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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Acknowledgement
 ~  References

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Year : 2003  |  Volume : 21  |  Issue : 4  |  Page : 257-261

Relationship of plasmid profile with the antibiotic sensitivity pattern of helicobacter pylori isolates from peptic ulcer disease patients in Chennai

Departments of Microbiology, Dr. ALM Post graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113, India

Correspondence Address:
Departments of Microbiology, Dr. ALM Post graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113, India

 ~ Abstract 

PURPOSE: One hundred and ten Helicobacter pylori isolates from peptic ulcer disease patients and matched controls were analysed for any possible relationship between the presence of cryptic plasmids and their antibiotic sensitivity pattern. METHODS: Antral biopsies of patients with gastric and duodenal ulcer, gastric cancer, non ulcer dyspepsia and matched controls were cultured for H.pylori. Antibiotic susceptibility and MIC analysis of the clinical isolates was done by E-test. Plasmid profiles of the isolates were analysed using mini ultra prep plasmid kit. RESULTS: Out of the 110 isolates tested, 89.1% isolates were resistant to metronidazole, 10.9 % were resistant to clarithromycin and 0.9% were resistant to multiple drugs. Isolates harbouring plasmids were seen in all the groups and constituted 5.4% of total isolates. CONCLUSIONS: The presence of plasmids in the clinical isolates of H.pylori did not have any correlation with their antibiotic resistance pattern.

How to cite this article:
Dharmalingam S, Rao U A, Jayaraman G, Thyagarajan S P. Relationship of plasmid profile with the antibiotic sensitivity pattern of helicobacter pylori isolates from peptic ulcer disease patients in Chennai. Indian J Med Microbiol 2003;21:257-61

How to cite this URL:
Dharmalingam S, Rao U A, Jayaraman G, Thyagarajan S P. Relationship of plasmid profile with the antibiotic sensitivity pattern of helicobacter pylori isolates from peptic ulcer disease patients in Chennai. Indian J Med Microbiol [serial online] 2003 [cited 2021 Feb 25];21:257-61. Available from:

Antibiotic resistance is an ever increasing problem associated with the treatment of most microbial infections including Helicobacter pylori infection which is shown to be the major cause of gastritis and peptic ulcer disease.[1],[2],[3] Chronic infection with H.pylori is an accepted risk factor in the development of gastric cancer.[4] Based on findings of several studies, currently recommended therapy for the eradication of H.pylori infection include a AQ (omeprazole, lanoprazole or pantoprazole) together with clarithromycin and either metronidazole or amoxycillin.[5],[6] Success rates of cure with the use of these combination therapy ranges from 85% to 95%. However the resistance to metronidazole or clarithromycin results in an increased failure rate of therapies.[7] Any global effort to eradicate H.pylori with antibiotics, however, also has to overcome the substantial problem of drug resistance. In examining the mechanisms of antimicrobial resistance of H.pylori, two factors were reported to be responsible. Extrinsic factors include extent of infection (microbial load or relative virulence of strains), immune status of individuals and compliance with treatment regimen. Intrinsic factors include biochemical and genetic basis of microbial resistance.[3]
In many pathogenic bacteria, plasmids frequently carry antibiotic resistance encoding genes (besides encoding virulence factors) allowing bacteria to survive antibiotic treatment beside encoding virulence factors. Virtually nothing is known about the significance of plasmid presence in H.pylori including their possible role in pathogenesis. Penfold et al first reported 48% of clinical isolates of Campylobacter pylori to possess plasmids,[8] while 8-32% positivity for plasmids in fresh clinical isolates of H.pylori has been reported subsequently.[9],[10],[11] To fill the lacuna of knowledge in this area among Indian isolates of H.pylori, the present study was conducted to look for endogenous plasmids in H.pylori isolates and to analyse them for any correlation with their antibiotic resistance pattern.

 ~ Materials and Methods Top

One hundred and ten H.pylori isolates were isolated from antral biopsy specimens obtained from patients attending the gastroenterology departments of Madras Medical College and Stanley Medical College, Chennai (duodenal ulcer -58, gastric ulcer -10, non ulcer dyspepsia-29, gastric cancer-6 and control -7). They were identified by Gram stain, positive urease, oxidase, catalase tests and other confirmatory tests as described.[12]
Antibiotic susceptibility testing by E-test
All clinical isolates of H.pylori were subcultured onto  Brucella More Details chocolate agar plates and antibiotic susceptibility was done using E-test strips obtained from AB Biodisk, Sweden. Briefly, H.pylori inoculum was prepared in brain heart infusion broth (Difco) and turbidity was adjusted to MacFarland No.2 standard. The inoculum was seeded onto Mueller Hinton blood agar with 7% defibrinated sheep blood, after the plates were dried in CO2 incubator at 370C for 5-10 minutes. The bottom edge of the E-test strip was placed against the agar surface, holding it at an angle to the surface with the MIC scale marks facing upwards and the concentration maximum near the periphery of the plate. The E-test strips used were for metronidazole, clarithromycin, amoxycillin, tetracycline and ciprofloxacin. Inoculated plates were incubated at 370C in a 12% CO2 atmosphere for 3-5 days. The MIC values were read at the end point of intersection between the zone edge and the E-test strip. The cut off values to decide susceptibility in E-test were 8 痢/mL for metronidazole, 4 痢/mL for tetracycline and amoxycillin, and 2 痢/mL for clarithromycin and ciprofloxacin. ATCC
H.pylori standard strain 26695 and metronidazole sensitive strain (HK 101) were used as positive controls.
Isolation of plasmid
For plasmid analysis, H. pylori isolates were grown in  Brucella More Details chocolate agar with antibiotics. The colonies were suspended in 300 無 of Tris EDTA buffer in a microcentrifuge tube. Plasmid isolation was done by mini ultra prep plasmid kit (AB gene, Epsom, Surrey KT 19 9AP, UK).
In brief, the culture was centrifuged for 30 seconds and supernatant discarded, 50 無 of suspension buffer (Tris, EDTA, RNase) was added to cell pellet and vortexed thoroughly. Lysis buffer (SDS/NaOH) 100 無 was added and mixed by inverting once. 325 無 of KAC/binding salt buffer was added and centrifuged for 1 minute at 10,000 g. The clear supernatant was transferred to a spin filter and centrifuged for 10 seconds at 10,000 g. 300 無 of wash solution (Tris, Ethanol, NaCl) was added and centrifuged for 2 minutes at 10,000 g. 50 無 of elusion buffer (100mM Tris HCl) was added onto the middle of the white spin filter membrane and centrifuged for 30 seconds. Plasmid DNA was thus collected in the tube.
Agarose gel electrophoresis
0.7% agarose gel was prepared and was used to run the plasmid DNA. E. coli pGEM3Zf+ control plasmid DNA was loaded at each run. Plasmid DNA was visualized after ethidium bromide staining in a UV transilluminator (biorad gel documentation system, USA).

 ~ Results Top

A total of 110 isolates were tested for antibiotic susceptibility by E-test. Twelve out of 110 (10.9%) strains were found to be sensitive to metronidazole, 89.1% (98/110) were sensitive to clarithromycin and 99.1% (109/110) were sensitive to both amoxycillin and ciprofloxacin. All the isolates tested were sensitive to tetracycline while 89.1% (98/110) isolates were resistant to metronidazole [Table - 1].
Our study revealed multiple resistance patterns amongst H. pylori isolates from all disease groups. 10.9% isolates were found to be resistant to metronidazole and clarithromycin. Only one isolate (0.9%) showed multiple resistance to metronidazole, clarithromycin, amoxycillin and ciprofloxacin [Table - 2].
When all the 110 H. pylori isolates were screened for plasmids, 6 isolates (5.4%) were found to harbour plasmids. The distribution pattern of plasmids in H.pylori isolated from different disease group was 5.1% in duodenal ulcer isolates, 10% in gastric ulcers, 3.4% in non-ulcer dyspepsias, and 16.6% in gastric cancers. None of the H.pylori isolates isolated from controls possessed plasmids [Table - 3], [Figure:1].
The plasmid positive isolates were analysed for their antibiotic sensitivity patterns revealed by E-test. All the plasmid positive isolates were resistant to metronidazole and sensitive to clarithromycin, amoxycillin and tetracycline. Metronidazole resistance was also seen in plasmid negative isolates. Therefore, no correlation between antibiotic resistance and plasmid profile was observed [Table - 4].

 ~ Discussion Top

In general, antimicrobial resistance can arise through acquisition of genetic material encoding enzymes that inactivate a particular antibiotic (eg: beta lactamase, chloramphenicol, acetyl transferase or kanamycin phosphotransferase), export antibiotics (eg: tetracycline antiport ) or alter the drug target through mutation (eg: penicillin binding protein or 23S rRNA in macrolide resistance).[3] Nitroimidazoles are metabolized by H.pylori by several nitroreductases of which an oxygen insensitive NADPH nitroreductase encoded by the rdx A gene is the most important. Null mutations in this gene is associated with resistance.[13],[14] Generally the resistance mechanism for macrolides is similar and involves transition base mutations in the 23 S subunit of r RNA. In H.pylori, these mutations occur at positions 2142 and 2143. Several studies have demonstrated that transition base mutations (adenineguanine) at these positions correlate with clarithromycin resistance.[15],[16]
Resistance against lactam antibiotics like amoxycillin is generally due to hydrolysis by a lactamase or by mutational modification of penicillin binding protein. They have shown that pbp1 mutations can affect amoxycillin resistance of 69 A/Amx r.
This indicates that mutations in more than one gene probably is required to render H.pylori amoxycillin resistant.[17],[18]
Several studies have suggested that quinolones such as ciprofloxacin can be used in combination therapy to treat H.pylori infection. A PCR based analysis of the gyr A gene of ciprofloxacin resistant mutants of H.pylori revealed mutations in four locations that resulted in amino acid substitutions.[19]
Following the first report of Penfold et al in 1988 demonstrating plasmids in 48% of clinical isolates of C.pylori,[8] several investigators have worked on the presence and significance of plasmids in H.pylori.
Owen et al examined biotypes, ribopatterns, whole cell protein patterns and plasmid profiles of paired H. pylori clinical isolates from 17 patients. Their results indicated that emergence of resistance to metronidazole in H. pylori was unlikely to be attributed to plasmid coded determinants because there was no direct association between emergence of resistance and acquisition of a plasmid.[11] Yakoob et al reported, strains of H. pylori may become resistant by mutations in the chromosomal genes by acquisition of exogenous DNA or by transformation. There is a theoretical possibility of foreign DNA acquisition by plasmids, transposons or integrons. Plasmids occur in approximately half of all the strains of H. pylori but there is no evidence that any of these plasmids confer antibiotic resistance.[20]
Hosaka et al correlated, the susceptibilities of 24 H. pylori isolates to 5 lactam antibiotics, 2 macrolides, 2 aminoglycosides, 2 quinolones, 2 tetracyclines, rifampicin and chloramphenicol that were tested for the presence of plasmids. All the isolates showed similar susceptibilities against lactam antibiotics. In some H. pylori strains, drug resistance was observed against amikacin, chloramphenicol and quinolones. From the molecular epidemiological study cryptic plasmids were detected from the 3 isolates among 24 strains tested. Thus, no correlation was observed between antibiotic resistance and plasmid profiles of H. pylori isolates.[21]
In our study, 89.1% of isolates were found to be metronidazole resistant, 10.9% of isolates were found to be resistant to clarithromycin and 0.9% of isolates were resistant to amoxycillin and ciprofloxacin. Out of 89.1% of metronidazole resistant isolates only 5.4% (6/110) showed cryptic plasmids. All plasmid positive isolates were sensitive to amoxycillin, clarithromycin, tetracycline and ciprofloxacin antibiotics using E-test. No significant correlation was observed between antibiotic resistance and plasmid content in these H. pylori isolates. Our study correlates well with above quoted reports.
This happens to be the first report from India on plasmid profiling in Indian isolates of H.pylori. Even though, the plasmid profile did not correlate with antibiotic resistance pattern of H.pylori in our study, additional investigations are needed to ascertain the role of plasmids in the pathophysiology of H. pylori related diseases both epidemiologically and with respect to potential virulence factors.

 ~ Acknowledgement Top

We gratefully acknowledge the Department of Biotechnology, Ministry of Science and Technology, for the financial support to this work. 

 ~ References Top

1.The Eurogast study group. Epidemiology and risk factors for Helicobacter pylori infection among 3194 asymptomatic subjects in 17 population. Gut 1993:34:1672-1676.   Back to cited text no. 1    
2.Dunn BE, Cohen H, Blaser MJ. Helicobacter pylori. Clin Microbiol Rev 1997;10(4): 720-741.  Back to cited text no. 2    
3.Hoffman PS. Antibiotic resistance mechanisms of Helicobacter pylori. Can J Gastroenterol 1999; 13(3):243-249.  Back to cited text no. 3    
4.Correa P, Fox J, Fontham E, Ruiz B, Lin YP, Zavala D, Taylor N, Mackinley D, de Lima E, Portilla H, et al. Helicobacter pylori and gastric carcinoma. Serum antibody prevalence in populations with contrasting cancer risk. Cancer 1990:66:2569-2574.  Back to cited text no. 4    
5.Salcedo JA, Al-Kawas F. Treatment of Helicobacter pylori infection. Arch Intern Med 1998;158:842-851.  Back to cited text no. 5    
6.Veldhuyzen van Zanten S, Hunt RH, Cockeram A, Schep G, Malatjalian D, Sidorov J, Matisko A, Jellell D. Adding once-daily omeprazole 20mg to metronidazole / amoxycillin treatment for Helicobacter pylori gastritis: A randomized, double-blind trial showing the importance of metronidazole resistance. Am J Gastroenterol 1998;93:5-10.  Back to cited text no. 6    
7.Megraud F. Resistance to Helicobacter pylori to antibiotics. Aliment Pharmacol Ther 1997;11(Suppl 1):43-53.  Back to cited text no. 7  [PUBMED]  
8.Penfold SS, Lastovica AJ, Elisha BG. Demonstration of plasmids in Campylobacter pylori. J Infect Dis 1988;157(4): 850-851.  Back to cited text no. 8    
9.Heuermann D, Haas R. Genetic organization of a small cryptic plasmid of Helicobacter pylori. Gene 1995;165:17-24.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Smith SI, Miehlke S, Oyedeji KS, Arigbabu AA, Coker AO. Fingerprinting of Nigerian Helicobacter pylori isolates by plasmid profile and PCR. J Basic Microbiol 2002:42(1):45-53.  Back to cited text no. 10    
11.Owen RJ, Bell GD, Desai M, Moreno M, Gant PW, Jones PH, Linton D. Biotype and molecular fingerprints of metronidazole resistant strains of Helicobacter pylori from antral gastric mucosa. J Med Microbiol 1993;38:6-12.   Back to cited text no. 11  [PUBMED]  
12.Thyagarajan SP, Ray P, Das BK, Ayyagari A, Khan AA, Dharmalingam S, Rao UA, Rajasambandam P, Ramathilagam B, Bhasin D, Sharma MP, Naik SR, Habibullah CM. Geographic difference in antimicrobial resistance pattern of Helicobacter pylori clinical isolates from Indian patients: a multicentric study. J Gastroenterol Hepatol (in Press).  Back to cited text no. 12    
13.van der Wouden EJ, Thijs JC, van Zwet AA, Kleibeuker JH. Nitroimdazole resistance in Helicobacter pylori: a review. Aliment Pharmacol Ther 2000;14(1):7-14.   Back to cited text no. 13    
14.Chowdhury A, Berg DE, Jeong JY, Mukhopadhyay AK, Nair BK. Metronidazole resistance in Helicobacter pylori. Magnitude, mechanism and implications for India. Indian J Gastroenterol 2002;21:23-28.  Back to cited text no. 14    
15.Taylor DE, Ge Z, Purych D, Lo T, Hiratsuks 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.  Back to cited text no. 15    
16.Xia HX, Fan XG, Talley NJ. Clarithromycin resistance in Helicobacter pylori and its clinical relavance. World J Gastroenterol 1999;5(3):263-266.  Back to cited text no. 16    
17.Gerrits MM, Schuijffel D, van Zwef AA, Kuipers EJ, Vanderbroucke-Grauls CMJE, Kusters JG. Alterations in penicillin-binding protein 1A confer resistance to -lactam antibiotics in Helicobacter pylori. Antimicrob Agents Chemother 2002; 46(7): 2229-2233.  Back to cited text no. 17    
18.Paul R, Postius S, Melchers K, Schafer KP. Mutations of Helicobacter pylori genes rdxA and pbp1 cause resistance against Metronidazole and Amoxycillin. Antimicrob Agents Chemother 2001;45(3):962-965.  Back to cited text no. 18    
19.Moore RA, Beckthold B, Wong S, Kureishi A, Bryan LE. Nucleotide sequence of the gyrA gene and characterization of ciprofloxacin-resistant mutants of Helicobacter pylori. Antimicrob Agents Chemother 1995;39:107-111.  Back to cited text no. 19    
20.Yakoob J, Fan X, Hu G, Liu L, Zhang Z. Antibiotic susceptibility of Helicobacter pylori in the Chinese population. J Gastroenterol Hepatol 2001;16:981-985.  Back to cited text no. 20    
21.Hosaka Y, Irinoda K, Nakano R, Kitasato H, Okamoto R, Saigenji K, Inoue M. Antibacterial activity of 16 antibiotics against Helicobacter pylori. Jpn J Antibiot 2000;53(10):623-630.  Back to cited text no. 21    
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