Abstract
Consecutive fluoroquinolone (FQ)-resistant isolates (n = 109) identified at the Pham Ngoc Thach Hospital for Tuberculosis, Ho Chi Minh City, Vietnam, were sequenced in the quinolone resistance-determining regions of the gyrA and gyrB genes and typed by large sequence polymorphism typing and spoligotyping to identify the Beijing genotype of Mycobacterium tuberculosis. Beijing genotype prevalence was compared with 109 consecutive isolates from newly presenting patients with pulmonary tuberculosis from the hospital outpatient department. Overall, 82.6% (n = 90/109) of isolates had mutations in gyrAB. Nine novel mutations were identified in gyrB (S486F, N538T, T539P, D500A, D500H, D500N, G509A, E540V, and E540D). The influence of these novel gyrB mutations on FQ resistance is not proven. The Beijing genotype was significantly associated with FQ resistance (odds ratio [OR], 2.39 [95% confidence interval {CI}, 1.34 to 4.25]; P = 0.003). Furthermore, Beijing genotype FQ-resistant isolates were significantly more likely than FQ-resistant isolates of other genotypes to have gyrA mutations (OR, 7.75 [95% CI, 2.84 to 21.15]; P = 0.0001) and high-level (>8 μg/ml) FQ resistance (OR, 11.0 [95% CI, 2.6 to 47.0]; P = 0.001). The underlying mechanism of the association of the Beijing genotype with high-level FQ resistance in this setting remains to be determined. The association of the Beijing genotype with relatively high-level FQ resistance conferred by specific gyrA mutations reported here is of grave concern given the epidemic spread of the Beijing genotype and the current hopes for shorter first-line treatment regimens based on FQs.
Fluoroquinolones (FQs) are the most promising antituberculous therapeutic agents to be developed in 40 years (9, 31). They are widely used for the treatment of multidrug-resistant (MDR) tuberculosis (TB) despite the lack of clinical trials evaluating optimal doses, duration, and combinations (10, 28, 31). Gatifloxacin is currently in phase III trials as a first-line agent to shorten existing treatment regimens from 6 to 4 months (ClinicalTrials.gov identification number NCT00216385 [http://clinicaltrials.gov/ct2/show/NCT00216385]), and moxifloxacin is in phase III trials as a first-line substitute for either ethambutol (ETH) (ClinicalTrials.gov identification number NCT00082173 [http://clinicaltrials.gov/show/NCT00082173]) or isoniazid (INH) (ISRCTN register number 85595810 [http://www.controlled-trials.com/ISRCTN85595810]; ClinicalTrials.gov identification number NCT00144417 [www.clinicaltrials.gov/show/NCT00144417]).
There is concern about levels of preexisting FQ-resistant TB in regions with high drug resistance rates because these drugs are often available over the counter and are additionally prescribed as broad-spectrum antibiotics for the treatment of undiagnosed respiratory infections (4, 5, 11, 17, 23, 27, 29).
Vietnam has some of the highest primary drug resistance rates for Mycobacterium tuberculosis in the world, with over 35% of primary isolates being resistant to one or more first-line drugs (21, 26). Despite this, MDR TB rates remain relatively low, at 2.7% nationally, and the National Tuberculosis Program has achieved World Health Organization (WHO) targets for the detection and cure of TB for the last 10 years (14). An expanded MDR TB management program (formally DOTS-PLUS) will be piloted in the near future; however, the success of standardized regimens will depend heavily on preexisting levels of resistance to the most effective second-line agents, the FQs. At present, no data exist on FQ-resistant TB in Vietnam.
In mycobacteria, the FQs bind to DNA gyrase and inhibit DNA replication. Reports in the literature show that the majority (approximately 60%) of FQ-resistant M. tuberculosis isolates carry mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene, and a small number have mutations in the gyrB gene (10). It was previously postulated that efflux pump mechanisms account for FQ resistance in isolates with wild-type gyrAB genes (6).
While adherence remains the single most important factor in the emergence of drug-resistant TB, a factor contributing to the high prevalence of INH and streptomycin (STR) resistance in the region may be the high prevalence of strains of Mycobacterium tuberculosis of the Beijing genotype (1-3). The Beijing genotype first attracted attention as being the genotype of the strain responsible (W strain) for several large outbreaks of MDR TB in the United States in the early 1990s (28). It is associated with drug resistance and MDR in Vietnam (1, 3).
This study investigated the prevalence of the Beijing genotype among FQ-resistant isolates from southern Vietnam and the associated genotypic mutations and MICs of ofloxacin.
MATERIALS AND METHODS
Samples.
One hundred nine consecutive isolates identified as being FQ resistant (ofloxacin at 2 μg/ml) at the Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases (PNT), Ho Chi Minh City, Vietnam, following clinician-initiated referral testing between 2005 and 2007 were collected. FQ susceptibility testing is not routine in the Vietnamese National Tuberculosis Programme, and these isolates were tested following a request from the treating clinician, usually following retreatment failure.
Isolates from 109 consecutive patients presenting to the PNT outpatient department with pulmonary tuberculosis in August 2008 were prospectively collected as a control group. The outpatient department is a routine clinic serving the local population as a district tuberculosis clinic.
Detailed clinical data were not available for the FQ-resistant isolates; however, isolates were known not to be duplicated or epidemiologically linked and were widely distributed from districts around southern Vietnam, and they were not part of a single outbreak (data not shown).
Drug susceptibility testing.
Isolates were tested at PNT by standard WHO 1% proportion methods on Lowenstein-Jensen medium for susceptibility to ofloxacin (2 μg/ml) (all FQ-resistant isolates), INH (0.2 μg/ml), rifampin (rifampicin) (RIF) (40 μg/ml), ETH (2 μg/ml), and STR (4 μg/ml) (first 40 patients only). The PNT microbiology laboratory is a TB reference laboratory that participates in WHO supranational quality control for testing of susceptibility to the first-line drugs kanamycin and ofloxacin. The concordance for external quality control of ofloxacin testing was 100%.
FQ-resistant isolates that had no mutations in gyrAB upon sequencing were given anonymized codes and resubmitted for FQ testing, with the technician being unaware of the original phenotypic report or the sequencing results.
MIC testing for ofloxacin was performed for the first 65 FQ-resistant isolates at concentrations of 2, 4, 6, 8, and 12 μg/ml using the Lowenstein-Jensen 1% proportion method. Technicians performing MIC testing were blinded to the sequencing data.
Sequencing.
Isolates were sequenced in the QRDR of the gyrA and gyrB genes. PCR products were purified with QIAquick PCR purification kits (Qiagen). Both strands were sequenced with CEQ dye terminator cycle sequencing quick-start kits (Beckman Coulter, Inc., Fullerton, CA) in a half-volume reaction mixture using primers GYRATBF (5′-CAG CTA CAT CGA CTA TGC GA-3′) and GYRATBR (5′-GGG CTT CGG TGT ACC TCA T-3′) for gyrA (30) and in-house-designed primers gyrB TBF1 (5′-ACG CGA AAG TCG TTGTGA A-3′) and gyrB TBR1 (5′-CGC TGC CAC TTG AGT TTG TA-3′) for gyrB.
The thermal cycling protocol for gyrA was 95°C for 20 s, 62°C for 20 s, and 60°C for 2 min for 30 cycles. For gyrB primers, the annealing temperature was 50°C. The cycle sequencing products were subsequently subjected to ethanol precipitation steps according to the manufacturer's instructions and sequenced using the CEQ8000 genetic analysis system (Beckman Coulter).
Spoligotyping.
Spoligotyping of isolates was performed according to standard protocols (16). Briefly, DNA was extracted by the cetyltrimethylammonium bromide method and diluted to a working solution of 15 ng/μl. PCR products were amplified with primers Dra (biotin labeled) and Drb, hybridized to the spoligotype membrane (Isogen Bioscience BV, Maarsen, The Netherlands), incubated with streptavidin-peroxidase, and detected with ECL (Amersham, United Kingdom). Results were analyzed using Bionumerics software (Applied Maths, Belgium). The Beijing genotype here is used to designate all “Beijing-type” variants, i.e., any isolate missing spacers 1 to 34, with at least three of spacers 35 to 43.
Typing of LSPs.
Isolates were typed for large sequence polymorphisms (LSPs) at RD105 (East Asian lineage including the Beijing genotype) and RD239 (Indo-Oceanic lineage) to assign major geographical clades, as previously described (8). Isolates with neither deletion were assigned to the Euro-American clade on the basis of spoligotyping patterns. It was previously shown that these are the major clades present in Vietnam (2).
Analysis.
Univariate logistic regression was used to estimate the odds ratio (OR) for the association of variables with Stata 9 (Statacorp, TX).
RESULTS
Sequencing.
One hundred nine consecutive FQ-resistant isolates were sequenced in the gyrA and gyrB QRDRs. Results are presented in Table 1. Overall, 82.6% (n = 90/109) of isolates had mutations in gyrAB other than S95T; all isolates carried the S95T polymorphism, which is not involved in FQ resistance (10). A total of 45.9% (n = 50/109) of isolates carried mutations at codon 94, with five different amino acid changes at this site: D94G (n = 29), D94A (n = 12), D94Y (n = 5), D94N (n = 3), and D94H (n = 1). Three isolates had two or more mutations at codon 94. A90V was the next most prevalent mutation, with 20.2% (n = 22/109) of isolates carrying this mutation. Nine novel mutations were identified in gyrB (S486F, D500A, D500H, D500N, G509A N538T, T539P, E540V, and E540D). The influence of these novel mutations on FQ resistance is not proven. Many isolates (34/109 isolates [31.2%]) (Table 1) had heterogeneous peaks upon sequencing at the mutation site, indicating that these isolates may be a mixture of sensitive and resistant strains from within a single individual.
TABLE 1.
Mutation patterns in gyrA and gyrB identified in 109 consecutive FQ-resistant isolates by sequencinga
| gyrA mutation | gyrB mutation | No. of isolates (%) | No. of isolates with heteropeak |
|---|---|---|---|
| A90V | Wild type | 19 (17.4) | 3 |
| A90V | T539P | 1 (0.9) | 0 |
| A90V | D500A | 1 (0.9) | 1 |
| A90V | N538T | 1 (0.9) | 1 |
| A90V D94G | Wild type | 6 (5.5) | 6 |
| A90V D94A | Wild type | 2 (1.8) | 2 |
| A90V D94A | N538T | 1 (0.9) | 1 |
| A90V S91P D94G D94A | N538T | 1 (0.9) | 1 |
| D94A | Wild type | 11 (10.1) | 3 |
| D94G | Wild type | 26 (23.8) | 10 |
| D94G | N538T | 1 (0.9) | 1 |
| D94G D94A | Wild type | 1 (0.9) | 1 |
| D94H | Wild type | 1 (0.9) | 0 |
| D94N | Wild type | 2 (1.8) | 0 |
| D94A | D500N | 1 (0.9) | 1 |
| D94Y | Wild type | 5 (4.6) | 0 |
| S91P | Wild type | 2 (1.8) | 0 |
| S91P D94G D94A | Wild type | 1 (0.9) | 1 |
| A90V G88A | Wild type | 1 (0.9) | 1 |
| Wild type | D500N | 1 (0.9) | 0 |
| Wild type | E540V | 1 (0.9) | 0 |
| Wild type | S486F | 1 (0.9) | 0 |
| Wild type | E540D | 1 (0.9) | 0 |
| Wild type | D500H G509A | 1 (0.9) | 1 |
| Wild type | Wild type | 19 (17.4) | 0 |
| Total | 109 (100) | 34 |
All isolates carried the S95T polymorphism, which is not involved in FQ resistance.
Isolates showing heteropeaks were sequenced on two separate occasions and showed the same results in both forward and reverse strands.
Variable-number tandem-repeat typing by high-resolution 12-locus mycobacterial interspersed repetitive unit typing showed the presence of only a single mycobacterial interspersed repetitive unit type in each heteropeak isolate, and no heteropeaks were observed on sequencing chromatograms in the katG or rpoB genes at mutated loci (data not shown), supporting the conclusion that these data represent true heteroresistance rather than laboratory contamination with a second strain.
Drug susceptibility profiles.
The first 40 FQ-resistant isolates were tested for susceptibility to INH, RIF, STR, and ETH. Ninety percent (n = 36/40) of isolates tested were MDR, with 70% (n = 28/40) being resistant to all four drugs. A total of 17.5% of isolates (n = 7/40) showed resistance to INH, RIF, and STR; 5% (n = 2/40) were resistant to INH, STR, and ETH; 2.5% (n = 1/40) were resistant to INH, RIF, and ETH; 2.5% (n = 1/40) were resistant to STR alone; and 2.5% (n = 1/40) were sensitive to all four first-line agents (Table 2).
TABLE 2.
First-line drug resistance profiles of 40 FQ-resistant isolates
| Resistance pattern | Sensitivity | No. of isolates (%) |
|---|---|---|
| INH, RIF, STR, ETH | None | 28 (70.0) |
| INH, RIF, STR | ETH | 7 (17.5) |
| INH, STR, ETH | RIF | 2 (5.0) |
| INH, RIF, ETH | STR | 1 (2.5) |
| STR | INH, RIF, ETH | 1 (2.5) |
| None | INH, RIF, STR, ETH | 1 (2.5) |
| Total | 40 (100) |
Typing.
Typing results for the FQ-resistant and control isolates are presented in Table 3. A total of 75.2% (n = 82/109) of FQ-resistant isolates were of the Beijing genotype, whereas only 56.0% (n = 61/109) of isolates from the control group were of the Beijing genotype. This was confirmed by an RD105 deletion by LSP typing. According to univariate logistic regression analysis, the Beijing genotype was significantly associated with FQ resistance (OR, 2.39 [95% confidence interval {CI}, 1.34 to 4.25]; P = 0.003). FQ-resistant isolates were less likely to be an Indo-Oceanic strain (OR, 0.35 [95% CI, 0.17 to 0.71]; P = 0.004) (Table 3).
TABLE 3.
LSP types and spoligotypes of 109 consecutive FQ-resistant isolates and 109 consecutive pulmonary control isolates from southern Vietnam
| LSP type | Spoligotype clade | Octal codea | No. of isolates |
OR (95% CI) | P value | |
|---|---|---|---|---|---|---|
| Control group | FQ resistant | |||||
| East Asian (Beijing) | 61 | 82 | 2.39 (1.34-4.25) | 0.003 | ||
| Beijing | 000000000003771 | 61 | 82 | |||
| Indo-Oceanic | 32 | 14 | 0.35 (0.17-7.11) | 0.004 | ||
| CAS1 | 703377400001771 | 1 | 0 | |||
| EA11-SOM | 077777777413731 | 1 | 0 | |||
| EA12-MANILA | 677777477413771 | 1 | 0 | |||
| EA14-VNM | 777777774413771 | 20 | 5 | |||
| EA15 | 777777777413771 | 4 | 3 | |||
| EA153 | 777777777413371 | 1 | 0 | |||
| LAM3 | 776177607760771 | 1 | 0 | |||
| MANU2 | 777777777763771 | 1 | 0 | |||
| ZERO | 777777000000011 | 2 | 1 | |||
| Unknown | NA | 0 | 5 | |||
| Euro-American | 14 | 13 | 0.92 (0.41-2.05) | 0.837 | ||
| H1 | 777777774020771 | 1 | 0 | |||
| H3 | 111111111100111 | 1 | 3 | |||
| H3T3 | 777737777720771 | 1 | 0 | |||
| S | 776377737760771 | 0 | 1 | |||
| T1 | 111111111110111 | 2 | 1 | |||
| T2 | 777737777760731 | 1 | 1 | |||
| U | 777777777777771 | 4 | 1 | |||
| X1 | 777776777760731 | 0 | 1 | |||
| LAM9 | 111111101110111 | 0 | 2 | |||
| Unknown | NA | 4 | 3 | |||
| Undetermined | 2 | 0 | ||||
| Unclassified | 774017777413771 | 2 | 0 | |||
| Total | 109 | 109 | ||||
For more on octal code, see reference 3a. NA, not applicable.
The prevalence of the Beijing genotype among the control group is similar to that among pulmonary TB patients in Vietnam reported previously in other studies (1).
Association of the Beijing genotype with a gyrA mutation and MIC.
Isolates of the Beijing genotype were significantly more likely to have a gyrA mutation (n = 72/82 [87.8%]) than FQ-resistant isolates of other genotypes (n = 13/27 [48.1%]) (OR, 7.75 [95% CI, 2.84 to 21.15]; P < 0.0001).
The MIC of ofloxacin was determined for the first 65 FQ-resistant isolates. One isolate failed to grow upon subculture; the MIC was available for 64 isolates. The median MIC for each mutation was determined, excluding isolates carrying multiple mutations (n = 6) or heteropeaks upon sequencing (n = 13) (Table 4). The median MIC for each mutation was 8.0 μg/ml for isolates carrying gyrA with the A90V mutation (n = 10), 8 μg/ml for gyrA with the D94A mutation (n = 6), ≥12 μg/ml for gyrA with the D94G mutation (n = 10), 12 μg/ml for gyrA with the D94N mutation (n = 1), ≥12 μg/ml for gyrA with the D94Y mutation (n = 1), 4 μg/ml for gyrB with the D500N mutation (n = 1), 12 μg/ml for gyrB with the E540D mutation (n = 1), and 4 μg/ml for isolates with no gyrA or gyrB mutation. The inclusion of isolates showing heteropeaks upon sequencing did not change the median MIC (Table 4). Seven of 14 isolates carrying wild-type gyrAB were resistant to ofloxacin at 2 μg/ml on two occasions but had an MIC result of 2 μg/ml after several subcultures. The reason for this result is unclear, but it is likely that the initial isolates were initially borderline resistant to ofloxacin and that a more susceptible population has been selected by subculture in the absence of drug pressure. Only one of these isolates was of the Beijing genotype; therefore, if these isolates are borderline FQ resistant, the inclusion of these isolates has reduced the observed association between the Beijing genotype and FQ resistance. It was not possible to determine if the background genotype influences the MIC conferred by a specific mutation in this study because the majority of gyrA mutants were of the Beijing genotype; the number of non-Beijing genotype isolates with an individual mutation was not sufficient to draw any conclusions.
TABLE 4.
Median MIC of ofloxacin of for gyrA and gyrB mutant strains detected among 64 FQ-resistant isolates
| Mutation | Excluding heteropeaksb |
Including heteropeaks |
||
|---|---|---|---|---|
| No. of isolates | Median MIC (μg/ml) (range) | No. of isolates | Median MIC (μg/ml) (range) | |
| A90V | 10 | 8 (4->12) | 13 | 8 (4->12) |
| D94A | 6 | 8 (4-8) | 7 | 8 (4-8) |
| D94G | 10 | 12 (12->12) | 19 | 12 (12->12) |
| D94N | 1 | 12 | 1 | 12 |
| D94Y | 1 | >12 | 1 | >12 |
| G88A | 0 | NA | 1 | 12 |
| D500Na | 1 | 4 | 1 | 4 |
| E540Da | 1 | 12 | 1 | 12 |
| No mutation | 14 | 4 (2-12) | 14 | 4 (2-12) |
| A90V D500Aa | 0 | NA | 1 | 8 |
| A90V T539Pa | 1 | >12 | 1 | >12 |
| A90V D94A | 0 | NA | 1 | 8 |
| A90V D94G | 0 | NA | 2 | 12 (12->12) |
| D94G D94A | 0 | NA | 1 | 12 |
| Total | 45 | 8 (2->12) | 64 | 8 (2->12) |
Mutation in gyrB.
NA, not applicable.
All subsequent analyses excluded isolates showing heteropeaks upon sequencing or carrying mutations at multiple loci. Forty-five FQ-resistant isolates with MIC data were therefore included.
Isolates of the Beijing genotype were significantly more likely to have relatively high-level FQ resistance (≥8 μg/ml) (OR, 11.0 [95% CI, 2.6 to 47.0]; P = 0.001) and also to have gyrA94 mutations (OR, 7.4 [95% CI, 1.42 to 38.7]; P = 0.017). Isolates with low-level FQ resistance (≤4 μg/ml) are more likely to have wild-type gyrAB (OR, 38.9 [95% CI, 6.9 to 219.1]; P = <0.001).
DISCUSSION
We have shown a high prevalence of isolates of the Beijing genotype among FQ-resistant isolates identified in Ho Chi Minh City, Vietnam (OR, 2.39 [95% CI, 1.34 to 4.25]; P = <0.003). Furthermore, these isolates are associated with gyrA mutations at locus 94 (OR, 7.4 [95% CI, 1.42 to 38.7]; P = 0.017).
A high frequency of isolates of the Beijing genotype among FQ-resistant isolates from Russia was also previously reported (20). No MIC data were available in the report of that study.
The Beijing genotype was previously associated with an increased prevalence of the katG315 mutation, which confers high-level INH resistance (25), in several regions of the world (13, 19). The Beijing genotype appears to have an increased ability to either develop or support this mutation over other genotypes; the mechanism remains to be elucidated and is being investigated by several groups (7, 22). It is possible that a similar mechanism is responsible for both the increased rate of katG315 mutants and FQ-resistant gyrA mutants among strains of the Beijing genotype. Another possibility is that strains of the Beijing genotype have a higher level of intrinsic resistance to FQs, allowing them to tolerate low levels of FQs and subsequently develop gyrA mutations, or that these mutations confer an advantage in the absence of drug pressure. Further studies are under way at our institute.
A relatively high frequency of multiple mutants was also seen among FQ-resistant isolates in Vietnam (11/109 [10.1%]) (this report) and China (53/110 [48%]) (24) compared to data from reports from other regions. No association was seen between double mutations and genotype in this study (OR, 2.57 [95% CI, 0.54 to 12.1]; P = 0.23), suggesting that this is not a consequence of the high frequency of isolates of the Beijing genotype in these regions.
Over half of the non-Beijing genotype FQ-resistant isolates (n = 14/27 [51.9%]) had no mutations at the gyrA or gyrB QRDRs, while only 12.2% (n = 10/82) of Beijing isolates were wild type for gyrA and gyrB. The non-gyrA and non-gyrB mutants in this study have low-level FQ resistance (≤4 μg/ml). The mechanism of FQ resistance in these isolates remains unknown but is probably an efflux pump mechanism and may also be present in FQ-resistant isolates of the Beijing genotype. Other possibilities include an upregulation of DNA gyrase binding proteins such as MfpA from M. tuberculosis (12). This protein is a member of the pentapeptide repeat family, which also includes the plasmid-encoded Qnr protein, which is responsible for FQ resistance in Klebsiella pneumoniae, Salmonella enterica serovar Typhi, and other pathogens.
The gyrA G88A mutation identified in this study was previously described and shown to confer FQ resistance. However, the site-directed mutagenesis study reported previously by Matrat et al. showed an MIC of 2 μg/ml (18), whereas our isolate has an MIC of 12 μg/ml, probably due to the presence of the A90V mutation. Nine novel mutations were identified in gyrB in this study, with MIC data available for four mutants. Two mutations (T539P and D500A) were present in isolates that also carried an A90V mutation. The T539P A90V isolate had a higher MIC (>12 μg/ml) than the median MIC of isolates carrying only an A90V mutation (8.0 μg/ml), and it is likely that the T539P mutation in gyrB is responsible, but this remains to be confirmed. The remaining two gyrB mutations were found in single isolates, D500N (MIC = 4 μg/ml) and E540D (MIC = 12 μg/ml).
Further studies of FQ resistance mechanisms are urgently required in light of the emergence of extensively drug-resistant TB worldwide (15). It is possible that isolates that are resistant to ofloxacin with a low MIC may still be successfully treated with gatifloxacin and moxifloxacin. The association of the Beijing genotype with relatively high-level FQ resistance conferred by specific gyrA mutations reported here is of grave concern given the epidemic spread of isolates of the Beijing genotype (7) and the current hopes for shorter first-line treatment regimens based on FQs (31).
Acknowledgments
We thank the staff at Pham Ngoc Thach Hospital and the Hospital for Tropical Diseases for their contribution to this work.
This work was funded by the Wellcome Trust of Great Britain.
Footnotes
Published ahead of print on 31 August 2009.
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