420 | 0 | 2 | 25 | 141 | 162 | 84 | 3 | 1 | 1 | 1 | 0 | 0 | >2 |
LVX | 420 | 3 | 17 | 139 | 216 | 39 | 3 | 1 | 2 | 0 | 0 | 0 | 0 | >1 |
MXF | 420 | 20 | 110 | 227 | 53 | 8 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | >0.5 |
CLR | 420 | 15 | 11 | 9 | 11 | 12 | 16 | 31 | 124 | 43 | 55 | 43 | 50 | >2 |
LZD | 420 | 7 | 28 | 125 | 39 | 204 | 16 | 1 | 0 | 0 | 0 | 0 | 0 | >1 |
ISP | 117 | 0 | 0 | 1 | 15 | 81 | 20 | 0 | 0 | 0 | 0 | 0 | 0 | >1 |
CIP, ciprofloxacin; CLR, clarithromycin; ISP, isepamicin; LVX, levofloxacin; LZD, linezolid; MXF, moxifloxacin; OFX, ofloxacin.
Drug . | Total number of isolates . | MIC (mg/L) . | Cut-off MIC for resistance . | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
0.06 . | 0.12 . | 0.25 . | 0.5 . | 1 . | 2 . | 4 . | 8 . | 16 . | 32 . | 64 . | 128 . | |||
OFX | 420 | 2 | 2 | 10 | 116 | 243 | 40 | 3 | 2 | 2 | 0 | 0 | 0 | >2 |
CIP | 420 | 0 | 2 | 25 | 141 | 162 | 84 | 3 | 1 | 1 | 1 | 0 | 0 | >2 |
LVX | 420 | 3 | 17 | 139 | 216 | 39 | 3 | 1 | 2 | 0 | 0 | 0 | 0 | >1 |
MXF | 420 | 20 | 110 | 227 | 53 | 8 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | >0.5 |
CLR | 420 | 15 | 11 | 9 | 11 | 12 | 16 | 31 | 124 | 43 | 55 | 43 | 50 | >2 |
LZD | 420 | 7 | 28 | 125 | 39 | 204 | 16 | 1 | 0 | 0 | 0 | 0 | 0 | >1 |
ISP | 117 | 0 | 0 | 1 | 15 | 81 | 20 | 0 | 0 | 0 | 0 | 0 | 0 | >1 |
Drug . | Total number of isolates . | MIC (mg/L) . | Cut-off MIC for resistance . | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
0.06 . | 0.12 . | 0.25 . | 0.5 . | 1 . | 2 . | 4 . | 8 . | 16 . | 32 . | 64 . | 128 . | |||
OFX | 420 | 2 | 2 | 10 | 116 | 243 | 40 | 3 | 2 | 2 | 0 | 0 | 0 | >2 |
CIP | 420 | 0 | 2 | 25 | 141 | 162 | 84 | 3 | 1 | 1 | 1 | 0 | 0 | >2 |
LVX | 420 | 3 | 17 | 139 | 216 | 39 | 3 | 1 | 2 | 0 | 0 | 0 | 0 | >1 |
MXF | 420 | 20 | 110 | 227 | 53 | 8 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | >0.5 |
CLR | 420 | 15 | 11 | 9 | 11 | 12 | 16 | 31 | 124 | 43 | 55 | 43 | 50 | >2 |
LZD | 420 | 7 | 28 | 125 | 39 | 204 | 16 | 1 | 0 | 0 | 0 | 0 | 0 | >1 |
ISP | 117 | 0 | 0 | 1 | 15 | 81 | 20 | 0 | 0 | 0 | 0 | 0 | 0 | >1 |
CIP, ciprofloxacin; CLR, clarithromycin; ISP, isepamicin; LVX, levofloxacin; LZD, linezolid; MXF, moxifloxacin; OFX, ofloxacin.
Patient group . | Overall (n = 420) . | First-line drug susceptibility . | FQ-resistant (n = 14) . | ||
---|---|---|---|---|---|
susceptible (n = 349) . | resistant, not MDR (n = 50) . | MDR (n = 21) . | |||
Age >65 years | 194 (46.2%) | 167 (47.9%) | 23 (46.0%) | 4 (19.0%) | 6 (42.9%) |
Male gender | 290 (69.0%) | 237 (67.9%) | 38 (76.0%) | 15 (71.4%) | 8 (57.1%) |
Extent of TB | |||||
pulmonary | 380 (90.5%) | 312 (89.4%) | 48 (96.0%) | 20 (95.2%) | 13 (92.9%) |
extrapulmonary | 23 (5.5%) | 23 (6.6%) | 0 | 0 | 1 (7.1%) |
both | 17 (4.0%) | 14 (4.0%) | 2 (4.0%) | 1 (4.8%) | 0 |
Sputum AFS-positive | 115 (27.4%) | 98 (28.1%) | 13 (26.0%) | 4 (19.0%) | 4 (28.6%) |
Underlying disease | |||||
diabetes mellitus | 81 (19.3%) | 64 (18.3%) | 11 (22.0%) | 6 (28.6%) | 4 (28.6%) |
malignancy | 71 (16.9%) | 61 (17.5%) | 5 (10.0%) | 5 (23.8%) | 3 (21.4%) |
using immunosuppressant | 22 (5.2%) | 16 (4.6%) | 4 (8.0%) | 2 (9.5%) | 1 (7.1%) |
ESRD under haemodialysis | 16 (3.8%) | 10 (2.9%) | 4 (8.0%) | 2 (9.5%) | 0 |
liver cirrhosis | 12 (2.9%) | 9 (2.6%) | 3 (6.0%) | 0 | 0 |
HIV/AIDS | 10 (2.4%) | 5 (1.4%) | 3 (6.0%) | 2 (9.5%) | 0 |
Patient group . | Overall (n = 420) . | First-line drug susceptibility . | FQ-resistant (n = 14) . | ||
---|---|---|---|---|---|
susceptible (n = 349) . | resistant, not MDR (n = 50) . | MDR (n = 21) . | |||
Age >65 years | 194 (46.2%) | 167 (47.9%) | 23 (46.0%) | 4 (19.0%) | 6 (42.9%) |
Male gender | 290 (69.0%) | 237 (67.9%) | 38 (76.0%) | 15 (71.4%) | 8 (57.1%) |
Extent of TB | |||||
pulmonary | 380 (90.5%) | 312 (89.4%) | 48 (96.0%) | 20 (95.2%) | 13 (92.9%) |
extrapulmonary | 23 (5.5%) | 23 (6.6%) | 0 | 0 | 1 (7.1%) |
both | 17 (4.0%) | 14 (4.0%) | 2 (4.0%) | 1 (4.8%) | 0 |
Sputum AFS-positive | 115 (27.4%) | 98 (28.1%) | 13 (26.0%) | 4 (19.0%) | 4 (28.6%) |
Underlying disease | |||||
diabetes mellitus | 81 (19.3%) | 64 (18.3%) | 11 (22.0%) | 6 (28.6%) | 4 (28.6%) |
malignancy | 71 (16.9%) | 61 (17.5%) | 5 (10.0%) | 5 (23.8%) | 3 (21.4%) |
using immunosuppressant | 22 (5.2%) | 16 (4.6%) | 4 (8.0%) | 2 (9.5%) | 1 (7.1%) |
ESRD under haemodialysis | 16 (3.8%) | 10 (2.9%) | 4 (8.0%) | 2 (9.5%) | 0 |
liver cirrhosis | 12 (2.9%) | 9 (2.6%) | 3 (6.0%) | 0 | 0 |
HIV/AIDS | 10 (2.4%) | 5 (1.4%) | 3 (6.0%) | 2 (9.5%) | 0 |
AFS, acid-fast smear; ESRD, end-stage renal disease; FQ, fluoroquinolone; MDR, multidrug resistant.
Patient group . | Overall (n = 420) . | First-line drug susceptibility . | FQ-resistant (n = 14) . | ||
---|---|---|---|---|---|
susceptible (n = 349) . | resistant, not MDR (n = 50) . | MDR (n = 21) . | |||
Age >65 years | 194 (46.2%) | 167 (47.9%) | 23 (46.0%) | 4 (19.0%) | 6 (42.9%) |
Male gender | 290 (69.0%) | 237 (67.9%) | 38 (76.0%) | 15 (71.4%) | 8 (57.1%) |
Extent of TB | |||||
pulmonary | 380 (90.5%) | 312 (89.4%) | 48 (96.0%) | 20 (95.2%) | 13 (92.9%) |
extrapulmonary | 23 (5.5%) | 23 (6.6%) | 0 | 0 | 1 (7.1%) |
both | 17 (4.0%) | 14 (4.0%) | 2 (4.0%) | 1 (4.8%) | 0 |
Sputum AFS-positive | 115 (27.4%) | 98 (28.1%) | 13 (26.0%) | 4 (19.0%) | 4 (28.6%) |
Underlying disease | |||||
diabetes mellitus | 81 (19.3%) | 64 (18.3%) | 11 (22.0%) | 6 (28.6%) | 4 (28.6%) |
malignancy | 71 (16.9%) | 61 (17.5%) | 5 (10.0%) | 5 (23.8%) | 3 (21.4%) |
using immunosuppressant | 22 (5.2%) | 16 (4.6%) | 4 (8.0%) | 2 (9.5%) | 1 (7.1%) |
ESRD under haemodialysis | 16 (3.8%) | 10 (2.9%) | 4 (8.0%) | 2 (9.5%) | 0 |
liver cirrhosis | 12 (2.9%) | 9 (2.6%) | 3 (6.0%) | 0 | 0 |
HIV/AIDS | 10 (2.4%) | 5 (1.4%) | 3 (6.0%) | 2 (9.5%) | 0 |
Patient group . | Overall (n = 420) . | First-line drug susceptibility . | FQ-resistant (n = 14) . | ||
---|---|---|---|---|---|
susceptible (n = 349) . | resistant, not MDR (n = 50) . | MDR (n = 21) . | |||
Age >65 years | 194 (46.2%) | 167 (47.9%) | 23 (46.0%) | 4 (19.0%) | 6 (42.9%) |
Male gender | 290 (69.0%) | 237 (67.9%) | 38 (76.0%) | 15 (71.4%) | 8 (57.1%) |
Extent of TB | |||||
pulmonary | 380 (90.5%) | 312 (89.4%) | 48 (96.0%) | 20 (95.2%) | 13 (92.9%) |
extrapulmonary | 23 (5.5%) | 23 (6.6%) | 0 | 0 | 1 (7.1%) |
both | 17 (4.0%) | 14 (4.0%) | 2 (4.0%) | 1 (4.8%) | 0 |
Sputum AFS-positive | 115 (27.4%) | 98 (28.1%) | 13 (26.0%) | 4 (19.0%) | 4 (28.6%) |
Underlying disease | |||||
diabetes mellitus | 81 (19.3%) | 64 (18.3%) | 11 (22.0%) | 6 (28.6%) | 4 (28.6%) |
malignancy | 71 (16.9%) | 61 (17.5%) | 5 (10.0%) | 5 (23.8%) | 3 (21.4%) |
using immunosuppressant | 22 (5.2%) | 16 (4.6%) | 4 (8.0%) | 2 (9.5%) | 1 (7.1%) |
ESRD under haemodialysis | 16 (3.8%) | 10 (2.9%) | 4 (8.0%) | 2 (9.5%) | 0 |
liver cirrhosis | 12 (2.9%) | 9 (2.6%) | 3 (6.0%) | 0 | 0 |
HIV/AIDS | 10 (2.4%) | 5 (1.4%) | 3 (6.0%) | 2 (9.5%) | 0 |
AFS, acid-fast smear; ESRD, end-stage renal disease; FQ, fluoroquinolone; MDR, multidrug resistant.
The rates of susceptibility to ofloxacin, ciprofloxacin, levofloxacin and moxifloxacin were 98.3%, 98.6%, 98.6% and 97.6%, respectively. Except for streptomycin resistance, first-line drug resistances were correlated with any FQ resistance (Table 3). This correlation was strongest for MDR isolates. Of the 420 isolates, 108 were isolated from clinical specimens collected after FQ exposure. The median duration of FQ exposure was 7 days (25th percentile: 2; 75th percentile: 15). Four (8.9%) of the 45 patients with previous FQ exposure >1 week and 5 (7.9%) of the 63 with previous FQ exposure ≤1 week had cavitation on chest images, compared with 50 (16.0%) of the 312 patients without FQ exposure (P = 0.047). Neither the previous exposure to FQs nor the duration of FQ exposure was correlated with the FQ susceptibility of M. tuberculosis isolates (Table 3).
. | . | Overall . | AnyR* . | MDR* . | Prior anti-TB treatment* . | Previous FQ use . | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
. | . | no (n = 349) . | yes (n = 71) . | no (n = 399) . | yes (n = 21) . | yes (n = 63) . | no (n = 357) . | never (n = 312) . | ≤1 week (n = 63) . | >1 week (n = 45) . | |
Any FQ resistance | Number of isolates | 14 | 8 | 6 | 10 | 4 | 5 | 9 | 9 | 3 | 2 |
% | 3.3 | 2.3 | 8.5 | 2.5 | 19.0 | 7.9 | 2.5 | 2.9 | 4.8 | 4.4 |
. | . | Overall . | AnyR* . | MDR* . | Prior anti-TB treatment* . | Previous FQ use . | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
. | . | no (n = 349) . | yes (n = 71) . | no (n = 399) . | yes (n = 21) . | yes (n = 63) . | no (n = 357) . | never (n = 312) . | ≤1 week (n = 63) . | >1 week (n = 45) . | |
Any FQ resistance | Number of isolates | 14 | 8 | 6 | 10 | 4 | 5 | 9 | 9 | 3 | 2 |
% | 3.3 | 2.3 | 8.5 | 2.5 | 19.0 | 7.9 | 2.5 | 2.9 | 4.8 | 4.4 |
AnyR, any-drug resistance; FQ, fluoroquinolone; MDR, multidrug resistance.
*Significant difference (P < 0.05) in χ2 between groups.
. | . | Overall . | AnyR* . | MDR* . | Prior anti-TB treatment* . | Previous FQ use . | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
. | . | no (n = 349) . | yes (n = 71) . | no (n = 399) . | yes (n = 21) . | yes (n = 63) . | no (n = 357) . | never (n = 312) . | ≤1 week (n = 63) . | >1 week (n = 45) . | |
Any FQ resistance | Number of isolates | 14 | 8 | 6 | 10 | 4 | 5 | 9 | 9 | 3 | 2 |
% | 3.3 | 2.3 | 8.5 | 2.5 | 19.0 | 7.9 | 2.5 | 2.9 | 4.8 | 4.4 |
. | . | Overall . | AnyR* . | MDR* . | Prior anti-TB treatment* . | Previous FQ use . | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
. | . | no (n = 349) . | yes (n = 71) . | no (n = 399) . | yes (n = 21) . | yes (n = 63) . | no (n = 357) . | never (n = 312) . | ≤1 week (n = 63) . | >1 week (n = 45) . | |
Any FQ resistance | Number of isolates | 14 | 8 | 6 | 10 | 4 | 5 | 9 | 9 | 3 | 2 |
% | 3.3 | 2.3 | 8.5 | 2.5 | 19.0 | 7.9 | 2.5 | 2.9 | 4.8 | 4.4 |
AnyR, any-drug resistance; FQ, fluoroquinolone; MDR, multidrug resistance.
*Significant difference (P < 0.05) in χ2 between groups.
Among the 420 M. tuberculosis isolates, 63 were isolated from clinical specimens in patients who had received prior anti-tuberculous treatment. Of them, 19 patients had a prior history of TB post complete treatment and 44 were currently receiving anti-tuberculous treatment. The regimens used were isoniazid + ethambutol + rifampicin + pyrazinamide in 35 patients, isoniazid + ethambutol + rifampicin in 4, isoniazid + rifampicin + pyrazinamide in 2 and FQ-containing anti-tuberculous regimens in the remaining 3. Prior anti-tuberculous treatment was correlated with FQ resistance (Table 3).
A total of 14 isolates were resistant to at least one of the tested FQs. Of them, six belonged to the Beijing family (strains that only hybridized to the last nine spacer oligonucleotides).20 All of the eight non-Beijing strains had unique patterns. Sequencing of the QRDRs was performed for the 14 FQ-resistant isolates and another 28 FQ-susceptible isolates. Of them, 41 isolates had gyrA 95T while only one strain had a gyrA 95S allele. As shown in Table 4, five (35.7%) FQ-resistant isolates had a point mutation in gyrA (four D94G and one A90V). Another one (7.1%) FQ-resistant isolate had a point mutation in gyrB (N538D). No mutations in gyrA and gyrB were found in the remaining 8 FQ-resistant isolates and the 28 FQ-susceptible isolates.
. | . | Isolate . | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
. | . | 1 . | 2 . | 3 . | 4 . | 5 . | 6 . | 7 . | 8 . | 9 . | 10 . | 11 . | 12 . | 13 . | 14 . |
MIC | OFX | 16 | 2.0 | 2.0 | 4.0 | 4.0 | 2.0 | 2.0 | 4.0 | 8.0 | 1.0 | 1.0 | 16 | 8.0 | 2.0 |
CIP | 32 | 2.0 | 4.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 4.0 | 4.0 | 0.5 | 8.0 | 16 | 2.0 | |
LVX | 8.0 | 0.5 | 1.0 | 1.0 | 0.5 | 1.0 | 2.0 | 2.0 | 1.0 | 2.0 | 1.0 | 4.0 | 8.0 | 1.0 | |
MXF | 8.0 | 1.0 | 0.5 | 1.0 | 0.25 | 1.0 | 0.5 | 0.5 | 1.0 | 1.0 | 1.0 | 1.0 | 4.0 | 1.0 | |
Mutation | gyrA | D94G | D94G | D94G | A90V | D94G | |||||||||
gyrB | N538D |
. | . | Isolate . | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
. | . | 1 . | 2 . | 3 . | 4 . | 5 . | 6 . | 7 . | 8 . | 9 . | 10 . | 11 . | 12 . | 13 . | 14 . |
MIC | OFX | 16 | 2.0 | 2.0 | 4.0 | 4.0 | 2.0 | 2.0 | 4.0 | 8.0 | 1.0 | 1.0 | 16 | 8.0 | 2.0 |
CIP | 32 | 2.0 | 4.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 4.0 | 4.0 | 0.5 | 8.0 | 16 | 2.0 | |
LVX | 8.0 | 0.5 | 1.0 | 1.0 | 0.5 | 1.0 | 2.0 | 2.0 | 1.0 | 2.0 | 1.0 | 4.0 | 8.0 | 1.0 | |
MXF | 8.0 | 1.0 | 0.5 | 1.0 | 0.25 | 1.0 | 0.5 | 0.5 | 1.0 | 1.0 | 1.0 | 1.0 | 4.0 | 1.0 | |
Mutation | gyrA | D94G | D94G | D94G | A90V | D94G | |||||||||
gyrB | N538D |
OFX, ofloxacin; CIP, ciprofloxacin; LVX, levofloxacin; MXF, moxifloxacin.
. | . | Isolate . | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
. | . | 1 . | 2 . | 3 . | 4 . | 5 . | 6 . | 7 . | 8 . | 9 . | 10 . | 11 . | 12 . | 13 . | 14 . |
MIC | OFX | 16 | 2.0 | 2.0 | 4.0 | 4.0 | 2.0 | 2.0 | 4.0 | 8.0 | 1.0 | 1.0 | 16 | 8.0 | 2.0 |
CIP | 32 | 2.0 | 4.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 4.0 | 4.0 | 0.5 | 8.0 | 16 | 2.0 | |
LVX | 8.0 | 0.5 | 1.0 | 1.0 | 0.5 | 1.0 | 2.0 | 2.0 | 1.0 | 2.0 | 1.0 | 4.0 | 8.0 | 1.0 | |
MXF | 8.0 | 1.0 | 0.5 | 1.0 | 0.25 | 1.0 | 0.5 | 0.5 | 1.0 | 1.0 | 1.0 | 1.0 | 4.0 | 1.0 | |
Mutation | gyrA | D94G | D94G | D94G | A90V | D94G | |||||||||
gyrB | N538D |
. | . | Isolate . | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
. | . | 1 . | 2 . | 3 . | 4 . | 5 . | 6 . | 7 . | 8 . | 9 . | 10 . | 11 . | 12 . | 13 . | 14 . |
MIC | OFX | 16 | 2.0 | 2.0 | 4.0 | 4.0 | 2.0 | 2.0 | 4.0 | 8.0 | 1.0 | 1.0 | 16 | 8.0 | 2.0 |
CIP | 32 | 2.0 | 4.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 4.0 | 4.0 | 0.5 | 8.0 | 16 | 2.0 | |
LVX | 8.0 | 0.5 | 1.0 | 1.0 | 0.5 | 1.0 | 2.0 | 2.0 | 1.0 | 2.0 | 1.0 | 4.0 | 8.0 | 1.0 | |
MXF | 8.0 | 1.0 | 0.5 | 1.0 | 0.25 | 1.0 | 0.5 | 0.5 | 1.0 | 1.0 | 1.0 | 1.0 | 4.0 | 1.0 | |
Mutation | gyrA | D94G | D94G | D94G | A90V | D94G | |||||||||
gyrB | N538D |
OFX, ofloxacin; CIP, ciprofloxacin; LVX, levofloxacin; MXF, moxifloxacin.
Discussion
This study found that clinical isolates of M. tuberculosis had an overall FQ resistance of 3.3% and MDR isolates had a resistance rate of 19%. These rates were lower than those found in previous studies from southern Taiwan (6.2% in general and 22.2% for MDR-TB)10 and the Philippines (35.3% in general and 51.4% in MDR-TB),21 but higher than those found in previous studies conducted from other countries (1.8% in general and 4.1% in MDR-TB in the United States and Canada;3 4.3% in MDR-TB in Russia22). Since the study was performed in a tertiary-care centre, our results could probably be biased towards drug-resistant isolates. However, because several studies demonstrated an association of prior FQ use with the emergence of FQ-resistant M. tuberculosis,9,23 the high FQ resistance rate in the Philippines and Taiwan may reflect the poor control of FQ use in these countries, where all FQs are available without a prescription in local drug stores.21 In our hospital, FQs were prescribed for a total of 7482 and 7987 patient-episodes in 2004 and 2005, respectively, more than 60% being used for respiratory infections. Our further analysis revealed no correlation between previous exposure of FQs and FQ susceptibility of M. tuberculosis isolates. A longer duration of FQ exposure (>1 week versus ≤1 week) was also not associated with a higher resistance rate (Table 3). Given the previously reported natural prevalence of spontaneous FQ-resistant mutants of M. tuberculosis was 2 × 10−6 to 1 × 10−8 per replicating organism,17 and the large burden of organisms in the pulmonary cavity (1 × 108 cfu/mL on average),24 the significantly lower rate of pulmonary cavitation in patients with previous FQ exposure than in those without (8.3% versus 16.0%, P = 0.047) in this study might explain the reduced in vivo selection of FQ-resistant mutants. The lack of available data on previous medication history outside our hospital, which may have led to failure to consider FQ use prior to the hospital visit, is another possible explanation for these discrepant findings. In addition, the number of isolates in this study may not have been large enough to demonstrate significant differences in FQ susceptibility.
The similar proportion of Beijing family (42.9%) among FQ-resistant isolates to that (44.4%) in the general TB population in Taiwan20 and the unique spoligotypes of all of the non-Beijing isolates in this study did not support the clonal spreading of an FQ-resistant strain. Similar to previous studies,10,25 mutation in the QRDR of gyrA was the most common cause of FQ resistance. Other mechanisms, such as mutations in areas of gyrA or gyrB outside of the QRDR, decreased cell-wall permeability to the drug, an active drug efflux pump, sequestration of drug or drug inactivation, probably accounted for the FQ resistance in other FQ-resistant isolates, as well as FQ-resistant isolate 1 and isolate 13 (Table 4).26,27
Although a previous study showed that there was no cross-resistance between FQs and other anti-tuberculous agents,28 the selection of an FQ-resistant subpopulation of M. tuberculosis requires an actively multiplying bacillary population large enough to contain spontaneous drug-resistant mutants.14 Therefore, FQ resistance is primarily seen in patients with drug-resistant TB, especially MDR-TB, treated with an FQ as the only active agent.3,10,14 Our finding that M. tuberculosis isolates resistant to any first-line anti-tuberculous drug (isoniazid, rifampicin and ethambutol) were more likely to have FQ resistance was consistent with these previous studies (Table 3). This association was strongest in MDR isolates. Because of their potent in vitro and in vivo activity,3,4,29 as well as excellent safety record in long-term therapy,30,31 FQs continue to be used for the treatment of TB primarily in cases involving resistance or intolerance to first-line anti-tuberculous drugs. They are also candidates for use as new first-line drugs.31 Therefore we recommend that in Taiwan or other areas with a high FQ resistance rate, FQ susceptibility should be routinely assessed for clinical isolates of M. tuberculosis, especially for drug-resistant isolates, and when patients have a prior history of TB or prior use of anti-tuberculous drugs.
Because MDR-TB has emerged as a threat to TB control worldwide,32 new anti-tuberculous agents with bactericidal mechanisms different from those of available first-line drugs are urgently needed. In addition to FQs, other classes of antimicrobial agents that display various degrees of in vitro and/or in vivo activities against M. tuberculosis include aminoglycosides,33,34 macrolides35 and oxazolidinones.36 Our results showed that 17.6%, 96.0% and 82.9% of the tested M. tuberculosis isolates were susceptible to clarithromycin, linezolid and isepamicin, respectively, if resistance was defined as an MIC of >2 mg/L for clarithromycin35 and >1 mg/L for linezolid36 and isepamicin.34
In conclusion, our study demonstrated that overall clinical isolates were very susceptible to FQs, with a resistance rate of 3.3%. First-line drug resistance, especially MDR, and prior anti-tuberculous treatment were significantly associated with FQ resistance, implying that FQ susceptibility should be routinely assessed in these special situations. Mutations in the QRDR of gyrA (35.7%) and gyrB (7.1%) were the major causes of FQ resistance.
Acknowledgements
This study was supported by the Institute for Biotechnology and Medicine Industry, Taiwan.
Transparency declarations
None to declare.