Genetic Diversity and Drug Resistance of Helicobacter pylori Strains in Isfahan, Iran

Document Type : Original Article


1 Department of Microbiology, School of Medicine and Iranian Research Center for Infectious Diseases, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Gastroenterology, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Bacteriology, Pasteur Institute of Iran, Tehran, Iran

4 Iranian Research Center for Infectious Diseases, Isfahan University of Medical Sciences, Isfahan, Iran


Resistance to antimicrobial agents, particularly metronidazole and clarithromycin, is frequently observed in Helicobacter pylori and may be associated with treatment failure. This resistance rate varies according to the population studied. The aim of this study was to assess the pattern of antimicrobial resistance of H. pylori isolates from dyspeptic patients in Isfahan.
Materials and Methods
Antral gastric biopsies from 230 dyspeptic patients were cultured. Susceptibility testing to commonly used antibiotics performed on pure cultures of 80 H. pylori-positive isolates by Modified Disk Diffusion Method (MDDM). Genomic DNA extracted and subjected for study of entire genomic pattern using Random Amplified Polymorphic DNA- Polymerase Chain Reaction (RAPD-PCR).
The overall rates of primary resistance were 30.0%, 8.75%, 6.25%, 3.75%, 3.75%, and 2.50% for metronidazole, ciprofloxacin, clarithromycin, azithromycin, tetracycline, and amoxicillin, respectively.  Multiple antibiotic resistances were observed in 8 of 27 resistant isolates (29.6%) that mainly were double resistance with the prevalence of 6.25%. No association between antimicrobial resistance and either the gender, age or clinical presentation of the patients were detected. In RAPD-PCR, great diversity observed in 27 resistant strains isolated from different patients and this heterogeneity was not significantly different from susceptible strains.
Primary H. pylori resistance to metronidazole in our population was lower than the developing world and even other parts of Iran, to ciprofloxacin was considerable in comparison with results in most other countries. Moreover, antibiotic resistance had no effect on genomic pattern of H. pylori isolates. Finally, pretreatment H. pylori isolates susceptibility testing is highly recommended.


1.Everhart JE. Recent developments in the epidemiology of Helicobacter pylori. Gastroenterol Clin North Am 2000; 29:559-578.
2. Alarcon T, Domingo D, Lopez-Brea M. Antibiotic resistance problems with Helicobacter pylori. Int J Antimicrob Agents 1999; 12:19-26.
3. Dore MP, Osato MS, Kwon DH, Graham DY, EI-Zaatari FAK. Demonstration of unexpected antibiotic resistance of genotypically identical Helicobacter pylori isolates. Clin Infect Dis 1998; 27:84-89.
4. Zschausch HC E, Han SR, Meyer HG, Maeurer MJ. No association between Helicobacter pylori genotypes and antibiotic resistance phenotypes within families. Helicobacter 2002; 7:364-366. 
5.  Glupczynski Y, Broutet N, Cantagrel A, Andersen LP, Alarcon T, Lopez-Bera M, et al. Comparison of the E test and agar dilution method for untimicrobial susceptibility testing of Helicobacter pylori. Eur J Clin Microbiol Infect Dis 2002; 21:549-552.
6. Rimbara E, Noguchi N, Tanabe M, Kawai T, Matsumoto Y, Sasatsu M. Susceptibilities to clarithromycine, amoxicillin and metronidazole  of Helicobacter pylori isolates from the antrum and corpus in Tokyo, Japan, 1995 -2001. ClinMicrobiol Infect 2005; 11:307-311.
7.  Samra Z, shmuely H, Niv Y, Dinari G, Passaro DJ, Geler A, et al. Resistance of Helicobacter pylori  isolated in Israel to metronidazole, clarithromycin, tetracycline, amoxicillin and cefixime. J Antimicrob chemother 2002; 49:1023 -1026.
8.  Hu CT, Wu C-C, Lin CY, Cheng CC, Su SC, Tseng YH, et al. Resistance rate to antibiotics of Helicobacter pylori isolates in eastern Taiwan. J Gastroenterol Hepatol 2007; 22:720-723.
9. Boyanova L, Stancheva I, Spassova Z, Katzarov N, Mitov I, Koumanova R.  Primary and combined resistance to four antimicrobial agents in Helicobacter pylori in sofia, Bulgaria. J Med Microbiol 2000; 49:415-418.
10. Wolle K, Leodolter A, Malfertheiner P, Konig W. Antibiotic susceptibility of Helicobacter pylori in Germany: stable primary resistance from 1995 to 2000. J MedMicrobiol2002; 51:705 -709.
11. Mishra KK, Srivastava S, Garg A, Ayyagari A. Antibiotic susceptibility of Helicobacter pylori clinical isolates: Comparative Evaluation of Disk-Diffusion and E-test methods. Current  Microbiol 2006; 53:329-334.
12.  Parsons HK, Carter MJ, Sanders DS, Winstanley T, Lobo AJ. Helicobacter pylori antimicrobial resistance in the United Kingdom: the effect of age, sex and socio –economic status. Aliment Pharmacol Ther 2001; 15:1473 -1478.
13.  Osato MS, Reddy R, Reddy SG, Penland RL, Graham DY. Comparison of the E test and the NCCLS- approved agar dilution method to detect metronidazole and clarithromycin resistant Helicobacter pylori. Int J Antimicrob Agents 2001; 17:39-44.
14.  Xia H, Keane CT, Beattie S, Omorain CA. Standardization of disk diffusion test and its clinical significance for susceptibility testing of metronidazole against Helicobacter pylori. Antimicrob Agents Chemother 1994; 38: 2357-2361.
15. Yakoob J, FAN XG, HU GL, Liu L, Zhang Z. Antibiotic susceptibility of Helicobacter pylori in the Chinese population. J Gastroenterol Hepatol 2001; 16:981-985.
16. Chaves S, Gadanho M, Tenreiro R, Cabrita J. Assessment of metronidazole susceptibility in Helicobacter pylori: statistical validation and error rate analysis of breakpoints determined by the disk diffusion test. J Clin Microbiol 1999; 37:1628-1631.
17.  Perna F, Gatta L, Figura N, Ricci C, Tampieri A, Holton J, et al. Susceptibility of Helicobacter pylori to metronidazole. Am J Gastroenterol 2003; 98:2157-2161.
18. Midolo PD, Turnidge J, Lambert JR, Bell JM. Validation of a modified Kirby –Bauer disk diffusion method for metronidazole susceptibility testing of Helicobacter pylori. Diagn Microbiol Infect Dis 1995; 21:135-140.
19. Boyanova L. Comparative evaluation of two methods for testing metronidazole susceptibility of Helicobacter pylori in routine practice. Diagn Microbiol Infect Dis 1999; 35:33-36.
20. Lang L, Garcia F. Comparison of E-test and disk diffusion assay to evaluate resistance of Helicobacter pylori isolates to amoxicillin, clarithromycin, metronidazole, and tetracycline in Costa Rica. Int J Antimicrob Agents 2004; 24:572-577.
21.  Kuipers EJ, Israel DA, Kusters JG, Gerrits MM, Weel J, Van der Ende A, et al. Quasispecies development of Helicobacter pylori observed in paired isolates obtained years apart from the same host. J Infect Dis 2000; 181: 273-282.
22. Hussain MA, Kauser F, Khan AA, Tiwari S, Habibullah CM, Ahmed N. Implications of molecular genotyping of Helicobacter pylori isolates from different human populations by genomic fingerprinting of enterobacterial repetitive intergenic consensus regions for strain identification and geographic evolution. J Clin Microbiol 2004; 42:2372-2378.
23. Finger SA, Velapatino B, Kosek M, Santivanez L, Dailidiene D, Quino W, et al. Effectiveness of enterobacterial repetitive intergenic consensus PCR and random amplified polymorphic DNA fingerprinting for Helicobacter pylori strain differentiation. Appl Environ Microbiol 2006; 72:4713-4716.
24. Yakoob J, Hu GL, Fan XG, Yang HX, Liu sh, Tan DM, et al. Diversity of Helicobacter pylori among Chinese persons with H. pylori infection. APMIS 2000; 108:482-486.
25. Xia HX, Windle HJ, Marshall DG, Smyth CJ, Keane CT, O´Morain CA. Recrudescence of Helicobacter pylori after apparently successful eradication: novel application of randomly amplified polymorphic DNA fingerprinting. Gut 1995; 37:30-34.
26.  Kim JW, Kim JG, Chae SL, Cha YJ, Park SM. High prevalence of multiple strain colonization of Helicobacter pylori in Korean patients: DNA diversity among clinical isolates from the gastric corpus, antrum and duodenum. Korean J Intern Med 2004; 19:1-9.
27.  Elviss NC, Owen RJ, Breathnach A, Palmer C, Shetty N. Helicobacter pylori antibiotic-resistance patterns and risk factors in adult dyspeptic patients from ethnically diverse populations in centeral and south London during 2000. J MedMicrobiol 2005; 54:567-574.
28.  Pitcher DG, Saunders NA, Owen RJ. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett  Appl Microbiol 1989; 8:151-156.
29.  Akopyanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res 1992; 20:5137-5142.
30. Mohammadi M, Doroud D, Mohajerani N, Massarrat S. Helicobacter pylori antibiotic resistance in Iran. World J Gastroenterol 2005; 7:6009-6013.
31.  Osato MS, Reddy R, Reddy SG, Penland RL, Malaty HM, Graham DY. Pattern of primary resistance of Helicobacter pylori to metronidazole or clarithromycin in the United States. Arch Intern Med 2001; 161:1217-1220.
32.  Duck WM, Sobel J, Pruckler JM, Song Q, Swerdlow D, Friedman C, et al. Antimicrobial resistance incidence and risk factors among Helicobacter pylori-infected persons, United States. Emerg Infect Dis 2004; 10:1088-94.
33.  Adamek RJ, Suerbaum S, Pfaffenbach B, Opferkuch W. Primary and acquaired Helicobacter pylori resistance to clarithromycin, metronidazole, and amoxicillin-influence on treatment outcome. Am J Gastroenterol 1998; 93:386-389.
34. Bruce MG, Bruden DL, Mcmahon BJ, Hennessy TW, Reasonover A, Morris J, et al. Alaska sentinel surveillance for antimicrobial resistance in Helicobacter pylori isolates from Alaska native persons, 1999-2003. Helicobacter 2006; 11:581-588.
35. Fukazawa K, Seki M, Satoh K, Sugano K. Antimicrobial resistance testing of Helicobacter pylori epsilometer test and disk diffusion test. Nippon Rinsho 1999; 57:76-80.
36. Decross AJ, Marshall BJ, Mccallum RW, Hoffman SR, Barrett LJ, Guerrant RL. Metronidazole susceptibility testing for Helicobacter pylori: comparison of disk, broth, and agar dilution methods and their clinical relevance. J Clin Microbiol 1993; 31:1971-1974.
37. Loivukene K, Maaroos HI, Kolk H, Kull I, Labotkin K, Mikelsaar M. Prevalence of antibiotic resistance of Helicobacter pylori isolates in Estonia during 1995-2000 in comparison to the consumption of antibiotics used in treatment regimens. ClinMicrobiol Infect 2002; 8:598-603.
38. Realdi G, Dore MP, Piana A, Atzei A, Carta M, Cugia L, et al. Pretreatment antibiotic resistance in Helicobacter pylori infection: results of three randomized controlled studies. Helicobacter 1999; 4:106-112.
39. Wreiber K, Olsson-Liljequist B, Engstrand L. Development of resistant Helicobacter pylori in Sweden. Tendency toward increasing resistance to clarithromycin. Lakartidningen 1999; 96:582-584.
40.  Van der Wouden EJ, Van Zwet AA, Vosmaer GD, Oom JA, DE Jong A, Kleibeuker JH. Rapid increase in the prevalence of metronidazole-resistant Helicobacter pylori in the Netherlands. Emerg Infect Dis 1997; 3:385-389.
41. Sharara AI, Chedid M, Araj GF, Barada KA, Mourad FH. Prevalence of Helicobacter pylori resistance to metronidazole, clarithromycin, amoxicillin and tetracycline in Lebanon. Int J Antimicrob Agents 2002; 19:155-158.
42. Westblom TU, Unge P. Drug resistance of Helicobacter pylori: memorandum from a meeting at the sixth international workshop on Campylobacter, Helicobacter and related organisms. J Infect Dis 1992; 165:974-975.
43. Falsafi T, Mobasheri F, Nariman F, Najafi M. Susceptibilities to different antibiotics of Helicobacter pylori strains isolated from patients at the pediatric medical center of Tehran, Iran. J Clin Microbiol 2004; 42:387-389.
44. Fallahi GM, Maleknejad S. Helicobacter pylori culture and antimicrobial resistance in Iran. Indian J Pediatr2007; 74:127-130.
45.  Roghani HS, Massarrat S, Pahlewanzadeh MR, Dashti M. Effect of two different doses of metronidazole and tetracycline in bismuth triple therapy on eradication of Helicobacter pylori and its resistant strains. Eur J Gastroenterol Hepatol 1999; 11:709-712.
46.  Megraud F. Resistance of Helicobacter pylori to antibiotics. Aliment PharmacolTher 1997; 1:43-53.
47. Yilmaz O, Demiray E. Clinical role and importance of fluorescence in situ hybridization method in diagnosis of Helicobacter pylori infection and determination of clarithromycin resistance in H. pylori eradication therapy. World J Gastroenterol 2007; 13:671-675.
48.   Kim JJ, Reddy R, Lee M, Kim JG, El-Zaatari FA, Osato MS, et al. Analysis of metronidazole, clarithromycin and tetracycline resistance of Helicobacter pylori isolates from Korea. J Antimicrob Chemother 2001; 47:459-461.
49.  Van der Ende A, Van Doorn LJ, Rooijakkers S, Feller M, Tytgat GNJ, Dankert J. Clarithromycin-susceptible and resistant Helicobacter pylori isolates with identical randomly amplified polymorphic DNA-PCR genotypes cultured from single gastric biopsy specimens prior to antibiotic therapy. J Clin Microbiol 2001; 39:2648-2651.
50.  Rafeey M, Ghotaslou R, Nikvash S, Ashrafy Hafez A. Primary resistance in Helicobacter pylori isolated in children from Iran. J Infect Chemother 2007; 13:291-295.
51. Siavoshi F, Safari F, Doratotaj D, Khatami GR, Fallahi GH, Mirnaseri MM. Antimicrobial resistance of Helicobacter pylori isolates from Iranian adults and children. Arch Iran Med 2006; 9:308-314.
52.  Buckley JM, Deltenre M. Therapy of Helicobacter pylori infection. Curr Opin Gastroenterol 1997; 13:56-62.