|Year : 2014 | Volume
| Issue : 4 | Page : 408-413
Emerging antimicrobial resistance pattern of Helicobacter pylori in central Gujarat
HB Pandya1, Harihar Har Agravat2, JS Patel3, NRK Sodagar4
1 Assistant Professor, , Shree? Purshottamdas Moti Bhai Patel. Patel College of Paramedical Science and Technology, Anand, Surendranagar, India
2 Dean, ?chiman bhai odhav bhai Shah Medical College, Surendranagar, India
3 Head of the Department, Purshottam Bhai Dhaya Bhai Patel Institute of Applied Sciences, Charotar University of Science and Technology, Changa, Gujarat, India
4 Department of Medical Microbiology, Shree? Purshottamdas Moti Bhai Patel. Patel College of Paramedical Science and Technology, Anand, Surendranagar, India
|Date of Submission||10-May-2013|
|Date of Acceptance||16-Oct-2014|
|Date of Web Publication||4-Oct-2014|
H B Pandya
Assistant Professor, , Shree? Purshottamdas Moti Bhai Patel. Patel College of Paramedical Science and Technology, Anand, Surendranagar
Source of Support: None, Conflict of Interest: None
Background: Antimicrobial resistance is a growing problem in H. pylori treatment. The study was intended to evaluate the prevalence of resistance amongst 80 H.pylori isolates cultured from biopsy taken during routine endoscopies in 2008-2011. Materials and Methods: 855 gastro duodenal biopsies were collected and cultured on H.pylori selective medium (containing Brucella agar and Columbia agar (Hi media), with Skirrow's supplement (antibiotic supplement) and 7% human blood cells). H.pylori was isolated from 80 specimens. The antimicrobial susceptibility of H.pylori isolates was carried out by the Kirby Bauer technique against metronidazole (5 µg), clarithromycin (15 µg), ciprofloxacin (5 µg), amoxicillin (10 µg), tetracycline (30 µg), erythromycin (15 µg), levofloxacin (5 µg), and furazolidone (50 µg) (Sigma- Aldrich, MO). Results: 83.8% isolates were resistant to metronidazole, 58.8% were resistant to Clarithromycin 72.5% were resistant to Amoxicillin, 50% to Ciprofloxacin and 53.8% to tetracycline. furazolidone, erythromycin and Levofloxacin showed only 13.8% resistance to H.pylori. Multi drug resistance with metronidazole+ clarithromycin+ tetracycline was 85%. For all the drugs Antimicrobial resistance rate was found higher in males compare to females. Metronidazole and amoxicillin resistance was found noteworthy in patients with duodenal ulcer (p = 0.018), gastritis (P = 0.00), and in reflux esophagitis (P = 0.00). clarithromycin and tetracycline resistance was suggestively linked with duodenitis (P = 0.018), while furazolidone, erythromycin and levofloxacin showed excellent sensitivity in patients with duodenitis (P value- 0.018), gastritis (P= 0.00) and reflux esophagitis (P = 0.00). Resistance with metronidazole (P = 0.481), clarithromycin (P= 0.261), amoxicillin (P = 0.276), tetracycline (P = 0.356), ciprofloxacin (P = 0.164) was not correlated well with Age-group and Gender of the patients. Conclusion: A very high percentage of patients were infected with metronidazole and clarithromycin resistant strains. The use of antibiotics for other indications seems to be the major risk factor for the development of primary resistance. High incidence should alarm the gastroenterologist while prescribing the eradication regimen.
Keywords: Amoxicillin, clarithromycin, Resistance, metronidazole, H.pylori
|How to cite this article:|
Pandya H B, Agravat HH, Patel J S, Sodagar N. Emerging antimicrobial resistance pattern of Helicobacter pylori in central Gujarat. Indian J Med Microbiol 2014;32:408-13
|How to cite this URL:|
Pandya H B, Agravat HH, Patel J S, Sodagar N. Emerging antimicrobial resistance pattern of Helicobacter pylori in central Gujarat. Indian J Med Microbiol [serial online] 2014 [cited 2019 Oct 14];32:408-13. Available from: http://www.ijmm.org/text.asp?2014/32/4/408/142256
| ~ Introduction|| |
The discovery of Helicobacter pylori species by Warren and Marshall have not only introduced a whole new group of bacteria to science but also revolutionised our concept of gastro duodenal pathology and diverted the worldwide attention from pH (H + ion concentration) to Hp (H. pylori).
Various epidemiological studies have demonstrated that this organism plays an aetiological role in the development of type B active chronic gastritis;  and is likely to be associated with duodenal ulcer in 70-100% of cases and with gastric ulcer in 50-90%. Eradication of this infection with appropriate antibiotic therapy has been shown to decrease ulcer recurrence <10% in one year versus a 60-100% one year recurrence with use of anti-ulcer medications alone. Recently, proton pump inhibitors (PPI)-based eradication regimens containing two antibiotics have been demonstrated to have high eradication rates (greater than 90%). 
Amoxicillin, clarithromycin (CLA) and metronidazole (MTZ) are the most frequently used antibiotics for the treatment of H. pylori infection. However, antibiotic resistance frequently causes failure of eradication of H. pylori. 
The resistance of H. pylori to the recently available antibiotic treatment regimens has been a growing problem. In developed countries, MTZ resistance is found in 10-50% of adult patients infected with H. pylori, whereas virtually all strains are resistant to the agent in developing countries. In contrast, although the rates of CLA resistance are relatively low, ranging from 2% to 15%, the rate of CLA resistance is increasing during recent years. 
Majority of the countries including India, lack a regional surveillance programme for anti-microbial susceptibility against H. pylori, and majority of them lack consistency in methodology, and conflicting results are thus not unexpected. Therapeutics recommendations are mostly based on either insufficient data or those obtained from other geographically un-related region.
This study was initiated to monitor the local pattern of anti-microbial resistance among the isolates of H. pylori in Anand District of Central Gujarat from 2008 to 2011.
| ~ Materials and Methods|| |
Patients and strains
A total of 855 consecutive, symptomatic patients (518 males and 337 females; age 10-90 years) attending the endoscopy department of Deep Surgical Hospital, Anand, Gujarat were included. A total of 432 patients with gastritis, 39 patients with duodenitis, 35 with duodenal/gastric ulcer and 349 with reflux esophagitis, based on endoscopic findings, were enrolled in this study. Patients having a history of previous gastric surgery, recent or active gastrointestinal bleeding, Patients taking aspirin or non-steroidal anti-inflammatory drugs (NSAIDS) in the past 4 weeks or are on PPI, patients with previous therapy to eradicate H. pylori or if the informed consent was not obtained were excluded from the study. The study was conducted during the period from February 2008 to August 2011.
Approval of Human research ethics committee (HREC) of H. M. Patel Center for Medical Care and Education, Pramukh Swami Medical College, Karamsad, was taken prior to initiation of the work. Study was done according to the principles of Helsinki Declaration. Dully filled consent form was obtained from all the patients participating in the study.
Two antral biopsies were collected from each symptomatic patient in fasting condition after giving local anesthetic spray in pharyngeal region. Biopsies were collected in sterile Brain-Heart Infusion broth (BHI, MV-210-Hi Media labs) with 20% glycerol for safe transfer to the microbiology laboratory. Biopsy specimen is transported immediately to the processing laboratory without delay. If delay in processing is unavoidable the biopsy specimen can be stored in refrigerator for an hour at -4°C before culture. H. pylori were isolated by streaking homogenised biopsies on Brucella More Details blood agar (Brucella Hi Veg Agar Base, procured from Hi Media labs) and Columbia chocolate agar (procured from Hi Media labs) augmented with 7-10% human defibrinated blood and Skirrow's selective supplement (vancomycin, 10 μg/ml; polymyxin B sulfate 2.5 IU/ml; trimethoprim lactate 5 μg/ml) (campylobacter supplement-III, Hi Media labs). These plates were incubated at 37°C in an anaerobic jar with providing gas pack kit (campylobacter gas generating kit BR 060A, Oxoid) for H. pylori, which provides suitable microaerophilic condition (5% O 2 ; 10% CO 2 ; 85% N 2 ) for 4-6 days. H. pylori isolates were identified by typical colony morphology (minute, translucent, round, convex colonies on Brucella blood agar and small, round, opaque, golden colonies on modified chocolate agar), characteristic gram negative spiral appearance, positive urease, oxidase and catalase test. 
Anti-microbial susceptibility testing of the isolates
A total of 80 clinical isolates of H. pylori were sub cultured in to Brucella blood agar to obtain a pure culture. Susceptibility test was performed using Kirby-Bauer disk diffusion method on Muller-Hinton agar plate (Merck, Darmstadt, Germany) supplemented with 10% human blood. A standard inoculum of H. pylori culture was suspended in BHI broth. The turbidity was adjusted equal to McFarland 3. The inoculum was seeded onto Muller-Hinton blood agar plate using sterile cotton wool swab, plates were then allow to dry, thereafter, antibiotics discs with the following drug contents: MTZ (5 μg), CLA (15 μg), ciprofloxacin (CP) (5 μg), amoxicillin (10 μg), tetracycline (TET) (30 μg), erythromycin (15 μg), levofloxacin (5 μg) and furazolidone (50 μg) (Sigma-Aldrich, MO) were placed on the plates. The plates were incubated at 37°C under micro-aerophilic conditions (Campylobacter gas generating kit, BR 060A, Oxoid) for 3-4 days. The results were interpreted as per Clinical Laboratory Standards Institute (CLSI) 2011 guidelines.  Resistance was determined by zone of growth inhibition ≤16 mm for MTZ, ≤25 mm for amoxicillin and a zone diameter ≤30 mm for CLA, CP, TET, furazolidone and erythromycin. Zone of inhibition were compared with recorded diameters of the control organism, H. pylori (NCTC 11638), to determine susceptibility or resistance.
A Pearson Chi-squared test was used to find out the significant correlations. P values of less than 0.05 were considered to be statistically significant. Data analyses were conducted using Statistical Package for Social Sciences (SPSS) version 15.0.
| ~ Results|| |
A total of 125 isolates (82 from males and 43 from females) were sub cultured, but only 80 H. pylori isolates were isolated in pure form and were subjected to anti-microbial susceptibility testing. MTZ showed a high level of resistance in our zone, followed by amoxicillin, CLA, TET and CP. Furazolidone, erythromycin and levofloxacin showed only 13.8% resistance each to H. pylori. A total of 85% of the strains were multi-drug resistant (MDR). They showed resistance to MTZ, CLA and TET [Table 1].
|Table 1: Primary and combined antimicrobial resistance pattern of H. pylori (n=80) |
Click here to view
Anti-microbial Resistance pertaining to various gastroduodenal diseases
100% resistant strains were found in the patients with gastric ulcer and duodenitis, and 89% resistant strains in patients with reflux esophagitis (P - 0.000) [Table 2].
80% resistant strains were found in the patients with duodenitis (P - 0.018, <0.05). 57.6% resistant strains were found in gastritis (P - 0.21), 58.3% resistant strains were found in reflux esophagitis patients (P - 0.15) [Table 2].
% resistant strains were found in the patients with duodenal ulcer (P - 0.018, <0.05). 69.7% resistant strains were found in gastritis (P - 0.000), 69.4% resistant strains were found in reflux esophagitis patients (P - 0.000), In patients with duodenitis and gastric ulcer resistance was found 100% but number of patients tested were very few to comment any association [Table 2].
|Table 2: Correlation of metronidazole, clarithromycin and amoxicillin resistance with various gastro duodenal disorders |
Click here to view
CP showed 60% resistance in duodenitis patients, followed by reflux patients [Table 3].
|Table 3: Correlation of ciprofloxacin, tetracycline, furazolidone/erythromycin/levofloxacin resistance with various gastro duodenal disorders |
Click here to view
% of TET resistant strains were found in the patients with duodenitis (P - 0.018, <0.05). 45.5% resistant strains were found in gastritis (P - 0.46), 55.6% resistant strains were found in reflux esophagitis patients (P - 0.33) [Table 3].
Furazolidone, erythromycin and levofloxacin
Remarkably, we found no strains resistant in duodenal ulcer patients, while only 20% and 15% strains were resistant in the patients with duodenitis and gastritis, respectively [Table 3]. Furazolidone, levofloxacin and erythromycin revealed excellent results in our study.
| ~ Discussion|| |
The geographical disparity in the resistance of H. pylori to anti-microbials agents is alleged to be associated to the level of use of the agents in different communities.  Antibiotics especially MTZ, CLA, ampicillin and TET are widely use in Anand district to treat other infections. In addition, antibiotics self-medication is encourage by free access and over the counter purchase and by an ineffective drug control policy.  This could be the responsible factor for very high level of resistance of H. pylori to MTZ (83.8%), amoxicillin (72.5%), CLA (58.8%), TET (53.8%) and CP (50%) in our precinct.
Primary resistance of H. pylori to MTZ is consistent with earlier research conducted in several areas. , Two centres from the western part of northern India have reported relatively lower prevalence of resistant strains (Delhi, 37.5% and Chandigarh, 38.2%).  However, a report from eastern India has shown high MTZ resistance (85%) in H. pylori.
A multi-centric study has also reported quite high prevalence of MTZ resistant H. pylori from south India (Hyderabad, 100%, Chennai, 88.2%) and from northern India (Lucknow, 68%). 
A considerable difference has been observed in the rate of primary MTZ resistance of H. pylori, which may be because of extensive use of this inexpensive antibiotic for the treatment of parasitic, genital and dental infections especially in developing countries. 
The design of the present study did not allow for determination of aetiology of high level resistance. However, we hypothesise that the widespread use of MTZ in Anand district for the treatment of numerous infectious disease led to the development of resistance. Studies have found the mechanism of MTZ resistance due to inactivation of gene (mutation) rdxA gene, which encodes for oxygen-insensitive nicotinamide adenine dinucleotide phosphate (NADPH) nitroreductase, which in turn is responsible for activation of prodrug,  but the present study is unable to corroborate the finding, as we did not test for the presence of rdxA gene in H. pylori isolates. Regardless of the reason, it is clear that MTZ should not be included in the treatment regimens for H. pylori in Anand district.
There is lot of geographical variation in resistance pattern. The prevalence of MTZ resistant H. pylori may be attributed to (i) indiscriminate use of drugs in different areas and (ii) lack of uniformity in identification of the H. pylori strains as well as drug susceptibility testing in different laboratories. 
We also found that 57.5% of CLA resistant strains were also resistant to MTZ. The reason for such double resistance is not clear but it has been reported by another investigator.  We also found that 85.1% of H. pylori strains were MDR, that is, resistant to MTZ, CLA as well as TET.
Prevalence of CLA resistance was high (58.8%) in our region compared with other studies. ,
Studies from Hyderabad and Mumbai also showed very high CLA resistance prevalence rate of 71%, 96% and 91%, respectively.  High resistance rate may be due to the fact that in Anand district, CLA is widely used in patients with duodenal ulcer, non-ulcer dyspepsia and abdominal discomfort without a laboratory-confirmed diagnosis of H. pylori infection.
CLA, a second-generation macrolide also used for the management of respiratory tract infections and dental infections. Higher frequency of CLA usage in developed countries may be one of the explanations for resistance. Another factor contributing to resistance is mutation of the H. pylori 235 rRNA genes and A2143G.  Detection of higher CLA resistant strains may be due to the fact that our study has been conducted recently as compared with other studies. However, emergence of CLA resistance in H. pylori strains isolated in the present study seems to be alarming since it is the key drug used in the first-line therapy.
CLA resistance combined with MTZ and TET was 85.1% and with MTZ and CP was 48.75%. This shows MDR strains are also emerging in Anand district. CLA is an expensive antimicrobial agent and perhaps this is the reason. Further surveillance should be considered because the use of CLA combined with other anti-microbial agents for H. pylori treatment has been increasing.
It is again very arduous to explain high resistance for amoxicillin (72.5%) in our zone. Although reports from central India (Mumbai, 73%) and southern India (Hyderabad, 80%) show quite similar levels of resistance against AMX, but the majority of the reports from northern India (Lucknow, Delhi, Chandigarh), eastern India (Kolkata) and also one city in southern India (Chennai) showed no amoxicillin-resistant isolates. 
The clinical significance of amoxicillin susceptibility testing has not been documented. Frequent use of this antibiotic for the treatment of respiratory and skin infections in our country may contribute to bacterial resistance.
Paul et al.,  proved that H. pylori pbpl gene mutation makes it resistant to amoxicillin.
In our zone, we got emerging resistance of H. pylori with TET (53.8%) and CP (50%). TET resistance was not reported until 1996, when Midolo and colleagues  reported TET resistant strains from Australia. Resistance rate for both was high as compared with other studies. ,
A reason for this high resistance may be that CP is often used in the treatment of urinary tract and respiratory infections  Although CP is not a drug of choice in the treatment of H. pylori infection, its combination with amoxicillin may be considered as an alternative in cases of resistance to first-line anti-microbial agents. 
With levofloxacin, erythromycin and furazolidone we got only 13.8% of resistance. Levofloxacin has better tissue penetration and fewer side effects than CP.  Furazolidone has been used to as an alternative to overcome MTZ resistance. In our study, resistance to furazolidone was 13.8%, which is almost equivalent (13%) to the result of Godoy et al. This rate was higher than the other studies.  The reason for the high rate of furazolidone resistance and factors leading to it are still unknown and deserve further investigation. Furazolidone is emerging as an alternative for therapeutic regimens in developing countries due to its low cost and prevalence of resistant strains. Susceptibility pattern of H. pylori to commonly used antibiotics varies from region to region. This huge variation in the sensitivity patterns among H. pylori strains from different parts of India could be due to the possibility that these strains acquired resistance to antibiotics differently. It is possible because inhabitants from different parts of India bear distinct genetic traits, which may allow this long-term coloniser of human gastric niche to co-evolve with different host populations. However, we do not ignore the possibility of interpreting antibiotic sensitivity patterns for H. pylori falsely due to variation in the assay technique and interpretation criteria. The wide discrepancy in the antibiotic sensitivity profiles of H. pylori within the same country undoubtedly suggest the urgent need for a systematic and consensus approach to determine the antibiogram of the strains before trying out drug regimens.
Our results strongly recommends a surveillance of antibiotic resistance every 4 years to assess the effectiveness of current treatment regimens and antibiotic sensitivity testing as a routine in patients not responding to current regimens
| ~ Acknowledgements|| |
The study was supported financially by the funds of our institution Shree P. M. Patel Paramedical College, Anand, Gujarat. The authors are also grateful to Dr. Parimal Salvi for providing them the biopsy samples.
All the authors would like to thank our institution Shree P. M. Patel Paramedical College, Anand, Gujarat, for providing us funds to successfully complete our research.
| ~ References|| |
|1.||Rauws EA, Langenberg W, Houthoff HJ, Zanen HC, Tytgat GN. Campylobacter pylori associated with chronic active antral gastritis. A prospective study of it prevalence and the effect of antibacterial and antiulcer treatment. Gastroenterology 1988;94:33-40. |
|2.||Dergisi GT. Diagnostic methods of Helicobacter pylori infection. Emel Öztürk 2008;50:60-4. |
|3.||Lind T, Veldhuyzen van Zanten S, Unge P, Spiller R, Bayerdörffer E, O'Morain C, et al. Eradication of Helicobacter pylori using one-week triple therapies combining omeprazole with two antimicrobials: The MACH I Study. Helicobacter 1996;1:138-44. |
|4.||Huang JQ, Hunt RH. Treatment after failure: The problem of "Non-responders". Gut 1999;45:140-4. |
|5.||Mendonça S, Ecclissato C, Sartori MS, Godoy AP, Guerzoni RA, Degger M, et al. Prevalence of Helicobacter pylori resistance to metronidazole, clarithromycin, amoxicillin, tetracycline, and furazolidone in Brazil. Helicobacter 2000;5:79-83. |
|6.||Destura RV, Labio ED, Barrett LJ, Alcantara CS, Gloria VI, Daez ML, et al. Laboratory diagnosis and susceptibility profile of Helicobacter pylori infection in the Philippines. Ann Clin Microbiol Antimicrob 2004;3:25. |
|7.||Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty First Informational Supplement. CLSI document M100-S21. Wayne: CLSI;2011. |
|8.||Bindayna KM. Antibiotic susceptibilities of Helicobacter pylori. Saudi Med J 2001;22:53-7. |
|9.||Kulsuntiwong P, Chomvarin C, Chaicumpar K, Namwat W, Kaewkes W, Mairiang P, et al. Antimicrobial susceptibility of helicobacter pylori isolated from gastric biopsies in dyspeptic patients. Southeast Asian J Trop Med Public Health 2008;39:1102-9. |
|10.||Mirza IA, Mirza SH, Ali AM. Antimicrobial susceptibility pattern of H. pylori in isolates from Northern Pakistan. Int J Pathol 2007;5:18-20. |
|11.||Datta S, Chattopadhyay S, Patra R, De R, Ramamurthy T, Hembram J, et al. Most Helicobacter pylori strains of Kolkata in India are resistant to MTZ but susceptible to other drugs commonly used for eradication and ulcer therapy. Aliment Pharmacol Ther 2005;22:51-7. |
|12.||Vakil N, van Zanten SV, Kahrilas P, Dent J, Jones R. Global Consensus Group. The Montreal definition and classification of gastroesophageal reflux disease: A global evidence based consensus. Am J Gastroenterol 2006;101:1900-20. |
|13.||Saha DR. Correlation of histology with genotypes of Helicobacter pylori isolated from cases of peptic ulcer, non- ulcer dyspepsia, gastric carcinoma and lymphoma. National Institute of Cholera And Enteric Diseases (NICED) Annual Report 2004-2005. |
|14.||Kobayashi T, Kikuchi S, Lin Y, Yagyu K, Obata Y, Ogihara A, et al. Trends in the incidence of gastric cancer in Japan and their associations with Helicobacter pylori infection and gastric mucosal atrophy. Gastric Cancer 2004;7:233-9. |
|15.||Singh V, Mishra S, Maurya P, Rao G, Jain AK, Dixit VK, et al. Drug resistance pattern and clonality in H. pylori strains. J Infect Dev Ctries 2009;3:130-6. |
|16.||Thyagarajan SP, Ray P, Das BK, Ayyagari A, Khan AA, Dharmalingam S, et al. Geographical difference in antimicrobial resistance pattern of Helicobacter pylori clinical isolates from Indian patients: Multicentric study. J Gastroenterol Hepatol 2003;18:1373-8. |
|17.||Matsumura M, Hikiba Y, Ogura K, Togo G, Tsukuda I, Ushikawa K, et al. Rapid detection of mutations in the 23S rRNA gene of Helicobacter pylori that confers resistance to clarithromycin treatment to the bacterium. J Clin Microbiol 2001;39:691-5. |
|18.||Paul R, Postius S, Melchers K, Schäfer KP. Mutations of the Helicobacter pylori genes rdxA and pbp1 cause resistance against metronidazole and amoxicillin. Antimicrob Agents Chemother 2001;45:962-5. |
|19.||Midolo PD, Lambert JR, Russell EG, Lin SK. A practical single sample dry latex agglutination test for Helicobacter pylori antibody detection. J Clin Pathol 1995;48:969-71. |
|20.||Boyanova L, Koumanova R, Gergova G, Popova M, Mitov I, Kovacheva Y, et al. Prevalence of resistant Helicobacter pylori isolates in Bulgarian children. J Med Microbiol 2002;51:786-90. |
|21.||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. Clin Microbiol Infect 2002;8:598-603. |
|22.||Godoy AP, Ribeiro ML, Benvengo YH, Vitiello L, Miranda Mde C, Mendonca S, et al. Analysis of antimicrobial susceptibility and virulence factors in Helicobacter pylori clinical isolates. BMC Gastroenterol 2003;3:20. |
|23.||Kawakami E, Machado RS, Ogata SK, Langner M, Fukushima E, Carelli AP, et al. Furazolidone-based triple therapy for H. pylori gastritis in children. World J Gastroenterol 2006;12:5544-9. |
[Table 1], [Table 2], [Table 3]