Prevalence and antimicrobial susceptibility profile of Helicobacter pylori clinical isolates of patients with chronic gastritis and peptic ulcer in Jordan Mohammad K. Abu-Sini1, Luay F. Abu-Qatouseh2, Osama Refai2, Penelope Shihab2, Rula M. Darwish1, Talal A. Aburjai1 1Faculty of Pharmacy, University of Jordan, Amman, Jordan 2Jordan Company for Antibody Production (MONOJO), Amman, Jordan Summary Introduction: Gastritis and peptic ulcer are considered major health problems worldwide. It has been reported that more than 80% of chronic active gastritis are due to the pathogenic bacterium Helicobacter pylori where persistent infection remains for decades. Successful treatment of H. pylori routinely requires the use of multiple agents with different mechanisms including compounds inhibiting acid secretion in conjunction with Antibiotics. However, recent data showed the emergence of resistant clinical strains particularly against metronidazole and clarithromycin. The aim of this study is to determine the prevalence of and the susceptibility of H. pylori isolates recovered from patients with chronic gastritis or peptic ulcer to several antimicrobial agents in vitro. Materials and Methods: A prospective study has been conducting on Jordanian patients attended the gastrointestinal unit of the Jordan university hospital starting from May 2012 with gastro-duodenal problems. Both Antral and corpus mucosal biopsies from the stomach of each patient were obtained, processed and tested by rapid urease test and cultured on selective media (Columbia blood Agar containing 7% laked horse blood and dent selective supplement). Presumptive H. pylori colonies were subsequently confirmed by biochemical tests including catalase, oxidase and rapid urease tests. The antimicrobial susceptibility testing was performed by standard agar diffusion methods according to CLSI. Subsequently, MICs were determined by E test and methods. Results: Among 60 symptomatic patients, 35% (21 patients) showed positive H. pylori infection by both rapid urease test and culture. The antibiotic susceptibility profile of 10 strains were tested and showed that all of the isolates were sensitive to amoxicillin and 90% to levofloxacin. Resistance to ciprofloxacin (MIC ≥ 4.0 µg/ml) and clarithromycin (MIC ≥ 2.0 µg/ml) were observed in 10% of the isolated while 80% of the strains were resistant to metronidazole (MIC ≥ 8.0 µg/ml). Conclusion: The present study showed that the prevalence of metronidazole resistance among clinical isolates of H. pylori is very high. Lower resistance to other antibiotics are reported. Concern should be taken into consideration when metronidazole is used for the treatment of H. pylori in our region. Introduction Helicobacter pylori (H. pylori), is a Gram negative curved rod bacterium considered as the most important etiological agent of the human peptic ulcer and one of the most common chronic bacterial pathogens of humans (Figure 1). It colonizes the gastric epithelial surface and withstands the stomach's hostile environment by microaerophilic growth capacity and the production of numerous virulence factors which may lead to chronic gastritis, peptic ulceration and gastric cancer in the later life (1). Infections with H. pylori in the developing countries have been reported to be higher than in developed countries. In Africa, 70 to 80 % are infected with the organism and 61 to 100 % harbour the organism in sub- Saharan Africa (2). Although accurate non-invasive methods such as the urea breath test, the stool antigen test, and serology are available, biopsy based invasive techniques including the rapid urease test, histology and culture, are required to confirm the infection (3). Moreover, isolation of H. pylori from gastric mucosal biopsy specimens is important to confirm H. pylori as the causative agent of gastritis and is a prerequisite for further studies of the organism addressing drug susceptibility testing, analysis and characterization of virulence factors, molecular epidemiology studying or other comparative studies (4). Treatment of gastric ulcer could be achieved by a combination of therapeutic agents such as antibiotics, bismuth subsalicylate, proton pump inhibitors and H2-blockers (5). H. pylori have developed resistance against most antibiotics, especially metronidazole, which limits their use in the treatment of infections. This problem is being encountered more in Africa (6). Figure 1: Morphology of H. pylori; small translucent colonies (upper), gram negative curved rods (lower). Results Twenty one patients (35%) among 60 patients with clinical symptoms were diagnosed with positive H. pylori infection by both rapid urease test and culture. Antimicrobial susceptibility of standard antibiotics against 10 strains were tested. All tested isolates were susceptible to amoxicillin with MIC range (003 - <0.015 µg/ml) (Table 1). Also 90% of the isolates were susceptible to levofloxacin with MIC range (0.008 - >32 µg/ml). Resistance to ciprofloxacin 30% (MIC ≥ 4.0 µg/ml) and clarithromycin 10% (MIC ≥ 2.0 µg/ml) were observed. Metronidazole had a reduced activity (MIC >256 µg/ml), 80% of the isolates were resistant to this compound (Figures 2 and 3). Sensitivity of clinical strains of H. pylori to standard antimicrobial agents (MIC µg/ml) Strain number Amoxicillin Levofloxacin Metronidazole Ciprofloxacin Clarithromycin St1 S St2 R (> 256) St3 R ( > 4.0) R (> 2.0) St4 St5 St6 St7 St8 R (> 32.0) R (> 16.0) St9 R (> 8.0) St10 Table 1: Antimicrobial susceptibilities of the clinical strains of H. pylori against standard antibiotics. Figure 3: Antimicrobial susceptibility tests [disc diffusion, MIC (E-test)] of standard antibiotics against clinical strains of H. pylori. Conclusion There is a reportable increase of resistance among the clinical strains of H. pylori toward the standard antimicrobial agents used in routine treatment course of gastritis. Care should be taken when metronidazole is used due to the dramatic resistance among the clinical strains. Amoxicillin and/or levofloxacin are potentially better alternatives to clarithromycin and metronidazole for the treatment of H. pylori infections. Materials and Methods This is a prospective study including inpatients and outpatients >18 years old selected from May 2012 , and consulting at the gastroenterology unit of the Jordan university hospital. This study involved patients with clinical symptoms which required a digestive endoscopy. Written and informed consent was obtained from all patients. Specimen Collection Antral and corpus mucosal biopsies from stomach were obtained by a gastroenterologist in Jordan University Hospital while making the endoscopic diagnosis of the patient. A biopsy from the antrum and another from the corpus were transferred into 4 ml aliquots containing 2% Proteose Peptone. This medium is considered as a transport medium. Sample Preparation and Culture Media Biopsy tissues were homogenized as follows: each biopsy for culture was homogenized using a tissue homogenizer. Aliquots of 100 µl of the homogenate were inoculated into Columbia blood agar containing 7% laked horse blood supplemented with Helicobacter pylori selective supplement (Dent). All supplements were added under aseptic conditions after sterilization of the media by autoclaving (121°C for 15 minutes). Identification of Helicobacter pylori H. pylori were identified on the basis of colonial morphology; gram staining and other conventional biochemical tests: catalase, oxidase and urease tests. Antimicrobial susceptibility testing The susceptibilities of the H. pylori to amoxicillin, metronidazole, clarithromycin, ciprofloxacin, and levofloxacin were determined by the disc diffusion method. H. pylori were grown on Columbia blood agar containing 7% laked horse blood at 37°C under microaerophilic conditions. Suspensions of fresh cultures were made in sterile PBS and turbidity was adjusted to 12X108 CFU/ml (corresponding to McFarland standards 4). Sterile swabs were used to inoculate plates of Columbia blood agar base containing 7% laked horse blood. Antibiotic discs including amoxicillin (25µg), metronidazole (5µg), clarithromycin (15µg), ciprofloxacin (5µg), and levofloxacin (5µg) were placed on the bacterial lawns and the plates were incubated at 37˚C under microaerophilic conditions for 7 days. After that zone of inhibition for each antibiotics were measured using calliper. Determination of Minimum Inhibitory Concentration (MIC) Susceptibility to antibiotics testing was performed via minimum inhibitory concentration (MIC) determination by E-test for amoxicillin, metronidazole, and levofloxacin according to the recommendations of Megraud et al., (7). The tested inoculum was adjusted to a turbidity of 4 McFarland standard. Sterile swabs were used to inoculate plates of Columbia blood agar base containing 7% laked horse blood. After 7 days of incubation at 37˚C in microaerophilic atmosphere, the MIC of each antibiotic was determined. References Tanih, N.F., Clarke, A.M., Mkwetshana, N., Green, E., Ndip, L.M., and Ndip, R.N. 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