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Antimicrob Agents Chemother. Oct 2009; 53(10): 4498–4500.
Published online Aug 17, 2009. doi:  10.1128/AAC.00287-09
PMCID: PMC2764174

Fluoroquinolone Resistance in Mycobacterium tuberculosis and Mutations in gyrA and gyrB[down-pointing small open triangle]

Abstract

This study evaluated cross-resistance of Mycobacterium tuberculosis strains to ofloxacin, moxifloxacin, and gatifloxacin and investigated the presence of mutations in gyrA and gyrB. Fluoroquinolone susceptibilities were determined for 41 M. tuberculosis strains by the proportion method on 7H11, and MICs were determined by the resazurin microtiter assay. Forty strains shared the same resistance results for the three fluoroquinolones. However, one strain, with an Asn-533 → Thr mutation in gyrB, was susceptible to ofloxacin but resistant to moxifloxacin and gatifloxacin.

Drug resistance is a major threat for the control of tuberculosis (TB). Multidrug-resistant (MDR) Mycobacterium tuberculosis strains resistant to at least rifampin (rifampicin) and isoniazid and extensively drug resistant TB caused by M. tuberculosis strains that in addition to being MDR are resistant to any fluoroquinolone and to at least one of three injectable second-line drugs, amikacin, kanamycin, or capreomycin, have now been reported worldwide (25).

Ofloxacin, ciprofloxacin, and levofloxacin have been used as second-line drugs for the treatment of drug-resistant TB or in patients with intolerance to one of the first-line drugs (24). A new generation of fluoroquinolones is under clinical evaluation, and these are being proposed as first-line regimens for shortening the duration of treatment (2, 12, 17). Moxifloxacin and gatifloxacin are promising drugs undergoing phase III trials (27), having previously shown favorable pharmacokinetic activity and low MICs against M. tuberculosis (5, 9, 14).

The main target of fluoroquinolones in M. tuberculosis is the DNA gyrase, encoded by gyrA and gyrB (26). Mutations in two short regions known as “quinolone resistance-determining regions” have been associated with fluoroquinolone resistance in M. tuberculosis (20). Mutations in Ala-90 and Asp-94 of gyrA have been the most commonly found (1, 7, 16). We looked for mutations gyrA and gyrB in a panel of fluoroquinolone-resistant M. tuberculosis strains and evaluated the possible correlation between gene mutations and resistance to ofloxacin, moxifloxacin, and gatifloxacin based on their MICs.

A total of 41 M. tuberculosis clinical isolates were studied. Twenty-six strains were MDR. Fresh subcultures were prepared on Löwenstein-Jensen medium and kept at 37°C for no longer than 4 weeks. Moxifloxacin (Bayer, Brussels, Belgium), gatifloxacin (Lupin Ltd., India), and ofloxacin (Sigma-Aldrich, Bornem, Belgium) stock solutions were prepared at 1 mg/ml in 0.1 N NaOH, filter sterilized, and stored at −20°C for less than 2 months. DNA was obtained from a loopful of culture resuspended in 200 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]), heat inactivated at 100°C for 10 min, and centrifuged at 10,000 × g at 4°C for 20 min. Two microliters of supernatant was used for amplification of the gyrA and gyrB genes using the primers (oligo 101) GyraseB-sense (5′-TAAGAGCGCCACCGACATCGGTGGATTG-3′) (positions 1404 to 1431 of gyrB) and (oligo 102) GyraseA-reverse (5′-GATGAAATCGACTGTCTCCTCGTCGATTTCCC-3′) (positions 428 to 459 of gyrA). The PCR product was 1,231 bp in size and was used as a template for the sequencing reaction, using the primers oligo 101 for the sense strand and (oligo 103) GyraseAB-Seq (5′-GTCGATTTCCCTCAGCATCTCCATC-3′) (positions 414 to 438 of gyrA) for the reverse strand. Cycling parameters were 5 min at 94°C, followed by 39 cycles of 45 s at 94°C, 45 s at 66°C, and 1 min at 72°C, followed by elongation at 72°C for 10 min. PCR products were sequenced with an automatic DNA sequencer (Applied Biosystems 3730 DNA analyzer). The ClustalX (version 1.83.1) and Genedoc (version 2.100) software programs were used for analysis. Mutations in gyrA and gyrB were identified using BLAST by comparison with the M. tuberculosis strain H37Rv sequence (GenBank accession no. NC_000962). Drug susceptibility testing was performed by the proportion method (PM) on 7H11 agar according to the standard procedure (11) with breakpoint concentrations of 2 mg/liter for ofloxacin and 0.5 mg/liter for moxifloxacin and gatifloxacin (18). MIC determinations were performed by using the resazurin microtiter assay as described previously (10).

Table Table11 shows the mutations in gyrA or gyrB correlated with the MICs obtained by resazurin microtiter assay and the drug resistance profiles of the strains determined by the PM on 7H11 agar. Out of 41 strains, 16 were susceptible and 25 resistant to ofloxacin. For moxifloxacin and gatifloxacin, 15 were susceptible and 26 resistant by as determined the proportion method. As determined by sequencing of gyrA and gyrB, 10 strains had an Ala-90 → Val mutation, four strains had an Asp-94 → Gly mutation, three strains (each) had Asp-94 → Tyr or Asp-94 → Ala, and one strain (each) had Asp-94 → Asn or Asp-89 → Asn. Additionally, one strain had an Asp-94 → His mutation in gyrA and a deletion of the codons 678 and 679 in gyrB, and another had a double point mutation, Ala-90 → Val plus Asp-94 → Asn, in gyrA. One additional finding was a strain with no mutation in gyrA or gyrB but resistant to the three drugs and another strain with an Asn-533 → Thr mutation in gyrB resistant to moxifloxacin and gatifloxacin but susceptible to ofloxacin. Strains with a Thr-80 → Ala (n = 1) or Pro-8 → Ala (n = 1) mutation, a Thr-80 → Ala plus Ala-90 → Gly double mutation (n = 1), or a mutation in the gyrA putative promoter (n = 2), as well as all wild-type strains (n = 10), were susceptible to the three drugs.

TABLE 1.
Mutations in gyrA and gyrB, MICs determined by REMA, and susceptibility profiles of M. tuberculosis strains determined by PMa

Cross-resistance among fluoroquinolones is of concern because they have a mode of action different from that of the classical first-line anti-TB drugs. Having been used widely for other infectious diseases, they are even available without prescription in several countries, increasing the burden of selective pressure and compromising their efficacy in the treatment of TB. In the present study, moxifloxacin and gatifloxacin were more active than ofloxacin, showing strong cross-resistance with the latter, as previously reported (19, 20). Twenty-four of the 26 moxifloxacin- and gatifloxacin-resistant strains had a mutation in gyrA, mostly at positions Ala-90 and Asp-94, corroborating previous reports (1, 7, 16, 21). This high frequency allows considering this region as a candidate marker for fluoroquinolone resistance detection and a possible indicator of an extensively drug-resistant case (3, 23). Mutations in gyrB have been less commonly found in M. tuberculosis, with Asp-495, Arg-516, and Asn-533 reported previously (6, 16). Herein we have shown that an Asn-533 → Thr mutation in gyrB was associated with resistance to moxifloxacin and gatifloxacin but interestingly not to ofloxacin. We can speculate that the absence of resistance to ofloxacin could be related to the fact that gyrB could be having more interaction with common structures of moxifloxacin and gatifloxacin, such as the methoxy derivative at C-8 or the methyl piperazine ring at position C-7, which are absent in ofloxacin. This finding stresses the need to look for gyrB mutations when determining resistance to moxifloxacin and gatifloxacin. Furthermore, one strain (08-0787) presented an Asp-94 → His mutation in gyrA and a deletion of the codons 678 and 679 in gyrB, while another strain (04-0519) had a double mutation, Ala-90 → Val and Asp-94 → Asn, that was related to high-level resistance, which is in agreement with previous reports, where multiple mutations are associated with high-level resistance (20). One strain without mutation in gyrA or gyrB was resistant to all three drugs. This situation has been reported previously with fluoroquinolone resistance (4, 7, 8) and is possibly explained by a mutation outside of the regions sequenced or another molecular alteration decreasing the accumulation of the drug inside the cell, such as an efflux mechanism (13, 15, 22).

In conclusion, we found almost full cross-resistance to ofloxacin, moxifloxacin, and gatifloxacin among strains, with the exception of one strain, with the mutation Asn-533 → Thr in gyrB, which was resistant to moxifloxacin and gatifloxacin but still susceptible to ofloxacin. Further studies are needed to confirm these findings among large numbers of clinical isolates of M. tuberculosis.

Acknowledgments

We are grateful to Bayer HealthCare Pharmaceuticals (Brussels, Belgium) for providing the moxifloxacin powder. The excellent technical assistance of Pim de RIjk is gratefully acknowledged.

A. Von Groll was partially supported by CNPq-Brazil (project no. 410468/2006-1) and Belgium Federal Government Grant Programmatorische Federale Overheidsdienst Wetenschapsbeleid contract no. BL/09/BR2. Pedro Almeida da Silva is a senior researcher with a fellowship from CAPES-Brazil. This study was partially funded by EC project HEALTH-F3-2007-201690 (FAST-XDR-DETECT).

We have no conflict of interest to declare.

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 August 2009.

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