Vol. 36, No. 1
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1992, p. 163-166
0066-4804/92/010163-04$02.00/0 Copyright © 1992, American Society for Microbiology
Role of Metronidazole Resistance in Therapy of Helicobacter pylori Infections HILPI RAUTELIN,l* KARI SEPPALA,2 OLLI-VEIKKO RENKONEN,1 UNTO VAINIO,3 AND TIMO U. KOSUNEN' Department of Bacteriology and Immunology, University of Helsinki, Haartmaninkatu 3,1 and Gastroenterological Unit, Second Department of Medicine, University of Helsinki,2 00290 Helsinki, and Central Military Hospital, 00300 Helsinki,3 Finland
Susceptibility to metronidazole was determined by disk diffusion tests for 559 strains of Helicobacter pylori isolated from patients. The overall metronidazole resistance was 26%. In males metronidazole-resistant strains made 18% of all H. pyloni strains, and in females the corresponding figure was 40% (P < 0.001). MICs of metronidazole were determined for H. pyloni strains from 86 patients undertaking triple therapy, i.e., treatment with colloidal bismuth subcitrate, amoxicillin, and metronidazole. Of the nonresponders who remained culture positive despite the therapy, 69% had strains with metronidazole MICs of .32 ,ig/ml before the therapy, and all nonresponders had metronidazole-resistant strains after the therapy. Metronidazole resistance was, however, also found in 27% of responders before therapy. To find whether the MICs of metronidazole for H. pyloni strains remained constant for longer periods, consecutive isolates sampled several years apart from the same patients were tested in parallel and no changes in the MICs were found. H. pylori was successfully eradicated by the triple therapy from 91% of patients with metronidazole-susceptible pretreatment strains and from 63% of patients with metronidazole-resistant strains before the therapy (P < 0.01). Although resistance to metronidazole has a significant role in treatment failures in H. pylori infections, high eradication rates can be achieved with the use of the present triple therapy even in populations with a high overall metronidazole resistance rate.
Helicobacter pylori is able to cause gastritis in previously healthy persons (12, 17). There is also strong evidence that H. pylori might be an important factor in peptic ulcer disease (24). Duodenal ulcer is a chronic disease with frequent relapses when traditionally treated with agents that reduce acid secretion in the stomach and provide symptomatic relief (11). Therefore, the possibility of treating these patients with antimicrobial agents effective in eradicating H. pylori which results in the improvement of the concomitant gastritis (14, 18, 23) and in remarkably fewer ulcer relapses (19), has gained great interest. However, despite the susceptibility of H. pylori to most antimicrobial agents in vitro (7, 13) such results have not necessarily predicted successful eradication of the bacterium in vivo. Treatment with single antimicrobial agents, such as erythromycin (14), fluoroquinolones (6, 15), and doxycycline (22), has failed to eradicate H. pylori. Also, better results obtained with other single antimicrobial agents, such as amoxicillin, bismuth salts, and nitrofurans, have been short lived and early relapses have occurred (16, 18). So far the most successful results for the eradication of H. pylori from the gastric mucosa have been obtained with a triple treatment consisting of amoxicillin or tetracycline, metronidazole or tinidazole, and a bismuth compound (2, 3). The occurrence of metronidazole-resistant H. pylori strains has been reported to be related to the earlier use of nitroimidazoles (1, 5). Not only have nitroimidazole-resistant H. pylori strains been isolated before the treatment of this infection, but also rapid development of resistance has been demonstrated in vitro (9) and in vivo (5, 8) during therapy with nitroimidazoles. This study was undertaken to determine the overall in vitro susceptibility to metronidazole *
of H. pylori strains isolated from Finnish patients undergoing endoscopy for various upper gastrointestinal symptoms. We also wanted to clarify whether the eradication rates of H. pylori in the triple-treated patients with metronidazole-resistant strains before therapy differed from the results for the patients with metronidazole-susceptible pretreatment strains and how often metronidazole resistance was found in strains isolated from patients not responding to the combined treatment.
(Results of this study were presented in part at the Third Workshop of the European Helicobacter pylori Study Group, 8 to 10 November 1990, Toledo, Spain.) MATERIALS AND METHODS Helicobacter strains. H. pylori strains from 86 patients, 35 women (age range, 38 to 77 years; mean, 54 years) and 51 men (age range, 24 to 76 years; mean, 51 years), who were later treated with daily doses of 120 mg of colloidal bismuth subcitrate four times daily, 500 mg of amoxicillin four times daily, and 500 mg of metronidazole three times daily for 2 weeks were studied. All patients had H. pylori-associated chronic gastritis. At least a single pretreatment isolate was available from all patients, and 12 patients had two to five H. pylori-positive pretreatment biopsy samples taken 1 to 46 months before treatment. The successful eradication of H. pylori was verified by the inability to culture H. pylori from gastric biopsies taken 4 weeks and 6 months after the treatment. H. pylori was also isolated from gastric biopsies of patients not undertaking triple therapy. Single H. pylori strains were available from a total of 559 patients (51 of these were included in the treatment group), 208 women (age range, 22 to 86 years; mean, 57 years) and 351 men (age range, 18 to 85
Corresponding author. 163
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Received 12 July 1991/Accepted 30 October 1991
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age FIG. 1. Distribution of H. pylori-positive men (n = 351) (A) and H. pylori-positive women (n = 208) (B) into different age groups, and the resistance (R) or susceptibility (S) of the H. pylori strains to metronidazole on the basis of the disk diffusion test (metronidazole, 16 ,ug).
47 years). The age distribution of the individuals is shown in Fig. 1. The strains were identified on the basis of colony appearance, Gram staining, and positive reactions in biochemical tests (catalase, oxidase, and urease). Susceptibility testing. MICs of metronidazole were determined for H. pylori strains from patients undertaking triple therapy; an agar dilution method with Brucella agar plates (BBL, Cockeysville, Md.) supplemented with whole horse blood (7%) was used. Consecutive isolates from the same patient were tested in parallel. Metronidazole (Sigma, St. Louis, Mo.) was dissolved, and serial dilutions were prepared in sterile water. Metronidazole-containing medium was prepared within 24 h prior to use and was stored at room temperature. The turbidity of suspensions of H. pylori strains grown in Mueller-Hinton broth (BBL) in an atmosphere of 5% 02, 10% Co2, and 85% N2 at 37°C for 24 h was adjusted to a MacFarland standard of 0.5 by using a Cobas Inocheck instrument (F. Hoffman-La Roche Ltd., Basel, Switzerland). A multipoint inoculator (Mast) was used to apply i0' CFU per spot. H. pylori NCTC 11637 and NCTC 11638 were used as controls on each plate. The plates were incubated in a microaerobic atmosphere for 48 h. The MIC was defined as the lowest concentration of metronidazole that inhibited visible growth completely. The break point of resistance (8 ,ug/ml) was determined as described in reference 17a.
Because metronidazole-susceptible and -resistant H. pylori strains could well be discriminated with the use of both the agar dilution method and the disk diffusion test (Fig. 2), the majority of the H. pylori strains were tested for susceptibility to metronidazole by disk diffusion with 16-Rg disks of metronidazole (Rosco Diagnostica, Taastrup, Denmark). Modified Thayer-Martin agar plates without antibiotics consisted of Mueller-Hinton agar (BBL), proteose peptone (Difco Laboratories, Detroit, Mich.), starch, and agar and were enriched with horse blood (8%), IsoVitaleX (BBL), and hemin. The agar plates were incubated for 48 h in a microaerobic atmosphere, and isolates with inhibition zone diameters smaller than 24 mm were considered resistant to metronidazole as established for anaerobes (4). Statistics. Statistical analysis was performed by using the chi-square test with Yates's correction when appropriate and the Fisher exact test (21). RESULTS
years; mean,
For 86 H. pylori strains, the susceptibility to metronidazole was determined by both the disk diffusion test and the agar dilution method. With both tests the susceptible and resistant strains could easily be discriminated from each other (Fig. 2). H. pylori strains for which the metronidazole MICs were 0.5 to 4 ,ug/ml had inhibition zone diameters ranging from 30 to 77 mm in the disk diffusion test, and all strains for which the metronidazole MICs were .32 p,g/ml had no inhibition zone. The overall resistance of 559 H. pylori strains to metronidazole determined by the disk diffusion test was 26%. In men metronidazole-resistant strains made up 18% of all strains. For the different age groups (Fig. 1A) the percentage of metronidazole-resistant strains in men was almost the same. In women 40% of all strains showed resistance to metronidazole (Fig. 1B), and thus metronidazole resistance was significantly more common than in men (P < 0.001). In the two youngest age groups the number of women was so limited that the difference in metronidazole resistance between the sexes did not reach statistical significance. However, in the following three age groups metronidazoleresistant strains were found significantly more often in women than in men (P < 0.001, P < 0.001, and P < 0.01) (Fig.1). For persons .66 years of age the difference was not significant. MICs of metronidazole were determined for the H. pylori
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MIC (ug/mi) of metronidazole FIG. 3. The MICs of metronidazole for H. pylori strains isolated from gastric mucosa of patients before treatment with amoxicillin, metronidazole, and colloidal bismuth subcitrate. H. pylori could be successfully eradicated in responders, whereas nonresponders remained culture positive for H. pylori despite the treatment. pretreatment strains isolated from 86 patients who later undertook triple treatment (Fig. 3). From 70 of these patients
H. pylori was successfully eradicated, whereas 16 patients (12 women) remained culture-positive despite the treatment. Of the responders and nonresponders, 27 and 69%, respectively, had pretreatment strains for which the metronidazole MICs were -32 ,ug/ml (P < 0.01). Before treatment, five patients were subjected to endoscopy at 1- to 3-month intervals after the first gastric biopsies were taken. MICs of metronidazole for these consecutive isolates did not change during the short pretherapy follow-up period; neither did the MICs of metronidazole for consecutive isolates taken during longer follow-up periods before therapy differ; three patients had two, and another four patients had three to five consecutive samples taken at intervals of 21 to 46 months before the triple treatment. All posttreatment isolates from nonresponders, two to five consecutive H. pylori-positive posttreatment isolates from a follow-up period of 2 to 18 months of nine nonresponders and a single posttreatment isolate from the remaining seven nonresponders, were metronidazole resistant, with MICs of .32 ,ug/ml.
DISCUSSION Until now the best results in the eradication of H. pylori from the gastric mucosa have been achieved by triple therapy in which nitroimidazoles have been an important component. For double therapies including a bismuth compound and an antibiotic not belonging to the nitroimidazoles, eradication rates have been only 40 to 60% (10, 18), whereas for triple therapies, including nitroimidazoles and bismuth salts, eradication rates of over 90% have been achieved (2, 3). Because eradication of H. pylori has not always been successful, reasons for such failures have been suggested to be due to its resistance to metronidazole (5, 8). In the present study, the susceptibility to metronidazole of H. pylori strains from 559 unselected patients was studied by disk diffusion. The overall metronidazole resistance (26%) of the present study was almost identical to the findings of Glupczynski et al. in Belgium (27%) (5). They reported, however, no significant difference between metronidazole resistance in women (25%) and men (30%), whereas we found that women har-
bored metronidazole-resistant strains more than twice as often as men did. The difference in metronidazole resistance between men and women was not restricted to any certain age group, even if statistical significance could not be reached in the two youngest age groups and in the oldest one. The difference in the metronidazole resistance rates between men and women could possibly be explained by the frequent use of nitroimidazoles in the treatment of gynecological infections. In the present study we also wanted to find whether the eradication results for the triple-treated patients with metronidazole-resistant pretreatment strains differed from the results obtained with the same therapy in patients with metronidazole-susceptible pretreatment strains. Metronidazole resistance was more than twice as common among persons who remained culture positive in spite of the therapy (69%) as it was among persons who were later treated successfully with the combination therapy (27%) (P < 0.01). Thus, metronidazole resistance clearly has an impact on the results of the triple therapy. Metronidazole resistance was also found to develop rather quickly, as shown by nonresponders who had metronidazole-susceptible pretreatment strains and who harbored resistant isolates after therapy. The percentage of women among the nonresponders (75%) was greater than one would have expected on the basis of their proportion of all those treated (41%) (P < 0.01); however, the more frequent occurrence of metronidazoleresistant strains isolated from women explains this finding. Two of the four men who did not respond to the triple therapy had metronidazole-resistant pretreatment strains, whereas 10 of 12 nonresponding women harbored metronidazole-resistant pretreatment strains. To find whether H. pylori isolates from the same patients differed in terms of their susceptibility to metronidazole, MICs of metronidazole were determined for these isolates in parallel. The MICs remained constant even in samples isolated several years apart. This could be due to infrequent spontaneous development of resistance and to the fact that the developed metronidazole resistance of H. pylori seems to remain unchanged for long periods. However, it was not confirmed that the consecutive isolates were identical. The eradication rate of H. pylori in our triple-treated patients was 81%. Eradication could also be -verified by significant drops in the specific H. pylori antibody titers (20) and by changes in the histological picture (23); however, this was not done in this study. For patients with metronidazolesusceptible pretreatment strains for eradication rate was 91%, and for those with resistant pretreatment strains the corresponding figure was 63% (P < 0.01). The eradication rate in the metronidazole-susceptible group is close to the best results obtained in the treatment of H. pylori was triple therapy (2, 3). In our metronidazole-resistant group, the eradication rate was higher than in studies using double therapies with bismuth compounds and amoxicillin (10, 18), although the different treatment protocols are not exactly comparable. Metronidazole resistance plays an important role in the failure to eradicate of H. pylori, but we could show that high eradication rates can be achieved with the use of the present triple therapy, even in populations with a high metronidazole resistance rate. ACKNOWLEDGMENTS The skillful technical assistance of Sirpa Juvonen and Eila Kelo is gratefully acknowledged. We are grateful to Martti Vaara, Anthony Moran, and Arda Kuikka for reading the manuscript.
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78(Suppl. 1):115-116. 11. Lane, M. R., and S. P. Lee. 1988. Recurrence of duodenal ulcer after medical treatment. Lancet i:1147-1149. 12. Marshall, B. J., J. A. Armstrong, D. B. McGechie, and R. J. Glancy. 1985. Attempt to fulfil Koch's postulates for pyloric campylobacter. Med. J. Aust. 142:436-443. 13. McNulty, C. A. M., and J. C. Dent. 1988. Susceptibility of clinical isolates of Campylobacter pylori to twenty-one antimi-
crobial agents. Eur. J. Clin. Microbiol. Infect. Dis. 7:565-569. 14. McNulty, C. A. M., J. C. Gearty, B. Crump, M. Davis, I. A. Donovan, V. Melikian, D. M. Lister, and R. Wise. 1986. Campylobacter pyloridis and associated gastritis: investigator blind, placebo controlled trial of bismuth salicylate and erythromycin ethylsuccinate. Br. Med. J. 293:645-649. 15. Mertens, J. C. C., W. Dekker, E. E. J. Ligtvoet, and P. Blok. 1989. Treatment failure of norfloxacin against Campylobacter pylori and chronic gastritis in patients with nonulcerative dyspepsia. Antimicrob. Agents. Chemother. 33:256-257. 16. Morgan, D., W. Kraft, M. Bender, and A. Pearson. 1988. Nitrofurans in the treatment of gastritis associated with Campylobacter pylori. Gastroenterology 95:1178-1184. 17. Morris, A., and G. Nicholson. 1987. Ingestion of Campylobacter pyloridis causes gastritis and raised fasting gastric pH. Am. J. Gastroenterol. 82:192-199. 17a.National Committee for Clinical Laboratory Standards. 1990. Standard MF-A. National Committee for Clinical Laboratory Standards, Villanova, Pa. 18. Rauws, E. A., W. Langenberg, H. J. Houthoff, H. C. Zanen, and G. N. Tytgat. 1988. Campylobacter pyloridis-associated chronic active antral gastritis. A prospective study of its prevalence and the effects of antibacterial and antiulcer treatment. Gastroenterology 94:33-40. 19. Rauws, E. A. J., and G. N. J. Tytgat. 1990. Cure of duodenal ulcer associated with eradication of Helicobacter pylori. Lancet 335:1233-1235. 20. Seppala, K., T. Kosunen, P. Sipponen, and V. Valtonen. 1990. Decrease of Helicobacter pylori antibodies reflects successful eradication therapy, abstr. p. 917. Abstr. 9th World Congress of Gastroenterology. 21. Swinscow, T. D. V. 1978. Statistics at square one, 3rd ed. The British Medical Association, London. 22. Unge, P., and H. Gnarpe. 1988. Pharmacokinetic, bacteriological and clinical aspects on the use of doxycycline in patients with active duodenal ulcer associated with Campylobacter pylori. Scand. J. Infect. Dis. Suppl. 53:70-73. 23. Valle, J., K. Seppala, P. Sipponen, and T. Kosunen. 1991. Disappearance of gastritis after eradication of Helicobacter pylori: a morphometric study. Scand. J. Gastroenterol. 26:10571065. 24. Wyatt, J. I., B. J. Rathbone, M. F. Dixon, and R. V. Heatley. 1987. Campylobacter pyloridis and acid induced gastric metaplasia in the pathogenesis of duodenitis. J. Clin. Pathol. 40:841848.
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REFERENCES 1. Becx, M. C. J. M., A. J. H. M. Janssen, H. A. L. Clasener, and R. W. de Koning. 1990. Metronidazole-resistant Helicobacter pylori. Lancet i:539-540. (Letter.) 2. Borody, T., P. Cole, S. Noonan, A. Morgan, G. Ossip, J. Maysey, and S. Brandl. 1988. Long-term Campylobacter pylori recurrence post-eradication. Gastroenterology 94:A43. 3. Borsch, G., U. Mai, and W. Opferkuch. 1988. Oral triple-therapy (OTT) may effectively eradicate Campylobacter pylori (C.p.) in man: a pilot study. Gastroenterology 94:A44. 4. Casals, J. B., and N. Pringler. 1990. Standardization of the agar diffusion method for susceptibility testing of anaerobes. SSAC Ann. Meet. Oslo, Norway. 5. Glupczynski, Y., A. Burette, E. de Koster, J.-F. Nyst, M. Deltenre, S. Cadranel, L. Bordeaux, and D. de Vos. 1990. Metronidazole resistance in Helicobacter pylori. Lancet 335: 976-977. (Letter.) 6. Glupczynski, Y., M. Labbe, A. Burette, M. Delmee, V. Avesani, and C. Bruck. 1987. Treatment failure of ofloxacin in Campylobacter pylori infection. Lancet i:1096. (Letter.) 7. Goodwin, C. S., P. Blake, and E. Blincow. 1986. The minimum inhibitory and bactericidal concentrations of antibiotics and anti-ulcer agents against Campylobacter pyloridis. J. Antimicrob. Chemother. 17:309-314. 8. Goodwin, C. S., B. J. Marshall, E. D. Blincow, D. H. Wilson, S. Blackbourne, and M. Phillips. 1988. Prevention of nitroimidazole resistance in Campylobacter pylori by coadministration of colloidal bismuth subcitrate: clinical and in vitro studies. J. Clin. Pathol. 41:207-210. 9. Haas, C. E., D. E. Nix, and J. J. Schentag. 1990. In vitro selection of resistant Helicobacter pylori. Antimicrob. Agents Chemother. 34:1637-1641. 10. Lambert, J. R., S. K. Lin, M. Schembri, L. Nicholson, and M. G. Korman. 1990. Helicobacter pylori therapy randomized study of denol/antibiotic combinations. Rev. Esp. Enferm. Apar. Dig.
ANTIMICROB. AGENTS CHEMOTHER.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1992, p. 163-166
0066-4804/92/010163-04$02.00/0 Copyright © 1992, American Society for Microbiology
Role of Metronidazole Resistance in Therapy of Helicobacter pylori Infections HILPI RAUTELIN,l* KARI SEPPALA,2 OLLI-VEIKKO RENKONEN,1 UNTO VAINIO,3 AND TIMO U. KOSUNEN' Department of Bacteriology and Immunology, University of Helsinki, Haartmaninkatu 3,1 and Gastroenterological Unit, Second Department of Medicine, University of Helsinki,2 00290 Helsinki, and Central Military Hospital, 00300 Helsinki,3 Finland
Susceptibility to metronidazole was determined by disk diffusion tests for 559 strains of Helicobacter pylori isolated from patients. The overall metronidazole resistance was 26%. In males metronidazole-resistant strains made 18% of all H. pyloni strains, and in females the corresponding figure was 40% (P < 0.001). MICs of metronidazole were determined for H. pyloni strains from 86 patients undertaking triple therapy, i.e., treatment with colloidal bismuth subcitrate, amoxicillin, and metronidazole. Of the nonresponders who remained culture positive despite the therapy, 69% had strains with metronidazole MICs of .32 ,ig/ml before the therapy, and all nonresponders had metronidazole-resistant strains after the therapy. Metronidazole resistance was, however, also found in 27% of responders before therapy. To find whether the MICs of metronidazole for H. pyloni strains remained constant for longer periods, consecutive isolates sampled several years apart from the same patients were tested in parallel and no changes in the MICs were found. H. pylori was successfully eradicated by the triple therapy from 91% of patients with metronidazole-susceptible pretreatment strains and from 63% of patients with metronidazole-resistant strains before the therapy (P < 0.01). Although resistance to metronidazole has a significant role in treatment failures in H. pylori infections, high eradication rates can be achieved with the use of the present triple therapy even in populations with a high overall metronidazole resistance rate.
Helicobacter pylori is able to cause gastritis in previously healthy persons (12, 17). There is also strong evidence that H. pylori might be an important factor in peptic ulcer disease (24). Duodenal ulcer is a chronic disease with frequent relapses when traditionally treated with agents that reduce acid secretion in the stomach and provide symptomatic relief (11). Therefore, the possibility of treating these patients with antimicrobial agents effective in eradicating H. pylori which results in the improvement of the concomitant gastritis (14, 18, 23) and in remarkably fewer ulcer relapses (19), has gained great interest. However, despite the susceptibility of H. pylori to most antimicrobial agents in vitro (7, 13) such results have not necessarily predicted successful eradication of the bacterium in vivo. Treatment with single antimicrobial agents, such as erythromycin (14), fluoroquinolones (6, 15), and doxycycline (22), has failed to eradicate H. pylori. Also, better results obtained with other single antimicrobial agents, such as amoxicillin, bismuth salts, and nitrofurans, have been short lived and early relapses have occurred (16, 18). So far the most successful results for the eradication of H. pylori from the gastric mucosa have been obtained with a triple treatment consisting of amoxicillin or tetracycline, metronidazole or tinidazole, and a bismuth compound (2, 3). The occurrence of metronidazole-resistant H. pylori strains has been reported to be related to the earlier use of nitroimidazoles (1, 5). Not only have nitroimidazole-resistant H. pylori strains been isolated before the treatment of this infection, but also rapid development of resistance has been demonstrated in vitro (9) and in vivo (5, 8) during therapy with nitroimidazoles. This study was undertaken to determine the overall in vitro susceptibility to metronidazole *
of H. pylori strains isolated from Finnish patients undergoing endoscopy for various upper gastrointestinal symptoms. We also wanted to clarify whether the eradication rates of H. pylori in the triple-treated patients with metronidazole-resistant strains before therapy differed from the results for the patients with metronidazole-susceptible pretreatment strains and how often metronidazole resistance was found in strains isolated from patients not responding to the combined treatment.
(Results of this study were presented in part at the Third Workshop of the European Helicobacter pylori Study Group, 8 to 10 November 1990, Toledo, Spain.) MATERIALS AND METHODS Helicobacter strains. H. pylori strains from 86 patients, 35 women (age range, 38 to 77 years; mean, 54 years) and 51 men (age range, 24 to 76 years; mean, 51 years), who were later treated with daily doses of 120 mg of colloidal bismuth subcitrate four times daily, 500 mg of amoxicillin four times daily, and 500 mg of metronidazole three times daily for 2 weeks were studied. All patients had H. pylori-associated chronic gastritis. At least a single pretreatment isolate was available from all patients, and 12 patients had two to five H. pylori-positive pretreatment biopsy samples taken 1 to 46 months before treatment. The successful eradication of H. pylori was verified by the inability to culture H. pylori from gastric biopsies taken 4 weeks and 6 months after the treatment. H. pylori was also isolated from gastric biopsies of patients not undertaking triple therapy. Single H. pylori strains were available from a total of 559 patients (51 of these were included in the treatment group), 208 women (age range, 22 to 86 years; mean, 57 years) and 351 men (age range, 18 to 85
Corresponding author. 163
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Received 12 July 1991/Accepted 30 October 1991
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FIG. 2. Relation between the MICs of metronidazole in the agar dilution method and the zone diameters in the disk diffusion test (metronidazole, 16 ,ug) based on the results with 86 H. pylori strains.
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age FIG. 1. Distribution of H. pylori-positive men (n = 351) (A) and H. pylori-positive women (n = 208) (B) into different age groups, and the resistance (R) or susceptibility (S) of the H. pylori strains to metronidazole on the basis of the disk diffusion test (metronidazole, 16 ,ug).
47 years). The age distribution of the individuals is shown in Fig. 1. The strains were identified on the basis of colony appearance, Gram staining, and positive reactions in biochemical tests (catalase, oxidase, and urease). Susceptibility testing. MICs of metronidazole were determined for H. pylori strains from patients undertaking triple therapy; an agar dilution method with Brucella agar plates (BBL, Cockeysville, Md.) supplemented with whole horse blood (7%) was used. Consecutive isolates from the same patient were tested in parallel. Metronidazole (Sigma, St. Louis, Mo.) was dissolved, and serial dilutions were prepared in sterile water. Metronidazole-containing medium was prepared within 24 h prior to use and was stored at room temperature. The turbidity of suspensions of H. pylori strains grown in Mueller-Hinton broth (BBL) in an atmosphere of 5% 02, 10% Co2, and 85% N2 at 37°C for 24 h was adjusted to a MacFarland standard of 0.5 by using a Cobas Inocheck instrument (F. Hoffman-La Roche Ltd., Basel, Switzerland). A multipoint inoculator (Mast) was used to apply i0' CFU per spot. H. pylori NCTC 11637 and NCTC 11638 were used as controls on each plate. The plates were incubated in a microaerobic atmosphere for 48 h. The MIC was defined as the lowest concentration of metronidazole that inhibited visible growth completely. The break point of resistance (8 ,ug/ml) was determined as described in reference 17a.
Because metronidazole-susceptible and -resistant H. pylori strains could well be discriminated with the use of both the agar dilution method and the disk diffusion test (Fig. 2), the majority of the H. pylori strains were tested for susceptibility to metronidazole by disk diffusion with 16-Rg disks of metronidazole (Rosco Diagnostica, Taastrup, Denmark). Modified Thayer-Martin agar plates without antibiotics consisted of Mueller-Hinton agar (BBL), proteose peptone (Difco Laboratories, Detroit, Mich.), starch, and agar and were enriched with horse blood (8%), IsoVitaleX (BBL), and hemin. The agar plates were incubated for 48 h in a microaerobic atmosphere, and isolates with inhibition zone diameters smaller than 24 mm were considered resistant to metronidazole as established for anaerobes (4). Statistics. Statistical analysis was performed by using the chi-square test with Yates's correction when appropriate and the Fisher exact test (21). RESULTS
years; mean,
For 86 H. pylori strains, the susceptibility to metronidazole was determined by both the disk diffusion test and the agar dilution method. With both tests the susceptible and resistant strains could easily be discriminated from each other (Fig. 2). H. pylori strains for which the metronidazole MICs were 0.5 to 4 ,ug/ml had inhibition zone diameters ranging from 30 to 77 mm in the disk diffusion test, and all strains for which the metronidazole MICs were .32 p,g/ml had no inhibition zone. The overall resistance of 559 H. pylori strains to metronidazole determined by the disk diffusion test was 26%. In men metronidazole-resistant strains made up 18% of all strains. For the different age groups (Fig. 1A) the percentage of metronidazole-resistant strains in men was almost the same. In women 40% of all strains showed resistance to metronidazole (Fig. 1B), and thus metronidazole resistance was significantly more common than in men (P < 0.001). In the two youngest age groups the number of women was so limited that the difference in metronidazole resistance between the sexes did not reach statistical significance. However, in the following three age groups metronidazoleresistant strains were found significantly more often in women than in men (P < 0.001, P < 0.001, and P < 0.01) (Fig.1). For persons .66 years of age the difference was not significant. MICs of metronidazole were determined for the H. pylori
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MIC (ug/mi) of metronidazole FIG. 3. The MICs of metronidazole for H. pylori strains isolated from gastric mucosa of patients before treatment with amoxicillin, metronidazole, and colloidal bismuth subcitrate. H. pylori could be successfully eradicated in responders, whereas nonresponders remained culture positive for H. pylori despite the treatment. pretreatment strains isolated from 86 patients who later undertook triple treatment (Fig. 3). From 70 of these patients
H. pylori was successfully eradicated, whereas 16 patients (12 women) remained culture-positive despite the treatment. Of the responders and nonresponders, 27 and 69%, respectively, had pretreatment strains for which the metronidazole MICs were -32 ,ug/ml (P < 0.01). Before treatment, five patients were subjected to endoscopy at 1- to 3-month intervals after the first gastric biopsies were taken. MICs of metronidazole for these consecutive isolates did not change during the short pretherapy follow-up period; neither did the MICs of metronidazole for consecutive isolates taken during longer follow-up periods before therapy differ; three patients had two, and another four patients had three to five consecutive samples taken at intervals of 21 to 46 months before the triple treatment. All posttreatment isolates from nonresponders, two to five consecutive H. pylori-positive posttreatment isolates from a follow-up period of 2 to 18 months of nine nonresponders and a single posttreatment isolate from the remaining seven nonresponders, were metronidazole resistant, with MICs of .32 ,ug/ml.
DISCUSSION Until now the best results in the eradication of H. pylori from the gastric mucosa have been achieved by triple therapy in which nitroimidazoles have been an important component. For double therapies including a bismuth compound and an antibiotic not belonging to the nitroimidazoles, eradication rates have been only 40 to 60% (10, 18), whereas for triple therapies, including nitroimidazoles and bismuth salts, eradication rates of over 90% have been achieved (2, 3). Because eradication of H. pylori has not always been successful, reasons for such failures have been suggested to be due to its resistance to metronidazole (5, 8). In the present study, the susceptibility to metronidazole of H. pylori strains from 559 unselected patients was studied by disk diffusion. The overall metronidazole resistance (26%) of the present study was almost identical to the findings of Glupczynski et al. in Belgium (27%) (5). They reported, however, no significant difference between metronidazole resistance in women (25%) and men (30%), whereas we found that women har-
bored metronidazole-resistant strains more than twice as often as men did. The difference in metronidazole resistance between men and women was not restricted to any certain age group, even if statistical significance could not be reached in the two youngest age groups and in the oldest one. The difference in the metronidazole resistance rates between men and women could possibly be explained by the frequent use of nitroimidazoles in the treatment of gynecological infections. In the present study we also wanted to find whether the eradication results for the triple-treated patients with metronidazole-resistant pretreatment strains differed from the results obtained with the same therapy in patients with metronidazole-susceptible pretreatment strains. Metronidazole resistance was more than twice as common among persons who remained culture positive in spite of the therapy (69%) as it was among persons who were later treated successfully with the combination therapy (27%) (P < 0.01). Thus, metronidazole resistance clearly has an impact on the results of the triple therapy. Metronidazole resistance was also found to develop rather quickly, as shown by nonresponders who had metronidazole-susceptible pretreatment strains and who harbored resistant isolates after therapy. The percentage of women among the nonresponders (75%) was greater than one would have expected on the basis of their proportion of all those treated (41%) (P < 0.01); however, the more frequent occurrence of metronidazoleresistant strains isolated from women explains this finding. Two of the four men who did not respond to the triple therapy had metronidazole-resistant pretreatment strains, whereas 10 of 12 nonresponding women harbored metronidazole-resistant pretreatment strains. To find whether H. pylori isolates from the same patients differed in terms of their susceptibility to metronidazole, MICs of metronidazole were determined for these isolates in parallel. The MICs remained constant even in samples isolated several years apart. This could be due to infrequent spontaneous development of resistance and to the fact that the developed metronidazole resistance of H. pylori seems to remain unchanged for long periods. However, it was not confirmed that the consecutive isolates were identical. The eradication rate of H. pylori in our triple-treated patients was 81%. Eradication could also be -verified by significant drops in the specific H. pylori antibody titers (20) and by changes in the histological picture (23); however, this was not done in this study. For patients with metronidazolesusceptible pretreatment strains for eradication rate was 91%, and for those with resistant pretreatment strains the corresponding figure was 63% (P < 0.01). The eradication rate in the metronidazole-susceptible group is close to the best results obtained in the treatment of H. pylori was triple therapy (2, 3). In our metronidazole-resistant group, the eradication rate was higher than in studies using double therapies with bismuth compounds and amoxicillin (10, 18), although the different treatment protocols are not exactly comparable. Metronidazole resistance plays an important role in the failure to eradicate of H. pylori, but we could show that high eradication rates can be achieved with the use of the present triple therapy, even in populations with a high metronidazole resistance rate. ACKNOWLEDGMENTS The skillful technical assistance of Sirpa Juvonen and Eila Kelo is gratefully acknowledged. We are grateful to Martti Vaara, Anthony Moran, and Arda Kuikka for reading the manuscript.
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This work was supported in part by a grant from the Finnish Medical Research Council.
78(Suppl. 1):115-116. 11. Lane, M. R., and S. P. Lee. 1988. Recurrence of duodenal ulcer after medical treatment. Lancet i:1147-1149. 12. Marshall, B. J., J. A. Armstrong, D. B. McGechie, and R. J. Glancy. 1985. Attempt to fulfil Koch's postulates for pyloric campylobacter. Med. J. Aust. 142:436-443. 13. McNulty, C. A. M., and J. C. Dent. 1988. Susceptibility of clinical isolates of Campylobacter pylori to twenty-one antimi-
crobial agents. Eur. J. Clin. Microbiol. Infect. Dis. 7:565-569. 14. McNulty, C. A. M., J. C. Gearty, B. Crump, M. Davis, I. A. Donovan, V. Melikian, D. M. Lister, and R. Wise. 1986. Campylobacter pyloridis and associated gastritis: investigator blind, placebo controlled trial of bismuth salicylate and erythromycin ethylsuccinate. Br. Med. J. 293:645-649. 15. Mertens, J. C. C., W. Dekker, E. E. J. Ligtvoet, and P. Blok. 1989. Treatment failure of norfloxacin against Campylobacter pylori and chronic gastritis in patients with nonulcerative dyspepsia. Antimicrob. Agents. Chemother. 33:256-257. 16. Morgan, D., W. Kraft, M. Bender, and A. Pearson. 1988. Nitrofurans in the treatment of gastritis associated with Campylobacter pylori. Gastroenterology 95:1178-1184. 17. Morris, A., and G. Nicholson. 1987. Ingestion of Campylobacter pyloridis causes gastritis and raised fasting gastric pH. Am. J. Gastroenterol. 82:192-199. 17a.National Committee for Clinical Laboratory Standards. 1990. Standard MF-A. National Committee for Clinical Laboratory Standards, Villanova, Pa. 18. Rauws, E. A., W. Langenberg, H. J. Houthoff, H. C. Zanen, and G. N. Tytgat. 1988. Campylobacter pyloridis-associated chronic active antral gastritis. A prospective study of its prevalence and the effects of antibacterial and antiulcer treatment. Gastroenterology 94:33-40. 19. Rauws, E. A. J., and G. N. J. Tytgat. 1990. Cure of duodenal ulcer associated with eradication of Helicobacter pylori. Lancet 335:1233-1235. 20. Seppala, K., T. Kosunen, P. Sipponen, and V. Valtonen. 1990. Decrease of Helicobacter pylori antibodies reflects successful eradication therapy, abstr. p. 917. Abstr. 9th World Congress of Gastroenterology. 21. Swinscow, T. D. V. 1978. Statistics at square one, 3rd ed. The British Medical Association, London. 22. Unge, P., and H. Gnarpe. 1988. Pharmacokinetic, bacteriological and clinical aspects on the use of doxycycline in patients with active duodenal ulcer associated with Campylobacter pylori. Scand. J. Infect. Dis. Suppl. 53:70-73. 23. Valle, J., K. Seppala, P. Sipponen, and T. Kosunen. 1991. Disappearance of gastritis after eradication of Helicobacter pylori: a morphometric study. Scand. J. Gastroenterol. 26:10571065. 24. Wyatt, J. I., B. J. Rathbone, M. F. Dixon, and R. V. Heatley. 1987. Campylobacter pyloridis and acid induced gastric metaplasia in the pathogenesis of duodenitis. J. Clin. Pathol. 40:841848.
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REFERENCES 1. Becx, M. C. J. M., A. J. H. M. Janssen, H. A. L. Clasener, and R. W. de Koning. 1990. Metronidazole-resistant Helicobacter pylori. Lancet i:539-540. (Letter.) 2. Borody, T., P. Cole, S. Noonan, A. Morgan, G. Ossip, J. Maysey, and S. Brandl. 1988. Long-term Campylobacter pylori recurrence post-eradication. Gastroenterology 94:A43. 3. Borsch, G., U. Mai, and W. Opferkuch. 1988. Oral triple-therapy (OTT) may effectively eradicate Campylobacter pylori (C.p.) in man: a pilot study. Gastroenterology 94:A44. 4. Casals, J. B., and N. Pringler. 1990. Standardization of the agar diffusion method for susceptibility testing of anaerobes. SSAC Ann. Meet. Oslo, Norway. 5. Glupczynski, Y., A. Burette, E. de Koster, J.-F. Nyst, M. Deltenre, S. Cadranel, L. Bordeaux, and D. de Vos. 1990. Metronidazole resistance in Helicobacter pylori. Lancet 335: 976-977. (Letter.) 6. Glupczynski, Y., M. Labbe, A. Burette, M. Delmee, V. Avesani, and C. Bruck. 1987. Treatment failure of ofloxacin in Campylobacter pylori infection. Lancet i:1096. (Letter.) 7. Goodwin, C. S., P. Blake, and E. Blincow. 1986. The minimum inhibitory and bactericidal concentrations of antibiotics and anti-ulcer agents against Campylobacter pyloridis. J. Antimicrob. Chemother. 17:309-314. 8. Goodwin, C. S., B. J. Marshall, E. D. Blincow, D. H. Wilson, S. Blackbourne, and M. Phillips. 1988. Prevention of nitroimidazole resistance in Campylobacter pylori by coadministration of colloidal bismuth subcitrate: clinical and in vitro studies. J. Clin. Pathol. 41:207-210. 9. Haas, C. E., D. E. Nix, and J. J. Schentag. 1990. In vitro selection of resistant Helicobacter pylori. Antimicrob. Agents Chemother. 34:1637-1641. 10. Lambert, J. R., S. K. Lin, M. Schembri, L. Nicholson, and M. G. Korman. 1990. Helicobacter pylori therapy randomized study of denol/antibiotic combinations. Rev. Esp. Enferm. Apar. Dig.
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