Antimicrobial-Resistance Profile of Helicobacter pylori, Obtained from Endoscopic Patients in Bahir Dar, North West Ethiopia

Background Antimicrobial resistance for Helicobacter pylori infection is a highly emerging problem throughout the world to treat gastric-associated diseases. People living in developing countries are more likely to acquire a Helicobacter pylori infection and less likely to gate treatment after infection due to poverty. Therefore, the current study was aimed at determining the magnitude and antibiotic-resistance profile of Helicobacter pylori isolated from patients who underwent endoscopic examination. Methods A cross-sectional study was conducted from January to May 2019 at endoscopy service-providing health facilities that are found in Bahir Dar, Ethiopia (Gamby teaching general hospital, Kidane Mihret specialty higher clinic, and Eyasta specialty higher clinic). Data were collected using a pretested, structured questionnaire. Antibiotic susceptibility of Helicobacter pylori isolates from gastric biopsies was determined. Data were analyzed using SPSS version 23. Results The 17.8% proportion of Helicobacter pylori was isolated from 135 endoscopy-examined patients (24/135). The majority of isolates (71% of 17/24) were from males, while only 29% of 7/24) were from females. Antimicrobial-resistance of Helicobacter pylori was high to all commonly prescribed antibiotics: amoxicillin and metronidazole (91.7%), clarithromycin and ciprofloxacin (66.7% each), and tetracycline (37.5%). Conclusion Helicobacter pylori isolates from the current study participants were rather low in number. But the highest antibiotic-resistance profile of Helicobacter pylori was observed. Therefore, these findings alarmingly indicate that routine antimicrobial susceptibility testing against Helicobacter pylori isolates is crucial for better patient management.


Introduction
Te Helicobacter pylori (H.pylori) is a Gram-negative, fourto-seven polar fagellated bacterium with spiral or curved morphology [1].It has a microaerophilic oxygen requirement.Te bacterium was frst isolated from the gastric culture in 1982 by Australian scientists Robin Warren and Barry Marshall [2].It is classifed under the kingdom Monera, domain Bacteria, phylum Proteobacteria, class Epsilonproteobacteria, order Campylobacterales, family Helicobactericeae, and genus Helicobacter [3].
H. pylori are the causative agent for gastric-associated diseases like gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma [4][5][6].Based on its global impact, the International Agency for Cancer Research and the World Health Organization (WHO) grouped H. pylori as a class I biological carcinogenic agent [7].
It is possible to be cured of the H. pylori infection with a proper treatment regimen.Among the treatment regimens, combinations containing clarithromycin and/or amoxicillin, metronidazole, tetracycline, and ciprofoxacin are the most commonly utilized antibiotics with proton pump inhibitors [6,8,9].But its antimicrobial resistance is worsening the treatment outcome [10,11].
Based on clarithromycin resistance, H. pylori was categorized as one of the World Health Organization (WHO) priority pathogens listed for research and development of new antibiotics [12].
Although global amoxicillin resistance is low, there are H. pylori strains with high levels of amoxicillin resistance that are related with beta-lactamase synthesis.Penicillinbinding protien1A point mutation has been associated to low-level amoxicillin resistance [13].
Te emergence of multidrug-resistant (MDR) strains of H. pylori is a global health concern.
Studies across the world are approving that H. pylori is becoming among the superbugs and recommending antimicrobial sensitivity testing prior to eradication therapy [14].
Particularly in low-and middle-income countries, the problem is worsening due to a lack of treatment options that include bismuth salt, including regimens, diagnostic tools, and skills for case detection [14][15][16].
Because of the worldwide increase in antibiotic resistance, most guidelines have recommended antimicrobial susceptibility testing to guide H. pylori eradication treatment [17].
However, due to the fastidious nature of H. pylori and the lack of a simple and cost-efective testing method, antimicrobial susceptibility testing for H. pylori is not available in Ethiopia.Profling regional or population-specifc antibiogram patterns is critical in directing the creation of effective empiric therapy regimens in such circumstances.As a result, the purpose of this study is to present the resistance profle of H. pylori in the study area.

Study Design and
Period.An institutional-based crosssectional study was undertaken on patients with gastricassociated disorders who had endoscopic examinations from January to May of 2019.

Sociodemographic and Clinical Data Collection.
During interviews, structured questioning was utilized to collect sociodemographic data from research participants.Participants' clinical state was also examined during endoscopic examinations by checking their patient charts and discussing with their physicians.Participants who were receiving eradication or antiacid therapy were barred from participation in the study.

Specimen Collection, Transport, and Processing.
Te lowest compliance for the Sydney protocol was obtained during biopsy specimen collection, according to the Sydney protocol.During an endoscopic examination of diferent anatomical areas of the stomach, two to four gastric biopsy specimens were taken from suspected gastric-associated diseased patients using sterile endoscope forceps [18].During specimen collection, any potential risk of an adverse outcome was assessed, and a clinical decision was made.To improve patient comfort and endoscopic performance, sedation for endoscopic procedures was maintained by delivering 1 ml (5 mg) of diazepam via intravenous root and 4-5 ml of lidocaine spray via rinsing the mouth.Biopsy specimens were instantly inoculated into brain-heart infusion broth and immediately delivered to Bahir Dar University's College of Medical and Health Sciences' Medical Microbiology Laboratory.After 48 hours of incubation, inoculated broths were subcultured onto Charcoal Cefoperazone Deoxycholate Agar (CCDA) and Campy Agar supplemented with 5% sheep blood [19].Secondary subculture was performed after 10 days of incubation for negative broths.In candle jars with a microaerobic environment (5% O 2 , 10% CO 2 , and 85% N 2 ), inoculated broth bottles and culture plates were incubated.Gasgenerating kits and anaerobic catalysts (OXOID, England) were used to keep the environment microaerobic.Subcultures were checked for bacterial development until the third day of inoculation.Furthermore, negative culture results were only issued after inspection of cultures every 24 hours for ten days of incubation.Te antimicrobial sensitivity test was the standardized Kirby-Bauer technique (disc difusion) test, which adhered to McNulty et al. recommendation.On Mueller-Hinton agar bases with 10% blood, a suspension equivalent to the McFarland standard was used.Ten, commonly used antibiotics were added, namely, metronidazole (MTZ 5 μg), amoxicillin (AMX 10 μg), tetracycline (TET 30 μg), erythromycin (ERY 15 μg), clarithromycin (CLT 5 μg), ciprofoxacin (CPR 5 μg), and amoxicillin with clavulanic or Augmentin (AUG 20/10 μg).

Bacterial Identifcation.
Te selective media that allow the growth of H. pylori species were used.A range of identifcation assays were used on preliminary bacterial colonies, including Gram staining, oxidase, catalase, and urease tests.Being microaerophilic and the need for selective and enriched media are features of H. pylori [20].
2.6.Quality Control.Te data gathering questionnaire was designed to be simple to use.Each participant's format was reviewed for completeness during the data collecting period.Standard operating procedures (SOPs) were also followed to ensure the quality of the research from preanalytical to postanalytical.In each sterilization operation, Geobacillus stearothermophilus (ATCC 7953) was autoclaved and incubated for growth detection.For the sterility test, 5% of each batch of media preparation was incubated overnight at 35-37 °C.Te Campylobacter jejuni (ATCC 33560) strain, which exhibits growth characteristics similar to those of H. pylori strains, was used to evaluate the culture media and incubation environment.Te majority of individuals in the culture-positive trial (75%, 18/24) were married.Furthermore, all of the culture-positive results (24/24) came from study participants with low income.

Antimicrobial-Resistance Profle of H. pylori Isolates.
All 24 isolates of H. pylori were tested for commonly used antimicrobials.Te antimicrobial susceptibility profle of H. pylori isolates showed resistance to more than one antimicrobial agent.For instance, antimicrobial-resistant H. pylori isolates were detected against amoxicillin and metronidazole (91.7% each), clarithromycin, ciprofoxacin, and erythromycin (66.7% each), while tetracycline was only at 37.5%.Resistance to commonly prescribed antibiotics was found to be more prevalent in patients with any type of gastritis, including gastric ulcers and atrophied gastritis.

Discussion
Antimicrobial resistance is currently a major global problem [21,22].As antibiotic resistance reduces treatment efcacy, it is time to consider routine susceptibility testing to guide individual patient treatment and monitor antibiotic resistance.Te burden of resistant strains of H. pylori remains a major challenge in the course of the treatment regimen [23][24][25].Triple or quadruple therapy is frequently prescribed by most clinicians.Tis strategy consists of two antibiotics plus a proton pump inhibitor or/and bismuth salt [26].Te current study also found that antimicrobial resistance is becoming more difcult in the study area.
In the current study, all H. pylori isolates from patients with gastric-associated diseases were resistant to more than one antibiotic.Majorities of H. pylori isolates were resistant to amoxicillin (91.7%), although global H. pylori resistance to amoxicillin is extremely contradictory and low [27,28].Tis might be an indication for evidence of the irrational use of amoxicillin for the treatment of diferent gastrointestinal infectious diseases.
In this study, the resistance rates of commonly prescribed antibiotics such as amoxicillin and metronidazole were 91.7% each, clarithromycin and ciprofoxacin were 66.7% each, and tetracycline was 37.5%.Similarly, a study conducted in the western part of Nigeria indicated that H. pylori isolates were resistant to common antibiotics: amoxicillin (66%), erythromycin (78%), metronidazole (95%), and tetracycline (100%) [29].
Te clarithromycin resistance fnding in this study indicates that the treatment regimen in the study area should be in concordance with the regimen protocol suggested for clarithromycin resistance >20% regions.Furthermore, WHO is alarmingly aware that H. pylori with clarithromycin resistance are the priority pathogens for new treatment protocol development [12].
However, a previous study conducted in the United States revealed that a lower proportion of H. pylori isolates (25.1%), clarithromycin (12.9%), and amoxicillin (0.9%) were resistant.In contrast to our fndings, the same study reported a very high susceptibility to amoxicillin [23].Furthermore, diferent studies reported a lower resistance proportion to amoxicillin [30].Studies indicated that highlevel amoxicillin resistance is associated with beta-lactamase production in H. pylori, and low-level amoxicillin resistance is linked to a point mutation on penicillin-binding pro-tien1A [13].It seems that there is a high prevalence of betalactamase producing H. pylori strains at the study area.Te broad use of this antibiotic to treat diferent infectious diseases could contribute to the development of more resistant strains.
In contrast to our study fndings, a study conducted on adult dyspeptic patients at Tikur Anbassa University Hospital, Ethiopia, indicated that all H. pylori isolates were sensitive to clarithromycin, tetracycline, and erythromycin, and only 6% of the isolates were resistant to amoxicillin, which was a much lower resistance rate than our study fnding [31].
In the present study, all strains of H. pylori isolates were multidrug resistant.Among all, thirteen (54.2%) are resistance for three drugs and three of all showed a MDR profle for six commonly prescribed antibiotics.From the study that was conducted to determine the frequency of MDR H. pylori strains in Iran, primary, double, and Canadian Journal of Infectious Diseases and Medical Microbiology multidrug resistance frequencies were assessed.Among the resistant strains: the rates of double and multiple drug resistance phenotypes were 22.6% (19/84) and 34.5% (29/84), respectively, which reviled lesser MDR prevalence compared with this study [32].

Limitations
Tis study considered only severely ill gastric-associated diseased participants who were sent for endoscopic examinations and might have a repeated eradication treatment history.But gastric complaints or patients in outpatient departments were being seen at growing rates which did not included in this study.Another limitation was the inability to get previously done culture-based studies on H. pylori infection antimicrobial resistance profles at the study area for better comparison.

Conclusions
Antimicrobial resistance of H. pylori isolates is so alarming that susceptibility testing before the initiation of treatment is vital for efective treatment of H. pylori-associated gastroduodenal diseases.Adherence to the World Health Organization's (WHO) recommended standard treatment regimens in regions with a history of high clarithromycin

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Canadian Journal of Infectious Diseases and Medical Microbiology 2.7.Data Processing and Analysis.Te collected data were validated for correctness before being entered into Epi Data software version 3.1.Te data were then imported into and analyzed with SPSS version 23.Data analysis was illustrated using frequency tables, and its relationship to relevant factors was examined.P values less than 0.05 were considered statistically signifcant.

Table 1 :
Antimicrobial-resistant profle of H. pylori isolates from patients attending endoscopic examination.Canadian Journal of Infectious Diseases and Medical Microbiology resistance will warrant better treatment outcomes.Further studies should be carried out on resistance mechanisms for better management of H. pylori-induced gastric-associated diseases.Further molecular investigations for the clarithromycin-resistance mechanism shall be conducted.Quinolones containing treatment regimens has to be also supported by antimicrobial sensitivity testing for better eradication treatment outcomes.