This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nahar, S.
Right arrow Articles by Rahman, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nahar, S.
Right arrow Articles by Rahman, M.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, October 2004, p. 4856-4858, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4856-4858.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Antimicrobial Susceptibility of Helicobacter pylori Strains Isolated in Bangladesh

Shamsun Nahar,1 Asish K. Mukhopadhyay,2 Rasel Khan,1 Mian Mashhud Ahmad,3 Simanti Datta,2 Santanu Chattopadhyay,2 Swapan Chandra Dhar,3 Shafiqul Alam Sarker,1 Lars Engstrand,4 Douglas E. Berg,5 G. Balakrish Nair,1 and Motiur Rahman1*

International Centre for Diarrhoeal Disease Research,1 Dhaka Medical College Hospital, Dhaka, Bangladesh,3 National Institute of Cholera and Enteric Disease, Calcutta, India,2 SMI, Stockholm, Sweden,4 Departments of Molecular Microbiology and Genetics, Washington University School of Medicine, St. Louis, Missouri5

Received 25 March 2004/ Returned for modification 28 April 2004/ Accepted 18 May 2004


arrow
ABSTRACT
 
Antimicrobial susceptibility of 120 Helicobacter pylori isolates to metronidazole, tetracycline, clarithromycin, and amoxicillin was determined, and 77.5, 15, 10, and 6.6% of the isolates, respectively, were resistant. Only rdxA inactivation and both rdxA and frxA inactivation were responsible for metronidazole resistance in 66% (8 of 12) and 33% (4 of 12) of the isolates, respectively.


arrow
TEXT
 
Eradication of Helicobacter pylori infection by treatment with two antimicrobial agents (clarithromycin and amoxicillin or metronidazole) and a proton pump inhibitor is recommended by various consensus groups (10, 16, 20). Antimicrobial resistance in H. pylori is a growing problem as it is the most important factor in determining treatment outcome. The prevalence of antimicrobial resistance varies with geographical regions (3, 25). Metronidazole resistance in H. pylori has been shown to be due to mutation in rdxA; mutation in frxA has also been shown to be associated with metronidazole resistance (11, 12, 23). In Bangladesh, the prevalences of H. pylori infection among infants, children, and adults are 61, 84, and 92%, respectively (1, 21, 22); however, information on antimicrobial susceptibility to commonly used drugs in H. pylori treatment is lacking. This study was conducted to evaluate (i) the prevalence of primary antibiotic resistance to commonly used antimicrobial agents and (ii) the genetic basis for metronidazole resistance in H. pylori isolates from Bangladesh.

Consecutive patients attending the Gastroenterology Department of Dhaka Medical College Hospital for upper gastrointestinal endoscopy were enrolled during 1999 to 2001. Diagnosis of peptic ulcer (PU) and non-ulcer dyspepsia (NUD) or gastritis was based on endoscopic examination of the stomach and duodenum. Biopsy samples were taken from each patient for culture.

Bacteria were grown in brain heart infusion agar with 7% sheep blood and incubated at 37°C in 5% O2, 10% CO2, and 85% N2 for 3 to 6 days. The MICs of amoxicillin, clarithromycin, metronidazole, and tetracycline for the isolates were determined by the agar dilution method as described elsewhere (18, 19). All tests were repeated twice, and H. pylori 26695 was used as a control. ß-Lactamase production was tested by the chromogenic cephalosporin method (6). The molecular mechanism of susceptibility and resistance to metronidazole was studied in 12 isolates. Metronidazole-susceptible (Mtzs) isolates were further studied (by inactivation of rdxA alone or rdxA and frxA for conversion into an Mtzr phenotype) by transformation of Mtzs isolates with plasmids pBS-rdxA-cam (rdxA::cat) and pBS-frxA-kan (frxA::kan) as described earlier (11, 12).

A total of 278 consecutive patients between 15 and 78 years of age were enrolled, and among them, 72.7% (202 patients) were male and 27.3% (76 patients) were female. Among the patients, 162 had PU and 116 had NUD and 62.6% (174 of 278) were culture positive for H. pylori. Among the culture-positive patients, 121 (69.5%) were male and 53 (30.4%) were female and 112 (64.3%) had PU and 62 (35.6%) had NUD. Of the 174 isolates, a total of 120 were available for antimicrobial susceptibility testing and 73.3% (88 of 120) and 26.6% (32 of 120) were from PU and NUD patients, respectively. Among the isolates, 77.5% (93 of 120), 15% (18 of 120), 10% (12 of 120), and 6.6% (8 of 120) were resistant to metronidazole, tetracycline, clarithromycin, and amoxicillin, respectively (Table 1). The range and distribution of MICs for the isolates are shown in Fig. 1. All amoxicillin-resistant isolates were ß-lactamase negative. Antimicrobial susceptibilities of the isolates collected from patients with PU and NUD and males and females were compared, and no significant difference (P ≤ 0.05) in antimicrobial resistance was observed among these groups (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Antibiotic susceptibility of 120 H. pylori strains isolated during 1999 to 2001 from Dhaka, Bangladesh



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 1. Distribution of MICs of clarithromycin (a), tetracycline (b), metronidazole (c), and amoxicillin (d) for 120 H. pylori isolates. The numbers above the bars represent the numbers of the isolates for which the particular MIC applies.

Inactivation of only rdxA was sufficient to confer the Mtzr phenotype in 66% (8 of 12) of isolates, 33% (4 of 12) of isolates were Mtzs, and inactivation of only frxA had little effect on Mtzs of all 12 isolates. Subsequent frxA inactivation of all rdxA-deficient strains increased the MIC of metronidazole from 16 µg/ml to 32 µg/ml for the eight strains which became resistant after only rdxA inactivation, and four strains which were sensitive after only rdxA inactivation reverted to the Mtzr phenotype (MIC, 32 µg/ml).

Resistance to metronidazole was the most common type of resistance, with worldwide rates of 10 to 90% (3, 25). The high prevalence (77%) of metronidazole resistance in Bangladesh might be due to frequent use of metronidazole for other intestinal and gynecological problems. Previous use of metronidazole has been shown to be associated with H. pylori resistance to this antimicrobial agent (17). Two types of Mtzs H. pylori were isolated in the present study: type I, requiring only inactivation of rdxA to become resistant; and type II, requiring inactivation of both rdxA and frxA to become resistant. Only frxA inactivation did not have any role in metronidazole resistance, as only subsequent inactivation of frxA in rdxA-inactivated isolates reverted from the Mtzs phenotype to the Mtzr phenotype and increased the MIC for the Mtzr phenotype. This is in contrast to the findings of Kwon et al., who interpreted that the resistant phenotype can obtained by inactivation either of frxA or rdxA (13). Thus, resistance to metronidazole in H. pylori is mainly due to mutation in the rdxA gene and results from de novo mutation in the resident rdxA gene, rather than lateral transfer of a mutant rdxA gene.

The reported prevalence of primary resistance to clarithromycin ranges between 0 and 15% in most countries (3, 25). Around 10% of the isolates in the present study were clarithromycin resistant. In Bangladesh, clarithromycin was introduced in the late 1990s, and it has been widely used for eradication of H. pylori. Previous use of macrolides has been shown to be associated with H. pylori resistance to clarithromycin (17).

Amoxicillin resistance was not considered important until recently identified in the United States, Canada, and Italy (7, 8). Amoxicillin is one of the most commonly used antimicrobial agents in Bangladesh in recent years. Although 6.6% of the isolates were resistant, none was positive for ß-lactamase. Amoxicillin resistance develops due to structural alterations in one of the penicillin-binding proteins (4, 5, 9) or changes in other proteins involved in cell wall synthesis (2, 15, 26), and the resistant phenotype may be lost due to freezing or storage. All isolates tested in the present study were frozen at least once, and the low prevalence of the resistance phenotype may be due to loss during storage. Primary resistance to tetracycline ranges between 5 and 59% in Asian countries (14, 24, 27). Around 15% of the isolates in the present study were tetracycline resistant, which is in agreement with an earlier finding from this region.

Therefore, it is reasonable to conclude that in our geographical area, antibiotic resistance is an emerging problem for the treatment of H. pylori-infected patients. The present study also demonstrates the need for continuous monitoring of the antimicrobial susceptibility in H. pylori for determination of optimal treatment regimens.


arrow
ACKNOWLEDGMENTS
 
This study was conducted at the ICDDR, B: Centre for Health and Population Research with the support of SIDA agreement no 2001-3970, component 75000255/A/7500260.

ICDDR, B acknowledges with gratitude the commitment of SIDA to the Centre's research efforts.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory Sciences Division, ICDDR, B, GPO Box-128, Dhaka-1000, Bangladesh. Phone: 871751-60. Fax: 880-2-872529/880-2-883116. E-mail: motiur{at}icddrb.org. Back


arrow
REFERENCES
 
    1
  1. Ahmad, M. M., M. Rahman, A. K. Rumi, S. Islam, F. Huq, M. F. Chowdhury, F. Jinnah, M. G. Morshed, M. S. Hassan, A. K. Khan, and M. Hasan. 1997. Prevalence of Helicobacter pylori in asymptomatic population—a pilot serological study in Bangladesh. J. Epidemiol. 7:251-254.[Medline]
    2. 2
  2. Costa, C. S., and D. N. Anton. 1993. Round-cell mutants of Salmonella typhimurium produced by transposition mutagenesis: lethality of rodA and mre mutations. Mol. Gen. Genet. 236:387-394.[CrossRef][Medline]
    3. 3
  3. Debets-Ossenkopp, Y. J., A. J. Herscheid, R. G. Pot, E. J. Kuipers, J. G. Kusters, and C. M. Vandenbrouck-Grauls. 1999. Prevalence of Helicobacter pylori resistance to metronidazole, clarithromycin, amoxycillin, tetracycline and trovafloxacin in The Netherlands. J. Antimicrob. Chemother. 43:511-515.[Abstract/Free Full Text]
    4. 4
  4. DeLoney, C. R., and N. L. Schiller. 2000. Characterization of an in vitro-selected amoxicillin-resistant strain of Helicobacter pylori. Antimicrob. Agents Chemother. 44:3368-3373.[Abstract/Free Full Text]
    5. 5
  5. Dore, M. P., D. Y. Graham, and A. R. Sepulveda. 1999. Different penicillin-binding protein profiles in amoxicillin-resistant Helicobacter pylori. Helicobacter 4:154-161.[CrossRef][Medline]
    6. 6
  6. Dore, M. P., M. S. Osato, G. Realdi, I. Mura, D. Y. Graham, and A. R. Sepulveda. 1999. Amoxycillin tolerance in Helicobacter pylori. J. Antimicrob. Chemother. 43:47-54.[Abstract/Free Full Text]
    7. 7
  7. Dore, M. P., A. R. Sepulveda, I. Mura, G. Realdi, M. S. Osato, and D. Y. Graham. 1997. Explanation for variability of omeprazole amoxycillin therapy? Tolerance of H. pylori to amoxycillin. Gastroenterology 112:A105.
    8. 8
  8. Fedorak, R., A. Archambault, R. Flamm, M. Osato, and D. Stamle. 1997. Antimicrobial susceptibility of H. pylori in Canada to three key antibiotics: metronidazole, clarithromycin, and amoxicillin. Gastroenterology 112:A115.
    9. 9
  9. Gerrits, M. M., D. Schuijffel, A. A. van Zwet, E. J. Kuipers, C. M. Vandenbroucke-Grauls, and J. G. Kusters. 2002. Alterations in penicillin-binding protein 1A confer resistance to ß-lactam antibiotics in Helicobacter pylori. Antimicrob. Agents Chemother. 46:2229-2233.[Abstract/Free Full Text]
    10. 10
  10. Graham, D. Y., W. A. de Boer, and G. N. Tytgat. 1996. Choosing the best anti-Helicobacter pylori therapy: effect of antimicrobial resistance. Am. J. Gastroenterol. 91:1072-1076.[Medline]
    11. 11
  11. Jeong, J.-Y., A. K. Mukhopadhyay, D. Dailidiene, Y. Wang, B. Velapatino, R. H. Gilman, A. J. Parkinson, G. B. Nair, B. C. Wong, S. K. Lam, R. Mistry, I. Segal, Y. Yuan, H. Gao, T. Alarcon, M. L. Brea, Y. Ito, D. Kersulyte, H. K. Lee, Y. Gong, A. Goodwin, P. S. Hoffman, and D. E. Berg. 2000. Sequential inactivation of rdxA (HP0954) and frxA (HP0642) nitroreductase genes causes moderate and high-level metronidazole resistance in Helicobacter pylori. J. Bacteriol. 182:5082-5090.[Abstract/Free Full Text]
    12. 12
  12. Jeong, J.-Y., A. K. Mukhopadhyay, J. K. Akada, D. Dailidiene, P. S. Hoffman, and D. E. Berg. 2001. Roles of FrxA and RdxA nitroreductases of Helicobacter pylori in susceptibility and resistance to metronidazole. J. Bacteriol. 183:5155-5162.[Abstract/Free Full Text]
    13. 13
  13. Kwon, D. H., F. A. El-Zaatari, M. Kato, M. S. Osato, R. Reddy, Y. Yamaoka, and D. Y. Graham. 2000. Analysis of rdxA and involvement of additional genes encoding NAD(P)H flavin oxidoreductase (FrxA) and ferredoxin-like protein (FdxB) in metronidazole resistance of Helicobacter pylori. Antimicrob. Agents Chemother. 44:2133-2142.[Abstract/Free Full Text]
    14. 14
  14. Kwon, D. H., J. J. Kim, M. Lee, Y. Yamaoka, M. Kato, M. S. Osato, F. A. El-Zaatari, and D. Y. Graham. 2000. Isolation and characterization of tetracycline-resistant clinical isolates of Helicobacter pylori. Antimicrob. Agents Chemother. 44:3203-3205.[Abstract/Free Full Text]
    15. 15
  15. Maki, H., and K. Murakami. 1997. Formation of potent hybrid promoters of the mutant llm gene by IS256 transposition in methicillin-resistant Staphylococcus aureus. J. Bacteriol. 179:6944-6948.[Abstract/Free Full Text]
    16. 16
  16. Malfertheiner, P., F. Megraud, C. O'Morain, A. P. Hungin, R. Jones, A. Axon, D. Y. Graham, G. Tytgat et al. 2002. Current concepts in the management of Helicobacter pylori infection—the Maastricht 2-2000 consensus report. Aliment. Pharmacol. Ther. 16:167-180.[Medline]
    17. 17
  17. McMahon, B. J., T. W. Hennessy, J. M. Bensler, D. L. Bruden, A. J. Parkinson, J. M. Morris, A. L. Reasonover, D. A. Hurlburt, M. G. Bruce, F. Sarco, and J. C. Butler. 2003. The relationship among previous antimicrobial use, antimicrobial resistance, and treatment outcomes for Helicobacter pylori infections. Ann. Intern. Med. 139:463-469.[Abstract/Free Full Text]
    18. 18
  18. National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards, Wayne, Pa.
    19. 19
  19. National Committee for Clinical Laboratory Standards. 1999. Performance standard antimicrobial susceptibility testing. National Committee for Clinical Laboratory Standards, Wayne, Pa.
    20. 20
  20. NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease. 1994. NIH, Consensus, and Conference: Helicobacter pylori in peptic ulcer disease. JAMA 272:65-69.[Abstract/Free Full Text]
    21. 21
  21. Sarker, S. A., D. Mahalanabis, P. Hildebrand, M. M. Rahaman, P. K. Bardhan, G. Fuchs, C. Beglinger, and K. Gyr. 1997. Helicobacter pylori: prevalence, transmission, and serum pepsinogen II concentrations in children of a poor periurban community in Bangladesh. Clin. Infect. Dis. 25:990-995.[Medline]
    22. 22
  22. Sarker, S. A., M. M. Rahman, D. Mahalanabis, P. K. Bardhan, P. Hildebrand, C. Beglinger, and K. Gyr. 1995. Prevalence of Helicobacter pylori infection in infants and family contacts in a poor Bangladesh community. Dig. Dis. Sci. 40:2669-2672.[CrossRef][Medline]
    23. 23
  23. Sisson, G., J. Y. Jeong, A. Goodwin, L. Bryden, N. Rossler, S. Lim-Morrison, A. Raudonikiene, D. E. Berg, and P. S. Hoffman. 2000. Metronidazole activation is mutagenic and causes DNA fragmentation in Helicobacter pylori and in Escherichia coli containing a cloned H. pylori rdxA+ (nitroreductase) gene. J. Bacteriol. 182:5091-5096.[Abstract/Free Full Text]
    24. 24
  24. Thyagarajan, S. P., et al. 2003. Geographical difference in antimicrobial resistance pattern of Helicobacter pylori clinical isolates from Indian patients: multicentric study. J. Gastroenterol. Hepatol. 18:1373-1378.[CrossRef][Medline]
    25. 25
  25. Toracchio, S., and L. Marzio. 2003. Primary and secondary antibiotic resistance of Helicobacter pylori strains isolated in central Italy during the years 1998-2002. Dig. Liver Dis. 35:541-545.[CrossRef][Medline]
    26. 26
  26. Wosten, M. M., E. E. Ishiguro, and B. A. van der Zeijst. 1997. Cloning and characterization of the lytB gene of Campylobacter jejuni. FEMS Microbiol. Lett. 157:117-121.[Medline]
    27. 27
  27. Wu, H., X. D. Shi, H. T. Wang, and J. X. Liu. 2000. Resistance of Helicobacter pylori to metronidazole, tetracycline and amoxycillin. J. Antimicrob. Chemother. 46:121-123.[Abstract/Free Full Text]

Journal of Clinical Microbiology, October 2004, p. 4856-4858, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4856-4858.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • De, R., Kundu, P., Swarnakar, S., Ramamurthy, T., Chowdhury, A., Nair, G. B., Mukhopadhyay, A. K. (2009). Antimicrobial Activity of Curcumin against Helicobacter pylori Isolates from India and during Infections in Mice. Antimicrob. Agents Chemother. 53: 1592-1597 [Abstract] [Full Text]  
  • Megraud, F., Lehours, P. (2007). Helicobacter pylori Detection and Antimicrobial Susceptibility Testing. Clin. Microbiol. Rev. 20: 280-322 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nahar, S.
Right arrow Articles by Rahman, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nahar, S.
Right arrow Articles by Rahman, M.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»