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Susceptibilities to clarithromycin, amoxycillin and metronidazole of Helicobacter pylori isolates from the antrum and corpus in Tokyo, Japan, 1995–2001
ORIGINAL ARTICLE
10.1111/j.1469-0691.2005.01099.x
Susceptibilities to clarithromycin, amoxycillin and metronidazole of Helicobacter pylori isolates from the antrum and corpus in Tokyo, Japan, 1995–2001 E. Rimbara1, N. Noguchi1, M. Tanabe1, T. Kawai2, Y. Matsumoto3 and M. Sasatsu1 1
Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Endoscopy Center, Tokyo Medical University and 3Hachioji Pharmaceutical Center Pharmacy, Hachioji, Tokyo, Japan
2
ABSTRACT The aim of this study was to determine the susceptibilities to clarithromycin, amoxycillin and metronidazole of Helicobacter pylori isolates from the antrum and corpus of Japanese patients examined during the period 1995–2001. There was an increase, from 6.2% in 1995 to 22.1% in 2000–2001, in the proportion of patients infected with clarithromycin-resistant H. pylori. Of patients infected with clarithromycin-resistant H. pylori, 39.1% were infected with both clarithromycin-susceptible and -resistant H. pylori. Furthermore, the MIC90 of clarithromycin for H. pylori rose from < 1 mg ⁄ L in 1995– 1998 to 8 mg ⁄ L in 1999. In contrast, the MIC90s of amoxycillin and metronidazole were £ 0.125 and 4 mg ⁄ L, respectively, throughout the study period. The results showed that, while most H. pylori isolates were susceptible to amoxycillin and metronidazole, resistance to clarithromycin among H. pylori isolates increased markedly in Japan during 1995–2001. The results also indicated a need to test the susceptibility of H. pylori isolates from more than two samples obtained from two different sites in the stomach of a single patient in order to diagnose the presence of clarithromycin-resistant H. pylori correctly. Keywords
Amoxycillin, antimicrobial resistance, clarithromycin, Helicobacter pylori, metronidazole, resistance
Original Submission: 25 May 2004;
Revised Submission: 26 August 2004;
Accepted: 11 December 2004
Clin Microbiol Infect 2005; 11: 307–311
INTRODUCTION Helicobacter pylori causes peptic ulcer and chronic gastritis [1,2], and has also been associated with gastric cancer and mucosal-associated lymphoid tissue lymphoma [3,4]. In Japan, H. pylori is usually eradicated by a triple therapy which includes amoxycillin, clarithromycin and a proton pump inhibitor [5]. Although this treatment is successful in 70–90% of cases [5–7], treatment failure can result from the presence of antimicrobial-resistant strains of H. pylori [8–10]. In such cases, clarithromycin is usually replaced by metronidazole [5,11]. However, infection with resistant strains of H. pylori results in a decreased eradication rate compared to that obtained with Corresponding author and reprint requests: M. Sasatsu, Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192–0392, Japan E-mail: [email protected]
susceptible strains [12–14]. In addition, mixed infections with H. pylori strains of different antibacterial susceptibilities have been reported [15–17]. For these reasons, information regarding the prevalence of antimicrobial-resistant H. pylori strains is important in predicting the therapeutic response. The present study investigated the susceptibilities to clarithromycin, amoxycillin and metronidazole of H. pylori isolates from gastric biopsies taken from two sites, the antrum and corpus, during the period 1995–2001.
MATERIALS AND METHODS Patients H. pylori isolates were obtained from 692 Japanese patients (469 male and 223 female; mean age 51.0 ± 12.1 years, range 12–77 years) who underwent upper endoscopy for evaluation of symptoms related to the upper gastrointestinal tract at Tokyo Medical University Hachioji Medical Center, or at Tokyo Medical University Hospital, Tokyo, Japan, between 1995 and 2001. The diagnoses included chronic gastritis, peptic
2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases
308 Clinical Microbiology and Infection, Volume 11 Number 4, April 2005
ulcer, mucosal-associated lymphoid tissue lymphoma, gastric polyps and gastric cancer. The samples were taken before the use of any eradication therapy. H. pylori infection was diagnosed by culture of gastric biopsy specimens obtained from the antrum and corpus. Informed consent was obtained from all patients. Culture H. pylori was cultured from gastric biopsy specimens on modified Skirrow agar (Nissui Pharmaceutical Co., Tokyo, Japan) under microaerophilic conditions (O2 5%, CO2 10%, N2 85%) for 3–7 days at 37C. H. pylori was identified by oxidase production and API Campy tests (bioMe´rieux, Marcy l’Etoile, France). Isolates were stored in Microbank vials (Pro-Laboratory Diagnostics, Richmond Hill, Ontario, Canada) or brain– heart infusion broth containing glycerol 20% v ⁄ v, either at ) 80C or at ) 198C in liquid nitrogen. Susceptibility testing The susceptibilities of H. pylori isolates to clarithromycin, amoxycillin and metronidazole were tested by the NCCLS agar dilution method [18]. Briefly, H. pylori isolates were subcultured for 3 days as described above, followed by the preparation of a 2 · McFarland standard in sterile saline. Aliquots (5 lL) of the suspension were then spotted on to Mueller–Hinton agar containing horse blood 5% v ⁄ v. MICs were determined after incubation for 3 days. H. pylori isolates were defined as resistant when the MICs of clarithromycin, amoxycillin and metronidazole were ‡ 1, ‡ 1 and ‡ 16 mg ⁄ L, respectively. H. pylori strain ATCC 43504 was used as a quality control organism. Statistical analysis Differences in the annual proportions of patients infected with antimicrobial-resistant H. pylori were tested by the v2 test, with p < 0.05 considered to be statistically significant.
RESULTS Incidence of patients with infection by antibacterial-resistant strains Table 1 shows the overall proportion of patients from whom antimicrobial-resistant H. pylori isolates were obtained, as well as the source of the isolates. The patients were divided into three groups: (1) Ra ⁄ Sb group, containing patients with antimicrobial-resistant H. pylori isolates from the antrum and antimicrobial-susceptible H. pylori isolates from the corpus; (2) Sa ⁄ Rb group, containing patients with antimicrobial-susceptible isolates from the antrum and antimicrobial-resistant isolates from the corpus; and (3) Ra ⁄ Rb group, containing patients with antimicrobial-resistant isolates from both antrum and corpus. Regardless of the above groups, the proportion of patients with clarithromycin-resistant H. pylori
Table 1. Proportions of patients infected with antimicrobial-resistant Helicobacter pylori according to the site of isolation (antrum and ⁄ or corpus) % resistance rate (number of patients) Antimicrobial agent
Ra/Sb
Sa/Rb
Ra/Rb
Clarithromycin Amoxycillin Metronidazole
1.8 (10 ⁄ 542) 0.4 (2 ⁄ 546) 0.2 (1 ⁄ 541)
2.8 (15 ⁄ 542) 0.0 (0 ⁄ 546) 0.6 (3 ⁄ 541)
7.2 (39 ⁄ 542) 0.2 (1 ⁄ 546) 0.7 (4 ⁄ 541)
S, sensitive; R, resistant. Ra ⁄ Sb group, patients with antimicrobial-resistant H. pylori isolates from the antrum and antimicrobial-susceptible H. pylori isolates from the corpus; Sa ⁄ Rb group, patients with antimicrobial-susceptible isolates from the antrum and antimicrobial-resistant isolates from the corpus; Ra ⁄ Rb group, patients with antimicrobial-resistant isolates from both antrum and corpus.
isolates was higher than the proportion of patients with amoxycillin- or metronidazoleresistant isolates. The patients with clarithromycin-resistant H. pylori isolates included 7.2% (39) of patients in the Ra ⁄ Rb group, compared with 1.8% (ten) patients in the Ra ⁄ Sb group, and 2.8% (15) patients in the Sa ⁄ Rb group. Thus, in total, 39.1% (25 ⁄ 64) of the patients with clarithromycinresistant isolates were infected with both clarithromycin-resistant and -susceptible H. pylori isolates. Similarly, two of three patients with amoxycillin-resistant H. pylori isolates, and four of eight patients with metronidazole-resistant H. pylori isolates, yielded both resistant and susceptible isolates. Annual proportion of patients with antimicrobial-resistant H. pylori The annual proportions of patients from whom antimicrobial-resistant H. pylori strains were isolated, from the antrum and ⁄ or the corpus, are shown in Table 2. The annual proportion of patients infected with isolates resistant to amoxycillin or metronidazole was < 5% during 1995– 2001. In contrast, the proportion of patients infected with clarithromycin-resistant H. pylori Table 2. Annual variation in the number of patients from whom antimicrobial-resistant Helicobacter pylori strains were isolated Number (%) of patients with H. pylori resistant to Year
Clarithromycin
Amoxycillin
Metronidazole
1995 1996 1997 1998 1999 2000–2001 Total
10 ⁄ 162 (6.2) 10 ⁄ 143 (7.0) 7 ⁄ 90 (7.8) 7 ⁄ 65 (10.8) 28 ⁄ 163 (17.2) 15 ⁄ 68 (22.1) 77 ⁄ 691 (11.1)
1 ⁄ 161 (0.6) 1 ⁄ 144 (0.7) 0 ⁄ 90 (0.0) 1 ⁄ 65 (1.5) 0 ⁄ 163 (0.0) 0 ⁄ 68 (0.0) 3 ⁄ 691 (0.4)
1 ⁄ 162 (0.6) 2 ⁄ 144 (1.4) 0 ⁄ 90 (0.0) 1 ⁄ 65 (1.5) 7 ⁄ 163 (4.3) 0 ⁄ 66 (0.0) 11 ⁄ 690 (1.6)
2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 11, 307–311
Rimbara et al. Susceptibility of H. pylori to antibacterial agents 309
rose significantly (p < 0.05) from 6.2% (95% CI, 3.4–11.0) in 1995 to 22.1% (95% CI, 13.8–33.3) in 2000–2001. Annual variation in MIC90 Fig. 1 shows the annual variation in MIC90s of clarithromycin, amoxycillin, and metronidazole. Although the MIC90 of clarithromycin for H. pylori isolates during 1995–1998 was < 1 mg ⁄ L, the
MIC90(mg/L)
(a)
64 32 16 8 4 2 1 0.5 0.25 0.125 0.063 0.031 0.016 0.008 1995
1996
1997
1998
1999
2000–2001
MIC90 in 1999 increased to 8 mg ⁄ L, which exceeded the recommended breakpoint of 1 mg ⁄ L. In contrast, the MIC90s of amoxycillin and metronidazole were < 0.125 and 4 mg ⁄ L, respectively, throughout the entire period of the study every year. The MIC50s of clarithromycin, amoxycillin and metronidazole were lower than the level of resistance. Multiresistant H. pylori Multiresistant isolates of H. pylori, i.e., resistant to more than two antimicrobial agents, including clarithromycin, were obtained from three patients, with two of these patients being infected with both clarithromycin- and amoxycillin-resistant H. pylori (clarithromycin and amoxycillin MICs of 32 and 1 mg ⁄ L, and 16 and 1 mg ⁄ L, respectively). The remaining patients yielded H. pylori isolates with clarithromycin and metronidazole MICs of 32 and 64 mg ⁄ L, respectively. Among patients with clarithromycin-resistant H. pylori, the proportion infected with multiresistant H. pylori was 3.9% (3 ⁄ 77).
MIC90(mg/L)
(b)
64 32 16 8 4 2 1 0.5 0.25 0.125 0.063 0.031 0.016 0.008
DISCUSSION
1995
1996
1997
1995
1996
1997
1998
1999
2000–2001
1998
1999
2000–2001
64 32 16 8 4 2 1 0.5 0.25 0.125 0.063 0.031 0.016 0.008
MIC90(mg/L)
(c)
Year
Fig. 1. Annual variation in MIC90s of (a) clarithromycin, (b) amoxycillin and (c) metronidazole for Helicobacter pylori isolates between 1995 and 2001. Squares, H. pylori isolates from the antrum; triangles, H. pylori isolates from the corpus.
This study investigated the susceptibilities of H. pylori isolates from the antrum and corpus to various antimicrobial agents, and the proportion of patients infected with resistant H. pylori, during the period 1995–2001. Among the patients with clarithromycin-resistant H. pylori, 39% yielded both clarithromycin-resistant and clarithromycinsusceptible H. pylori isolates. Kim et al. [16] reported the possibility that genomic alterations in pre-existing strains result in the simultaneous presence of antimicrobial-susceptible and -resistant strains in the same patient. Although no genotypic analysis was performed, it is possible that this explanation could account for the observations in the present study. Furthermore, Kawai et al. [19] reported that mixed infection by clarithromycin-susceptible and -resistant H. pylori strains could prevent eradication of H. pylori by the triple therapy and result in the selection of the resistant strains. Therefore, the present results emphasise the need to test the susceptibility of bacteria isolated from both the gastric antrum and corpus of the same patient in order to diagnose clarithromycin-resistant H. pylori infection correctly.
2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 11, 307–311
310 Clinical Microbiology and Infection, Volume 11 Number 4, April 2005
The data indicated that the proportion of patients with clarithromycin-resistant H. pylori has increased markedly in Japan, especially when compared to the proportion of patients with amoxycillin- and metronidazole-resistant H. pylori. Previous studies in Japan [20,21] have also reported an increase in clarithromycin-resistant H. pylori in recent years. Clarithromycin resistance in H. pylori has been associated with point mutations in domain V of the 23S rRNA gene, which is the binding site of macrolides, including clarithromycin [22–24]. Macrolides are used widely for the treatment of infections caused by Mycoplasma spp. [25] and Gram-positive bacteria [26], and are also used currently in Japan for the treatment of diffuse panbronchiolitis caused by Pseudomonas aeruginosa [27]. It has been reported in Japan since 1998 that eradication therapy involving clarithromycin is a safe and effective regimen [7]. As a result, clinical use of macrolides, including clarithromycin, has increased significantly in recent years. The significant increase in the MIC90 of clarithromycin for H. pylori between 1998 and 1999 is therefore probably associated with the increased use of clarithromycin in Japan. The total proportion of metronidazole-resistant H. pylori isolates in the present study was 1.6%, which is lower than the proportions of 12.4% and 9% reported by Kato et al. [21] and Perez et al. [20], respectively. However, Kato et al. [21] also showed the presence of regional differences in the proportions of metronidazole-resistant isolates in Japan, and these regional differences might also account for the relatively low proportion of metronidazoleresistant H. pylori isolates in the present study. Multiresistant H. pylori isolates were also observed in the present study, and all such multiresistant H. pylori isolates were resistant to clarithromycin. Since the frequency of amoxycillin- and metronidazole-resistant H. pylori was far lower than that of clarithromycin-resistant H. pylori, it seems likely that multiresistance results from the acquisition of amoxycillin and metronidazole resistance by clarithromycinresistant H. pylori isolates. In conclusion, the present study demonstrated an increased frequency of clarithromycin-resistant H. pylori isolates and the presence of multiresistant H. pylori isolates in Japan. These results suggest a need to develop alternatives to the clarithromycin-containing eradication therapy used currently.
ACKNOWLEDGEMENTS We thank A. Tomizawa for her contribution to the study. We also thank Y. Suzuki and N. Hasegawa for technical support. This work was supported by a grant for private universities from the Ministry of Education, Science, Sports, and Culture, Japan, and by a grant from the Promotion and Mutual Aid Corporation for Private Schools of Japan.
REFERENCES 1. Graham DY, Lew GM, Klein PD et al. Effect of treatment of Helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer. A randomized, controlled study. Ann Intern Med 1992; 116: 705–708. 2. Kokkola A, Sipponen P, Rautelin H et al. The effect of Helicobacter pylori eradication on the natural course of atrophic gastritis with dysplasia. Aliment Pharmacol Ther 2002; 16: 515–520. 3. Bayerdorffer E, Neubauer A, Rudolph B et al. Regression of primary gastric lymphoma of mucosa-associated lymphoid tissue type after cure of Helicobacter pylori infection. MALT Lymphoma Study Group. Lancet 1995; 345: 1591– 1594. 4. Uemura N, Okamoto S, Yamamoto S et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 2001; 345: 784–789. 5. Asaka M, Satoh K, Sugano K et al. Guidelines in the management of Helicobacter pylori infection in Japan. Helicobacter 2001; 6: 177–186. 6. Dore MP, Piana A, Carta M et al. Amoxycillin resistance is one reason for failure of amoxycillin–omeprazole treatment of Helicobacter pylori infection. Aliment Pharmacol Ther 1998; 12: 635–639. 7. Suzuki J, Mine T, Kobayasi I, Fujita T. Assessment of a new triple agent regimen for the eradication of Helicobacter pylori and the nature of H. pylori resistance to this therapy in Japan. Helicobacter 1998; 3: 59–63. 8. Poon SK, Chang CS, Su J et al. Primary resistance to antibiotics and its clinical impact on the efficacy of Helicobacter pylori lansoprazole-based triple therapies. Aliment Pharmacol Ther 2002; 16: 291–296. 9. Queiroz DM, Dani R, Silva LD et al. Factors associated with treatment failure of Helicobacter pylori infection in a developing country. J Clin Gastroenterol 2002; 35: 315–320. 10. Broutet N, Tchamgoue S, Pereira E, Lamouliatte H, Salamon R, Megraud F. Risk factors for failure of Helicobacter pylori therapy—results of an individual data analysis of 2751 patients. Aliment Pharmacol Ther 2003; 17: 99–109. 11. Murakami K, Sato R, Okimoto T et al. Efficacy of triple therapy comprising rabeprazole, amoxicillin and metronidazole for second-line Helicobacter pylori eradication in Japan, and the influence of metronidazole resistance. Aliment Pharmacol Ther 2003; 17: 119–123. 12. Megraud F, Lehn N, Lind T et al. Antimicrobial susceptibility testing of Helicobacter pylori in a large multicenter trial: the MACH 2 study. Antimicrob Agents Chemother 1999; 43: 2747–2752. 13. Adamek RJ, Suerbaum S, Pfaffenbach B, Opferkuch W. Primary and acquired Helicobacter pylori resistance to clarithromycin, metronidazole, and amoxicillin—influence on treatment outcome. Am J Gastroenterol 1998; 93: 386–389.
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14. Tankovic J, Lamarque D, Lascols C, Soussy CJ, Delchier JC. Impact of Helicobacter pylori resistance to clarithromycin on the efficacy of the omeprazole–amoxicillin–clarithromycin therapy. Aliment Pharmacol Ther 2001; 15: 707–713. 15. Wong BC, Wang WH, Berg DE et al. High prevalence of mixed infections by Helicobacter pylori in Hong Kong: metronidazole sensitivity and overall genotype. Aliment Pharmacol Ther 2001; 15: 493–503. 16. Kim JJ, Kim JG, Kwon DH. Mixed-infection of antibiotic susceptible and resistant Helicobacter pylori isolates in a single patient and underestimation of antimicrobial susceptibility testing. Helicobacter 2003; 8: 202–206. 17. Ikezawa K, Kashimura H, Kojima M et al. Pretreatment antimicrobial susceptibilities of paired gastric Helicobacter pylori isolates: antrum versus corpus. Helicobacter 1999; 4: 218–221. 18. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing; 12th informational supplement. M100-S12. Villanova, PA: NCCLS, 2002. 19. Kawai T, Kawakami K, Kudo T et al. Correlation between serum pepsinogen levels and gastric mucosal histological findings before and after Helicobacter pylori eradication therapy. Digest Endosc 2004; 16: 122–128. 20. Perez Aldana L, Kato M, Nakagawa S et al. The relationship between consumption of antimicrobial agents and the prevalence of primary Helicobacter pylori resistance. Helicobacter 2002; 7: 306–309.
21. Kato M, Yamaoka Y, Kim JJ et al. Regional differences in metronidazole resistance and increasing clarithromycin resistance among Helicobacter pylori isolates from Japan. Antimicrob Agents Chemother 2000; 44: 2214–2216. 22. Stone GG, Shortridge D, Flamm RK et al. Identification of a 23S rRNA gene mutation in clarithromycin-resistant Helicobacter pylori. Helicobacter 1996; 1: 227–228. 23. Versalovic J, Shortridge D, Kibler K et al. Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori. Antimicrob Agents Chemother 1996; 40: 477–480. 24. Taylor DE, Ge Z, Purych D, Lo T, Hiratsuka K. Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations. Antimicrob Agents Chemother 1997; 41: 2621–2628. 25. Hardy RD, Rios AM, Chavez-Bueno S et al. Antimicrobial and immunologic activities of clarithromycin in a murine model of Mycoplasma pneumoniae-induced pneumonia. Antimicrob Agents Chemother 2003; 47: 1614–1620. 26. Langtry HD, Brogden RN. Clarithromycin. A review of its efficacy in the treatment of respiratory tract infections in immunocompetent patients. Drugs 1997; 53: 973–1004. 27. Yanagihara K, Tomono K, Imamura Y et al. Effect of clarithromycin on chronic respiratory infection caused by Pseudomonas aeruginosa with biofilm formation in an experimental murine model. J Antimicrob Chemother 2002; 49: 867–870.
2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 11, 307–311
10.1111/j.1469-0691.2005.01099.x
Susceptibilities to clarithromycin, amoxycillin and metronidazole of Helicobacter pylori isolates from the antrum and corpus in Tokyo, Japan, 1995–2001 E. Rimbara1, N. Noguchi1, M. Tanabe1, T. Kawai2, Y. Matsumoto3 and M. Sasatsu1 1
Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Endoscopy Center, Tokyo Medical University and 3Hachioji Pharmaceutical Center Pharmacy, Hachioji, Tokyo, Japan
2
ABSTRACT The aim of this study was to determine the susceptibilities to clarithromycin, amoxycillin and metronidazole of Helicobacter pylori isolates from the antrum and corpus of Japanese patients examined during the period 1995–2001. There was an increase, from 6.2% in 1995 to 22.1% in 2000–2001, in the proportion of patients infected with clarithromycin-resistant H. pylori. Of patients infected with clarithromycin-resistant H. pylori, 39.1% were infected with both clarithromycin-susceptible and -resistant H. pylori. Furthermore, the MIC90 of clarithromycin for H. pylori rose from < 1 mg ⁄ L in 1995– 1998 to 8 mg ⁄ L in 1999. In contrast, the MIC90s of amoxycillin and metronidazole were £ 0.125 and 4 mg ⁄ L, respectively, throughout the study period. The results showed that, while most H. pylori isolates were susceptible to amoxycillin and metronidazole, resistance to clarithromycin among H. pylori isolates increased markedly in Japan during 1995–2001. The results also indicated a need to test the susceptibility of H. pylori isolates from more than two samples obtained from two different sites in the stomach of a single patient in order to diagnose the presence of clarithromycin-resistant H. pylori correctly. Keywords
Amoxycillin, antimicrobial resistance, clarithromycin, Helicobacter pylori, metronidazole, resistance
Original Submission: 25 May 2004;
Revised Submission: 26 August 2004;
Accepted: 11 December 2004
Clin Microbiol Infect 2005; 11: 307–311
INTRODUCTION Helicobacter pylori causes peptic ulcer and chronic gastritis [1,2], and has also been associated with gastric cancer and mucosal-associated lymphoid tissue lymphoma [3,4]. In Japan, H. pylori is usually eradicated by a triple therapy which includes amoxycillin, clarithromycin and a proton pump inhibitor [5]. Although this treatment is successful in 70–90% of cases [5–7], treatment failure can result from the presence of antimicrobial-resistant strains of H. pylori [8–10]. In such cases, clarithromycin is usually replaced by metronidazole [5,11]. However, infection with resistant strains of H. pylori results in a decreased eradication rate compared to that obtained with Corresponding author and reprint requests: M. Sasatsu, Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192–0392, Japan E-mail: [email protected]
susceptible strains [12–14]. In addition, mixed infections with H. pylori strains of different antibacterial susceptibilities have been reported [15–17]. For these reasons, information regarding the prevalence of antimicrobial-resistant H. pylori strains is important in predicting the therapeutic response. The present study investigated the susceptibilities to clarithromycin, amoxycillin and metronidazole of H. pylori isolates from gastric biopsies taken from two sites, the antrum and corpus, during the period 1995–2001.
MATERIALS AND METHODS Patients H. pylori isolates were obtained from 692 Japanese patients (469 male and 223 female; mean age 51.0 ± 12.1 years, range 12–77 years) who underwent upper endoscopy for evaluation of symptoms related to the upper gastrointestinal tract at Tokyo Medical University Hachioji Medical Center, or at Tokyo Medical University Hospital, Tokyo, Japan, between 1995 and 2001. The diagnoses included chronic gastritis, peptic
2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases
308 Clinical Microbiology and Infection, Volume 11 Number 4, April 2005
ulcer, mucosal-associated lymphoid tissue lymphoma, gastric polyps and gastric cancer. The samples were taken before the use of any eradication therapy. H. pylori infection was diagnosed by culture of gastric biopsy specimens obtained from the antrum and corpus. Informed consent was obtained from all patients. Culture H. pylori was cultured from gastric biopsy specimens on modified Skirrow agar (Nissui Pharmaceutical Co., Tokyo, Japan) under microaerophilic conditions (O2 5%, CO2 10%, N2 85%) for 3–7 days at 37C. H. pylori was identified by oxidase production and API Campy tests (bioMe´rieux, Marcy l’Etoile, France). Isolates were stored in Microbank vials (Pro-Laboratory Diagnostics, Richmond Hill, Ontario, Canada) or brain– heart infusion broth containing glycerol 20% v ⁄ v, either at ) 80C or at ) 198C in liquid nitrogen. Susceptibility testing The susceptibilities of H. pylori isolates to clarithromycin, amoxycillin and metronidazole were tested by the NCCLS agar dilution method [18]. Briefly, H. pylori isolates were subcultured for 3 days as described above, followed by the preparation of a 2 · McFarland standard in sterile saline. Aliquots (5 lL) of the suspension were then spotted on to Mueller–Hinton agar containing horse blood 5% v ⁄ v. MICs were determined after incubation for 3 days. H. pylori isolates were defined as resistant when the MICs of clarithromycin, amoxycillin and metronidazole were ‡ 1, ‡ 1 and ‡ 16 mg ⁄ L, respectively. H. pylori strain ATCC 43504 was used as a quality control organism. Statistical analysis Differences in the annual proportions of patients infected with antimicrobial-resistant H. pylori were tested by the v2 test, with p < 0.05 considered to be statistically significant.
RESULTS Incidence of patients with infection by antibacterial-resistant strains Table 1 shows the overall proportion of patients from whom antimicrobial-resistant H. pylori isolates were obtained, as well as the source of the isolates. The patients were divided into three groups: (1) Ra ⁄ Sb group, containing patients with antimicrobial-resistant H. pylori isolates from the antrum and antimicrobial-susceptible H. pylori isolates from the corpus; (2) Sa ⁄ Rb group, containing patients with antimicrobial-susceptible isolates from the antrum and antimicrobial-resistant isolates from the corpus; and (3) Ra ⁄ Rb group, containing patients with antimicrobial-resistant isolates from both antrum and corpus. Regardless of the above groups, the proportion of patients with clarithromycin-resistant H. pylori
Table 1. Proportions of patients infected with antimicrobial-resistant Helicobacter pylori according to the site of isolation (antrum and ⁄ or corpus) % resistance rate (number of patients) Antimicrobial agent
Ra/Sb
Sa/Rb
Ra/Rb
Clarithromycin Amoxycillin Metronidazole
1.8 (10 ⁄ 542) 0.4 (2 ⁄ 546) 0.2 (1 ⁄ 541)
2.8 (15 ⁄ 542) 0.0 (0 ⁄ 546) 0.6 (3 ⁄ 541)
7.2 (39 ⁄ 542) 0.2 (1 ⁄ 546) 0.7 (4 ⁄ 541)
S, sensitive; R, resistant. Ra ⁄ Sb group, patients with antimicrobial-resistant H. pylori isolates from the antrum and antimicrobial-susceptible H. pylori isolates from the corpus; Sa ⁄ Rb group, patients with antimicrobial-susceptible isolates from the antrum and antimicrobial-resistant isolates from the corpus; Ra ⁄ Rb group, patients with antimicrobial-resistant isolates from both antrum and corpus.
isolates was higher than the proportion of patients with amoxycillin- or metronidazoleresistant isolates. The patients with clarithromycin-resistant H. pylori isolates included 7.2% (39) of patients in the Ra ⁄ Rb group, compared with 1.8% (ten) patients in the Ra ⁄ Sb group, and 2.8% (15) patients in the Sa ⁄ Rb group. Thus, in total, 39.1% (25 ⁄ 64) of the patients with clarithromycinresistant isolates were infected with both clarithromycin-resistant and -susceptible H. pylori isolates. Similarly, two of three patients with amoxycillin-resistant H. pylori isolates, and four of eight patients with metronidazole-resistant H. pylori isolates, yielded both resistant and susceptible isolates. Annual proportion of patients with antimicrobial-resistant H. pylori The annual proportions of patients from whom antimicrobial-resistant H. pylori strains were isolated, from the antrum and ⁄ or the corpus, are shown in Table 2. The annual proportion of patients infected with isolates resistant to amoxycillin or metronidazole was < 5% during 1995– 2001. In contrast, the proportion of patients infected with clarithromycin-resistant H. pylori Table 2. Annual variation in the number of patients from whom antimicrobial-resistant Helicobacter pylori strains were isolated Number (%) of patients with H. pylori resistant to Year
Clarithromycin
Amoxycillin
Metronidazole
1995 1996 1997 1998 1999 2000–2001 Total
10 ⁄ 162 (6.2) 10 ⁄ 143 (7.0) 7 ⁄ 90 (7.8) 7 ⁄ 65 (10.8) 28 ⁄ 163 (17.2) 15 ⁄ 68 (22.1) 77 ⁄ 691 (11.1)
1 ⁄ 161 (0.6) 1 ⁄ 144 (0.7) 0 ⁄ 90 (0.0) 1 ⁄ 65 (1.5) 0 ⁄ 163 (0.0) 0 ⁄ 68 (0.0) 3 ⁄ 691 (0.4)
1 ⁄ 162 (0.6) 2 ⁄ 144 (1.4) 0 ⁄ 90 (0.0) 1 ⁄ 65 (1.5) 7 ⁄ 163 (4.3) 0 ⁄ 66 (0.0) 11 ⁄ 690 (1.6)
2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 11, 307–311
Rimbara et al. Susceptibility of H. pylori to antibacterial agents 309
rose significantly (p < 0.05) from 6.2% (95% CI, 3.4–11.0) in 1995 to 22.1% (95% CI, 13.8–33.3) in 2000–2001. Annual variation in MIC90 Fig. 1 shows the annual variation in MIC90s of clarithromycin, amoxycillin, and metronidazole. Although the MIC90 of clarithromycin for H. pylori isolates during 1995–1998 was < 1 mg ⁄ L, the
MIC90(mg/L)
(a)
64 32 16 8 4 2 1 0.5 0.25 0.125 0.063 0.031 0.016 0.008 1995
1996
1997
1998
1999
2000–2001
MIC90 in 1999 increased to 8 mg ⁄ L, which exceeded the recommended breakpoint of 1 mg ⁄ L. In contrast, the MIC90s of amoxycillin and metronidazole were < 0.125 and 4 mg ⁄ L, respectively, throughout the entire period of the study every year. The MIC50s of clarithromycin, amoxycillin and metronidazole were lower than the level of resistance. Multiresistant H. pylori Multiresistant isolates of H. pylori, i.e., resistant to more than two antimicrobial agents, including clarithromycin, were obtained from three patients, with two of these patients being infected with both clarithromycin- and amoxycillin-resistant H. pylori (clarithromycin and amoxycillin MICs of 32 and 1 mg ⁄ L, and 16 and 1 mg ⁄ L, respectively). The remaining patients yielded H. pylori isolates with clarithromycin and metronidazole MICs of 32 and 64 mg ⁄ L, respectively. Among patients with clarithromycin-resistant H. pylori, the proportion infected with multiresistant H. pylori was 3.9% (3 ⁄ 77).
MIC90(mg/L)
(b)
64 32 16 8 4 2 1 0.5 0.25 0.125 0.063 0.031 0.016 0.008
DISCUSSION
1995
1996
1997
1995
1996
1997
1998
1999
2000–2001
1998
1999
2000–2001
64 32 16 8 4 2 1 0.5 0.25 0.125 0.063 0.031 0.016 0.008
MIC90(mg/L)
(c)
Year
Fig. 1. Annual variation in MIC90s of (a) clarithromycin, (b) amoxycillin and (c) metronidazole for Helicobacter pylori isolates between 1995 and 2001. Squares, H. pylori isolates from the antrum; triangles, H. pylori isolates from the corpus.
This study investigated the susceptibilities of H. pylori isolates from the antrum and corpus to various antimicrobial agents, and the proportion of patients infected with resistant H. pylori, during the period 1995–2001. Among the patients with clarithromycin-resistant H. pylori, 39% yielded both clarithromycin-resistant and clarithromycinsusceptible H. pylori isolates. Kim et al. [16] reported the possibility that genomic alterations in pre-existing strains result in the simultaneous presence of antimicrobial-susceptible and -resistant strains in the same patient. Although no genotypic analysis was performed, it is possible that this explanation could account for the observations in the present study. Furthermore, Kawai et al. [19] reported that mixed infection by clarithromycin-susceptible and -resistant H. pylori strains could prevent eradication of H. pylori by the triple therapy and result in the selection of the resistant strains. Therefore, the present results emphasise the need to test the susceptibility of bacteria isolated from both the gastric antrum and corpus of the same patient in order to diagnose clarithromycin-resistant H. pylori infection correctly.
2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 11, 307–311
310 Clinical Microbiology and Infection, Volume 11 Number 4, April 2005
The data indicated that the proportion of patients with clarithromycin-resistant H. pylori has increased markedly in Japan, especially when compared to the proportion of patients with amoxycillin- and metronidazole-resistant H. pylori. Previous studies in Japan [20,21] have also reported an increase in clarithromycin-resistant H. pylori in recent years. Clarithromycin resistance in H. pylori has been associated with point mutations in domain V of the 23S rRNA gene, which is the binding site of macrolides, including clarithromycin [22–24]. Macrolides are used widely for the treatment of infections caused by Mycoplasma spp. [25] and Gram-positive bacteria [26], and are also used currently in Japan for the treatment of diffuse panbronchiolitis caused by Pseudomonas aeruginosa [27]. It has been reported in Japan since 1998 that eradication therapy involving clarithromycin is a safe and effective regimen [7]. As a result, clinical use of macrolides, including clarithromycin, has increased significantly in recent years. The significant increase in the MIC90 of clarithromycin for H. pylori between 1998 and 1999 is therefore probably associated with the increased use of clarithromycin in Japan. The total proportion of metronidazole-resistant H. pylori isolates in the present study was 1.6%, which is lower than the proportions of 12.4% and 9% reported by Kato et al. [21] and Perez et al. [20], respectively. However, Kato et al. [21] also showed the presence of regional differences in the proportions of metronidazole-resistant isolates in Japan, and these regional differences might also account for the relatively low proportion of metronidazoleresistant H. pylori isolates in the present study. Multiresistant H. pylori isolates were also observed in the present study, and all such multiresistant H. pylori isolates were resistant to clarithromycin. Since the frequency of amoxycillin- and metronidazole-resistant H. pylori was far lower than that of clarithromycin-resistant H. pylori, it seems likely that multiresistance results from the acquisition of amoxycillin and metronidazole resistance by clarithromycinresistant H. pylori isolates. In conclusion, the present study demonstrated an increased frequency of clarithromycin-resistant H. pylori isolates and the presence of multiresistant H. pylori isolates in Japan. These results suggest a need to develop alternatives to the clarithromycin-containing eradication therapy used currently.
ACKNOWLEDGEMENTS We thank A. Tomizawa for her contribution to the study. We also thank Y. Suzuki and N. Hasegawa for technical support. This work was supported by a grant for private universities from the Ministry of Education, Science, Sports, and Culture, Japan, and by a grant from the Promotion and Mutual Aid Corporation for Private Schools of Japan.
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2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 11, 307–311