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Neelain.edu.sd

Clarithromycin-Resistant Helicobacter Pylori Strains among
Dyspeptic Patients in Sudan
Nazar Abdalazeem*1, Hassan Abdul-Aziz1, Adam Ahmed Adam2, Waleed Hussein Omer2 and *1Ahfad University for Women, Omdurman, Sudan 1Alribat University, Academic Affairs 2Al-Neelain University, Faculty of Medicine and Al-Neelain Medical Research Centre, Sudan Corresponding author: P.O.Box:167, Omdurman, Sudan Tel 00249912476762. ABSTRACT
Introduction: The study aimed at characterizing the mutations in 23S rRNA gene related to
Clarithromycin-resistant Helicobacter pylori strains among dyspeptic patients in Khartoum State.
Methods:
Two hundred gastric biopsies were obtained by endoscopy from 200 patients with
dyspepsia. DNA was extracted from culture isolated and relevant mutations in 23S rRNA gene
were detected.
Results: Out of the 200 biopsies, H. pylori was isolated from 48 (24%) biopsies. Twelve of them
were found to be resistant to Clarithromycin. Eight of the resistant strains had both A2143G and
A2142G by using restriction enzymes Bsa1and Bbs1. Sequencing the remaining four isolates by
PCR detected A2140G mutation.
Conclusions: In conclusion, Clarithromycin-resistant H. pylori in Sudan may be the main cause
of treatment failure aiming at eradication of the bacterium from patients. Such a finding may
necessitate the need for other treatment regimens. More collaborated research in this field is
needed.

Keywords: Helicobacter pylori, Clarithromycin resistance.
INTRODUCTION
bacterium that causes chronic gastritis and peptic ulcer disease . Infection with H. pylori can be effectively treated by the Clarithromycin is a macrolide that binds to combination of a proton pump inhibitor in the 23S rRNA components of the bacterial addition Clarithromycin and amoxicillin. ribosome. Resistant strains emerge due to failure of such binding because of modification of the target site by point mutations in the peptidyl transferase region macrolide resistance, but the most common H. pylori contains two copies of the 23S (A2143G)(3, 4). The commonest mutation site rRNA gene8. Several points mutations have MATERIALS AND METHODS
Study Design:The study is a descriptive
Study Area: The study was carried out in
analytic cross–sectional hospital based one. the GIT Units in Alribat Teaching Hospital, Al Neelain Medical Centre and Asia Study Objective: The study aimed at
characterizing the mutations in 23S rRNA gene related to Clarithromycin-resistant Study Population: Any dyspeptic patient
Helicobacter pylori strains among dyspeptic study area had an equal chance to be enrolled. Specific Objectives:
Study Sample: The sample included 200
Helicobacter pylori detection among dyspeptic patients undergoing upper Selection Criteria: A valid verbal consent
Ethical Consideration: Each participant
was informed about his/her role in study and consent. The caretakers validated the consent of the insane and underage participants. The specimens collected were Collection and transportation of
Culturing of specimen: A Specimen from
specimens: Upper gastrointestinal
containing agar base (brain heart infusion affected sites. Each biopsy was transferred agar), selective supplement (Skirrow) and immediately to a buffered normal saline as a transport medium and processed in no more microaerophilic kits (campylobacter system quality control of antibiotic susceptibility Identification of isolated bacterial
growth: A- Macro examination: Growths of
DNA Extraction: DNA was extracted from
observed after 3 to 5 days on the brain heart infusion agar plated with specimens from according to manufacturer's instructions examination of gram-stained films from the C- Biochemical test: Bacteria growths were further identified by the biochemical tests of cytochrome oxidase, catalase, and urease transferred to a clean epindorf tube containing 99% isopropanol. Antimicrobial sensitivity test: For this
of H. pylori were regrown on brain heart mutations
and Skirrow. A suspension from 3-day-old Clarithromycin resistance in the 23S
bacterial cells of each isolate was prepared rRNA gene:
in brain heart infusion broth (2 ml) equivalent to the McFarland turbidity PCR Amplification:
standard. The suspensions were spread on the surface of the brain heart agar base with cotton swabs. The plates were briefly dried clarithromycin (15 micrograms), were added to each plate and incubated microaerobically at 37 ºC for 3-5 days. One plate without antibiotic disk was also incubated in each Amplification was carried out in a thermal cycler (Thermo Hybaid, MBS 0.5 S). The inhibition zone diameter was measured PCR cycling conditions consisted of one in millimeters, with a ruler. Resistance was cycle at 94°C for five min., 40 cycles at determined by a zone of growth inhibition 94°C for one min, 55°C for one min. and 72°C for 1 min, and one cycle at 72°C for clarithromycin. Greater zones of complete growth inhibition indicated the presence of susceptible strains. H. pylori strain ATCC 26695 was used as a reference strain for the RFLP Analysis:
detect restriction site that correspond to the Data Analysis: All data were analyzed by
SPSS soft ware programme.
of them were resistant to Clarithromycin. The male participants constituted 108 (54%) Eight of the resistant isolates showed point mutation at both positions of A2143G and participants’ age ranged between 10 and 89 enzymes. The remaining four isolates were Out of the 200 gastric biopsies, 116 (58%) found to have point mutation at position were positive for H. pylori. Out of the 200 cultured specimens, 48 showed growths; 12 Table (2) Susceptibility of the culture isolates to Clarithromycin-by culture A2142G by BsaI and BbsI restriction enzymes A to G mutation at position 2140 by PCR sequencing (Figure-1) Detection of mutation A2143G by BSa1 restriction digestion, the restriction Fragments of the 267-bp PCR products were analyzed by electrophoresis on a 3% agarose gel, Lanes (A) 50-bp DNA Step Ladder molecular size markers (Promega). Lanes (B) and (C), PCR products of H. pylori strains digested with BSaI. Lanes (D), PCR products of the control strain A2143G H. pylori strains digested with BsaI restriction enzymes. Lanes (E) and (F), PCR products of H. pylori strains dos not digest with BSaI. (Figure.2) Detection of mutation A21432G by Bbs1 restriction digestion, the restriction Fragments of the 267-bp PCR products were analyzed by electrophoresis on a 3% agarose gel, Lanes (A) 50-bp DNA Step Ladder molecular size markers (Promega). Lanes (B) and (C), PCR products of H. pylori strains digested with BbsI. Lanes (D), PCR products of the control strain A2142G H. pylori strains digested with BbsI restriction enzymes. Lanes (E) and (F), PCR products of H. pylori strains does not digest with BbsI. DISCUSSION
The prevalence of Helicobacter pylori in this study was 24%. Azim et al in a similar study clarithromycin. The primary risk factor for in Sudan reported a prevalence of 50% by resistance to clarithromycin is previous Iran reported a culture isolate rate of 31.94% infected with resistant or co-infected with Helicobacter pylori differs in different resistant and sensitive strains should be elucidated. Such a difference may be related to the clinical differences of the patients and a workable quick and accurate method for susceptibility to clarithromycin within 24 Helicobacter pylori isolates were found to be resistant to clarithromycin. As far as the In case of a clarithromycin resistance rate of published data can provide, this study was resistance was reported from Israel (7 ). Mohammad et al in Iran reported 22.62% prevalence of Helicobacter pylori resistance to clarithromycin which was comparable to the finding in this study (6). Clarithromycin is an important component of the triple therapy aiming at eradication of Helicobacter pylori from infected patients. the main cause of treatment failure. Mutations in 23S rRNA gene are the well recognized aetiology in the emergence of Helicobacter pylori resistant strains and A2143G is the commonest. Eight clarithromycin-resistant isolates showed A2143G and A2142G mutations. More than one mutation in the 23S rRNA may make the situation worse. The DNAs of four resistant isolates were not digested by the restriction enzymes in use. Their sequencing by PCR showed A2140G mutation. This finding was not previously reported with the mutations of in the 23S rRNA gene. The liberal use of macrolides for treatment of different infections may urge emergence of resistant
Conclusion: Helicobacter pylori resistance to clarithromycin is becoming a real
problem in treatment of patients with peptic ulcer disease. More studies in this field
are needed to make the situation clear for treatment options.
Acknowledgement: We are grateful to the staff in Al-Neelain Medical Research
centre and the endoscopic units of Al-Ribat Alwatani and Asia Hospitals for their
great help.
Disclosure: There is nothing to disclose.

REFERENCE

1. Blaser MJ. Ecology of Helicobacter pylori in the human stomach, J Clin Invest. 2. Dunn BE, Cohen H, Blaser MJ. Helicobacter pylori. Clinical Microbiology 3. Verlasovic J, Osato M, Spakovsky, Dore M, Reddy R, Stone G, et al. Point of mutations in the 23S rRNA gen of Helicobacter pylori associated with different levels of clarithromycin resistance. J Antimicrob Chemother 1997; 40: 283-6. 4. Taylor D, Ge Z, Purych D, Lo T and Hiratsuka K. Cloning and Sequence Analysis of Two copies of a 23 S rRNA Gene from Helicobacter pylori and Association of Clarithromycin Resistance with 23S RNAr Mutations. Antimicrob Agents Chemother 2008; 41: 2621-8. 5. Azim Mirghani YA, Ahmed S, Ahmed M, Ismail MO, Fedail SS, Kamel M, Saidia H., Detection of Helicobacter pylori in endoscopic biopsies in Suda1994 Oct;24(4):161-3. 6. Mohammad Kargar1, Maryam Baghernejad1, Abbas Doosti2 and Sadegh Ghorbani-Dalini1, Clarithromycin resistance and 23S rRNA mutations in Helicobacter pylori isolates in Iran, African Journal of Microbiology Research April, 2011,Vol. 5(8) pp. 853-856 7. Noam Zevit, Itzhak Levy, Haim Shmuely, Zmira Samra and Jacob Yahav, Antibiotic resistance of Helicobacter pylori in Israeli children, Scandinavian Journal of Gastroenterology, May 2010, Vol. 45, No. 5 , Pages 550-555

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