Gatifloxacin Resistance and Mutations in gyrA after Unsuccessful Helicobacter pylori Eradication in Japan
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《抗菌试剂及化学方法》
1.Department of Internal Medicine,2.Center for Integrated Medical Research,3.Department of Emergency Medicine,4.Keio University School of Medicine, Department of Gastroenterology, Kitasato Institute Hospital,5.Mitsubishi Kagaku Bio-Clinical Laboratories, Tokyo, Japan
ABSTRACT
A high resistance rate (47.9%) to gatifloxacin (GAT; 8-methoxy fluoroquinolone) in Helicobacter pylori (H. pylori) strains from 48 Japanese patients is observed after unsuccessful H. pylori eradication. A significant association between MICs for GAT equal to or above 1 μg/ml and mutations of the gyrA gene of H. pylori was demonstrated.
Resistance to antibiotics is the major cause of the failure to eradicate Helicobacter pylori. For such cases, alternative regimens need to be developed.
Perri et al. have reported an unacceptable low eradication rate of only 68% with a 7-day regimen of levofloxacin (LVX), amoxicillin (AMX), and pantoprazole (12). However, Sharara et al. have recently reported an excellent eradication rate of 92% with a 7-day regimen of gatifloxacin (GAT), AMX, and rabeprazole (14). It is important to note the superior in vitro activity of GAT over that of LVX against H. pylori (1). Therefore, GAT-based triple therapy might be a promising option for an alternative treatment regimen.
Several studies have shown that the "quinolone resistance-determining region" (QRDR) of the gyrA gene plays a critical role in the resistance of H. pylori to ciprofloxacin (CIP) (8, 15). H. pylori does not contain genes for topoisomerase IV, an important fluoroquinolone target in other bacteria (15). The present study demonstrated a correlation between MICs to GAT and mutations of the gyrA and gyrB genes in H. pylori isolated from Japanese patients after unsuccessful eradication of infection.
A total of 48 patients (32 males and 16 females; mean age, 57.8) with H. pylori infection after treatment failure were enrolled at Keio University Hospital between September 2004 and June 2005. Of the total, 42 patients had one treatment failure, 4 patients had two treatment failures, and 2 patients had three treatment failures (first-line treatment, triple therapy with clarithromycin [CLR], AMX, and proton pump inhibitor [PPI]; second-line treatment, triple therapy with metronidazole [MNZ], AMX, and PPI; and third-line treatment, triple therapy with LVX, AMX, and PPI). All patients underwent upper gastrointestinal endoscopy and gastric biopsy at Keio University Hospital.
The susceptibility of H. pylori isolates to CRL, GAT, and MNZ was determined by the agar dilution method according to the guidelines established by the CLSI (formerly NCCLS) (9). Isolates were considered resistant to MNZ if the MIC of the drug was 8 μg/ml and resistant to CLR and GAT if the MIC of these drugs was 1 μg/ml (3, 7, 10). The resistance rates to GAT, CLR, and MNZ were 47.9%, 79.2%, and 6.3%, respectively. The GAT resistance rates were 22.2% (2/9) in the strains susceptible to both CLR and MNZ, 50% (18/36) in the strains resistant to CLR but susceptible to MNZ, 100% (1/1) in the strains resistant to MNZ but susceptible to CLR, and 100% (2/2) in the strains resistant to both CLR and MNZ.
Studies in Europe have suggested that the primary resistance rate might be as low as 8% for CIP (2, 16) and that only 9% of the isolates show resistance to CIP after failure of primary eradication (4, 13). In contrast, our drug susceptibility tests showed a high resistance rate to GAT. Therefore, quinolone-resistant strains may be more prevalent in Japan than in Europe, due to wider use of fluoroquinolones.
We amplified by PCR and sequenced the QRDRs of gyrA (from codon 38 to 154) and gyrB (from codon 392 to 500) genes: gyrA (forward), 5'-TTTRGCTTATTCMATGAGCGT-3'; gyrA (reverse), 5'-GCAGACGGCTTGGTARAATA-3'; gyrB (forward), 5'-YGCAAAAGCCAGAGAAGCCA-3'; and gyrB (reverse), 5'-ACATGCCCTTGTTCAATCAGC-3'). The sequences obtained were compared with the published sequences of the H. pylori gyrA and gyrB genes (GenBank accession no. L29481) (3). Twenty-two of the 23 (95.7%) GAT-resistant strains had point mutations in the gyrA gene at codon 87 Asn or 91 Asp. On the other hand, only 1 of the 25 (4.0%) susceptible strains had the mutation, with a substitution at amino acid 87 (Tables 1 and 2). The sensitivity and specificity of detection of mutations in the gyrA gene in the GAT-resistant strains were 95.7% and 95.7%, respectively. Significant association was observed between mutations in the gyrA gene and resistance of the strains to GAT (P < 0.001). Mutations of the gyrB gene, on the other hand, were not encountered in either the susceptible or the resistant strains. The examination of mutations in the gyrA gene may be useful as a marker of GAT resistance.
Although many isolates bear the same gyrA mutation, their GAT MICs vary over an eightfold range. It might be due to the other mechanisms determining GAT MICs, such as an active multidrug efflux mechanism (5). Since the CmeABC efflux pump is linked to the acquired fluoroquinolone resistance in Campylobacter isolates (6), active efflux systems may also be involved in H. pylori susceptibility to GAT.
Four GAT- and CLR-susceptible strains were serially plated onto GAT (0.12 μg/ml)- or CLR (0.015 μg/ml)-containing agar with increasing agar density. Although none of the strains plated on CLR-containing agar developed resistance to GAT until the 10th generation of repeated culture, all four strains plated on GAT-containing agar developed GAT resistance and three strains showed mutations of gyrA, suggesting that a high incidence of GAT resistance could not arise through mutation of susceptible bacteria during the course of therapy with CLR, AMX, and PPI and could be due to primary infection with resistant bacteria or to history of fluoroquinolone treatment.
Oram and Fisher reported that examination of silent nucleotide changes in the gyrA genes revealed four different patterns of DNA polymorphism in Escherichia coli (11). In the present study, compared with the reported sequence (GenBank accession no. L29481), there were CT substitutions at position 108, 132, 210, 396, and 438, GA substitutions at positions 276, 277, 291, and 318, TC substitutions at positions 183 and 225, and AG substitutions at positions 135 and 366. While KS0149 and KS0150 shared the same patterns of polymorphism, the other strains showed different patterns, indicating that these strains were not genetically related and that each strain developed GAT resistance during the previous fluoroquinolone usage.
In conclusion, a high resistance rate to GAT in H. pylori strains from Japanese patients is observed after unsuccessful eradication. Mutation in the gyrA gene of H. pylori was significantly associated with MICs for GAT equal to or above 1 μg/ml. Although GAT may be one of the promising candidates for third-line therapy, the choice of GAT should be made after drug susceptibility tests or gyrA gene analysis.
REFERENCES
Bauernfeind, A. 1997. Comparison of the antibacterial activities of the quinolones Bay 12-8039, gatifloxacin (AM 1155), trovafloxacin, clinafloxacin, levofloxacin and ciprofloxacin. J. Antimicrob. Chemother. 40:639-651.
Boyanova, L., A. Mentis, M. Gubina, E. Rozynek, G. Gosciniak, S. Kalenic, V. Goral, L. Kupcinskas, B. Kantarceken, A. Aydin, A. Archimandritis, D. Dzierzanowska, A. Vcev, K. Ivanova, M. Marina, I. Mitov, P. Petrov, A. Ozden, and M. Popova. 2002. The status of antimicrobial resistance of Helicobacter pylori in eastern Europe. Clin. Microbiol. Infect. 8:388-396.
Fujimura, S., S. Kato, K. Iinuma, and A. Watanabe. 2004. In vitro activity of fluoroquinolone and the gyrA gene mutation in Helicobacter pylori strains isolated from children. J. Med. Microbiol. 53:1019-1022.
Heep, M., M. Kist, S. Strobel, D. Beck, and N. Lehn. 2000. Secondary resistance among 554 isolates of Helicobacter pylori after failure of therapy. Eur. J. Clin. Microbiol. Infect. Dis. 19:538-541.
Kutschke, A., and B. L. M. de Jonge. 2005. Compound efflux in Helicobacter pylori. Antimicrob. Agents Chemother. 49:3009-3010.
Luo, N., O. Sahin, J. Lin, L. O. Michel, and Q. Zhang. 2003. In vivo selection of Campylobacter isolates with high levels of fluoroquinolone resistance associated with gyrA mutations and the function of the CmeABC efflux pump. Antimicrob. Agents Chemother. 47:390-394.
Masaoka, T., H. Suzuki, K. Kurabayashi, A. G. Kamiya, and H. Ishii. 2004. Second-line treatment of Helicobacter pylori infection after dilution agar methods and PCR-RFLP analysis. Aliment. Pharmacol. Ther. 20(Suppl. 1):68-73.
Moore, R. A., B. Beckthold, S. Wong, A. Kureishi, and L. E. Bryan. 1995. Nucleotide sequence of the gyrA gene and characterization of ciprofloxacin-resistant mutants of Helicobacter pylori. Antimicrob. Agents Chemother. 39:107-111.
National Committee for Clinical Laboratory Standards. 2004. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 6th ed. Approved standard M7-A6. National Committee for Clinical Laboratory Standards, Wayne, Pa.
National Committee for Clinical Laboratory Standards. 2004. Performance standards for antimicrobial susceptibility testing; 14th informational supplement (aerobic dilution). M100-S14. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Oram, M., and L. M. Fisher. 1991. 4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction. Antimicrob. Agents Chemother. 35:387-389.
Perri, F., V. Festa, A. Merla, F. Barberani, A. Pilotto, and A. Andriulli. 2003. Randomized study of different ‘second-line’ therapies for Helicobacter pylori infection after failure of the standard ‘Maastricht triple therapy’. Aliment. Pharmacol. Ther. 18:815-820.
Pilotto, A., M. Franceschi, M. Rassu, G. Leandro, L. Bozzola, F. Furlan, and F. Di Mario. 2000. Incidence of secondary Helicobacter pylori resistance to antibiotics in treatment failures after 1-week proton pump inhibitor-based triple therapies: a prospective study. Dig. Liver Dis. 32:667-672.
Sharara, A. I., H. F. Chaar, E. Racoubian, O. Moukhachen, K. A. Barada, F. H. Mourad, and G. F. Araj. 2004. Efficacy of two rabeprazole/gatifloxacin-based triple therapies for Helicobacter pylori infection. Helicobacter 9:255-261.
Tankovic, J., C. Lascols, Q. Sculo, J.-C. Petit, and C.-J. Soussy. 2003. Single and double mutations in gyrA but not in gyrB are associated with low- and high-level fluoroquinolone resistance in Helicobacter pylori. Antimicrob. Agents Chemother. 47:3942-3944.
Toro, C., J. Garcia-Samaniego, J. Carbo, A. Iniguez, T. Alarcon, M. Lopez-Brea, and M. Baquero. 2001. [Prevalence of primary Helicobacter pylori resistance to eight antimicrobial agents in a hospital in Madrid]. Rev. Esp. Quimioter. 14:172-176.(Toshihiro Nishizawa,Hidek)
ABSTRACT
A high resistance rate (47.9%) to gatifloxacin (GAT; 8-methoxy fluoroquinolone) in Helicobacter pylori (H. pylori) strains from 48 Japanese patients is observed after unsuccessful H. pylori eradication. A significant association between MICs for GAT equal to or above 1 μg/ml and mutations of the gyrA gene of H. pylori was demonstrated.
Resistance to antibiotics is the major cause of the failure to eradicate Helicobacter pylori. For such cases, alternative regimens need to be developed.
Perri et al. have reported an unacceptable low eradication rate of only 68% with a 7-day regimen of levofloxacin (LVX), amoxicillin (AMX), and pantoprazole (12). However, Sharara et al. have recently reported an excellent eradication rate of 92% with a 7-day regimen of gatifloxacin (GAT), AMX, and rabeprazole (14). It is important to note the superior in vitro activity of GAT over that of LVX against H. pylori (1). Therefore, GAT-based triple therapy might be a promising option for an alternative treatment regimen.
Several studies have shown that the "quinolone resistance-determining region" (QRDR) of the gyrA gene plays a critical role in the resistance of H. pylori to ciprofloxacin (CIP) (8, 15). H. pylori does not contain genes for topoisomerase IV, an important fluoroquinolone target in other bacteria (15). The present study demonstrated a correlation between MICs to GAT and mutations of the gyrA and gyrB genes in H. pylori isolated from Japanese patients after unsuccessful eradication of infection.
A total of 48 patients (32 males and 16 females; mean age, 57.8) with H. pylori infection after treatment failure were enrolled at Keio University Hospital between September 2004 and June 2005. Of the total, 42 patients had one treatment failure, 4 patients had two treatment failures, and 2 patients had three treatment failures (first-line treatment, triple therapy with clarithromycin [CLR], AMX, and proton pump inhibitor [PPI]; second-line treatment, triple therapy with metronidazole [MNZ], AMX, and PPI; and third-line treatment, triple therapy with LVX, AMX, and PPI). All patients underwent upper gastrointestinal endoscopy and gastric biopsy at Keio University Hospital.
The susceptibility of H. pylori isolates to CRL, GAT, and MNZ was determined by the agar dilution method according to the guidelines established by the CLSI (formerly NCCLS) (9). Isolates were considered resistant to MNZ if the MIC of the drug was 8 μg/ml and resistant to CLR and GAT if the MIC of these drugs was 1 μg/ml (3, 7, 10). The resistance rates to GAT, CLR, and MNZ were 47.9%, 79.2%, and 6.3%, respectively. The GAT resistance rates were 22.2% (2/9) in the strains susceptible to both CLR and MNZ, 50% (18/36) in the strains resistant to CLR but susceptible to MNZ, 100% (1/1) in the strains resistant to MNZ but susceptible to CLR, and 100% (2/2) in the strains resistant to both CLR and MNZ.
Studies in Europe have suggested that the primary resistance rate might be as low as 8% for CIP (2, 16) and that only 9% of the isolates show resistance to CIP after failure of primary eradication (4, 13). In contrast, our drug susceptibility tests showed a high resistance rate to GAT. Therefore, quinolone-resistant strains may be more prevalent in Japan than in Europe, due to wider use of fluoroquinolones.
We amplified by PCR and sequenced the QRDRs of gyrA (from codon 38 to 154) and gyrB (from codon 392 to 500) genes: gyrA (forward), 5'-TTTRGCTTATTCMATGAGCGT-3'; gyrA (reverse), 5'-GCAGACGGCTTGGTARAATA-3'; gyrB (forward), 5'-YGCAAAAGCCAGAGAAGCCA-3'; and gyrB (reverse), 5'-ACATGCCCTTGTTCAATCAGC-3'). The sequences obtained were compared with the published sequences of the H. pylori gyrA and gyrB genes (GenBank accession no. L29481) (3). Twenty-two of the 23 (95.7%) GAT-resistant strains had point mutations in the gyrA gene at codon 87 Asn or 91 Asp. On the other hand, only 1 of the 25 (4.0%) susceptible strains had the mutation, with a substitution at amino acid 87 (Tables 1 and 2). The sensitivity and specificity of detection of mutations in the gyrA gene in the GAT-resistant strains were 95.7% and 95.7%, respectively. Significant association was observed between mutations in the gyrA gene and resistance of the strains to GAT (P < 0.001). Mutations of the gyrB gene, on the other hand, were not encountered in either the susceptible or the resistant strains. The examination of mutations in the gyrA gene may be useful as a marker of GAT resistance.
Although many isolates bear the same gyrA mutation, their GAT MICs vary over an eightfold range. It might be due to the other mechanisms determining GAT MICs, such as an active multidrug efflux mechanism (5). Since the CmeABC efflux pump is linked to the acquired fluoroquinolone resistance in Campylobacter isolates (6), active efflux systems may also be involved in H. pylori susceptibility to GAT.
Four GAT- and CLR-susceptible strains were serially plated onto GAT (0.12 μg/ml)- or CLR (0.015 μg/ml)-containing agar with increasing agar density. Although none of the strains plated on CLR-containing agar developed resistance to GAT until the 10th generation of repeated culture, all four strains plated on GAT-containing agar developed GAT resistance and three strains showed mutations of gyrA, suggesting that a high incidence of GAT resistance could not arise through mutation of susceptible bacteria during the course of therapy with CLR, AMX, and PPI and could be due to primary infection with resistant bacteria or to history of fluoroquinolone treatment.
Oram and Fisher reported that examination of silent nucleotide changes in the gyrA genes revealed four different patterns of DNA polymorphism in Escherichia coli (11). In the present study, compared with the reported sequence (GenBank accession no. L29481), there were CT substitutions at position 108, 132, 210, 396, and 438, GA substitutions at positions 276, 277, 291, and 318, TC substitutions at positions 183 and 225, and AG substitutions at positions 135 and 366. While KS0149 and KS0150 shared the same patterns of polymorphism, the other strains showed different patterns, indicating that these strains were not genetically related and that each strain developed GAT resistance during the previous fluoroquinolone usage.
In conclusion, a high resistance rate to GAT in H. pylori strains from Japanese patients is observed after unsuccessful eradication. Mutation in the gyrA gene of H. pylori was significantly associated with MICs for GAT equal to or above 1 μg/ml. Although GAT may be one of the promising candidates for third-line therapy, the choice of GAT should be made after drug susceptibility tests or gyrA gene analysis.
REFERENCES
Bauernfeind, A. 1997. Comparison of the antibacterial activities of the quinolones Bay 12-8039, gatifloxacin (AM 1155), trovafloxacin, clinafloxacin, levofloxacin and ciprofloxacin. J. Antimicrob. Chemother. 40:639-651.
Boyanova, L., A. Mentis, M. Gubina, E. Rozynek, G. Gosciniak, S. Kalenic, V. Goral, L. Kupcinskas, B. Kantarceken, A. Aydin, A. Archimandritis, D. Dzierzanowska, A. Vcev, K. Ivanova, M. Marina, I. Mitov, P. Petrov, A. Ozden, and M. Popova. 2002. The status of antimicrobial resistance of Helicobacter pylori in eastern Europe. Clin. Microbiol. Infect. 8:388-396.
Fujimura, S., S. Kato, K. Iinuma, and A. Watanabe. 2004. In vitro activity of fluoroquinolone and the gyrA gene mutation in Helicobacter pylori strains isolated from children. J. Med. Microbiol. 53:1019-1022.
Heep, M., M. Kist, S. Strobel, D. Beck, and N. Lehn. 2000. Secondary resistance among 554 isolates of Helicobacter pylori after failure of therapy. Eur. J. Clin. Microbiol. Infect. Dis. 19:538-541.
Kutschke, A., and B. L. M. de Jonge. 2005. Compound efflux in Helicobacter pylori. Antimicrob. Agents Chemother. 49:3009-3010.
Luo, N., O. Sahin, J. Lin, L. O. Michel, and Q. Zhang. 2003. In vivo selection of Campylobacter isolates with high levels of fluoroquinolone resistance associated with gyrA mutations and the function of the CmeABC efflux pump. Antimicrob. Agents Chemother. 47:390-394.
Masaoka, T., H. Suzuki, K. Kurabayashi, A. G. Kamiya, and H. Ishii. 2004. Second-line treatment of Helicobacter pylori infection after dilution agar methods and PCR-RFLP analysis. Aliment. Pharmacol. Ther. 20(Suppl. 1):68-73.
Moore, R. A., B. Beckthold, S. Wong, A. Kureishi, and L. E. Bryan. 1995. Nucleotide sequence of the gyrA gene and characterization of ciprofloxacin-resistant mutants of Helicobacter pylori. Antimicrob. Agents Chemother. 39:107-111.
National Committee for Clinical Laboratory Standards. 2004. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 6th ed. Approved standard M7-A6. National Committee for Clinical Laboratory Standards, Wayne, Pa.
National Committee for Clinical Laboratory Standards. 2004. Performance standards for antimicrobial susceptibility testing; 14th informational supplement (aerobic dilution). M100-S14. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Oram, M., and L. M. Fisher. 1991. 4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction. Antimicrob. Agents Chemother. 35:387-389.
Perri, F., V. Festa, A. Merla, F. Barberani, A. Pilotto, and A. Andriulli. 2003. Randomized study of different ‘second-line’ therapies for Helicobacter pylori infection after failure of the standard ‘Maastricht triple therapy’. Aliment. Pharmacol. Ther. 18:815-820.
Pilotto, A., M. Franceschi, M. Rassu, G. Leandro, L. Bozzola, F. Furlan, and F. Di Mario. 2000. Incidence of secondary Helicobacter pylori resistance to antibiotics in treatment failures after 1-week proton pump inhibitor-based triple therapies: a prospective study. Dig. Liver Dis. 32:667-672.
Sharara, A. I., H. F. Chaar, E. Racoubian, O. Moukhachen, K. A. Barada, F. H. Mourad, and G. F. Araj. 2004. Efficacy of two rabeprazole/gatifloxacin-based triple therapies for Helicobacter pylori infection. Helicobacter 9:255-261.
Tankovic, J., C. Lascols, Q. Sculo, J.-C. Petit, and C.-J. Soussy. 2003. Single and double mutations in gyrA but not in gyrB are associated with low- and high-level fluoroquinolone resistance in Helicobacter pylori. Antimicrob. Agents Chemother. 47:3942-3944.
Toro, C., J. Garcia-Samaniego, J. Carbo, A. Iniguez, T. Alarcon, M. Lopez-Brea, and M. Baquero. 2001. [Prevalence of primary Helicobacter pylori resistance to eight antimicrobial agents in a hospital in Madrid]. Rev. Esp. Quimioter. 14:172-176.(Toshihiro Nishizawa,Hidek)