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
and Skirrow. A suspension from 3-day-old
Clarithromycin resistance in the 23S
bacterial cells of each isolate was prepared
in brain heart infusion broth (2 ml) equivalent to the McFarland turbidity
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
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.
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
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