Molecular characterization and antibiotic susceptibility profiles of Helicobacter pylori isolated from patients with Gastrodeudenal diseases in Jordan

Introduction: Helicobacter pylori is a major cause of more than 80% of chronic active gastritis and other gastrodeudonal diseases worldwide. 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 gastrodeudonal diseases to several antimicrobial agents. Materials and Methods: A prospective study has been conducting on Jordanian patients attended the gastrointestinal unit of the Jordan university hospital starting from 2014-2015 with gastroduodenal diseases. Antral and corpus mucosal biopsies from the stomach of each patient were used for the isolation of H. pylori on selective culture media. Presumptive H. pylori colonies were subsequently confirmed by biochemical tests and standard 16S rDNA PCR assay. The antimicrobial susceptibility testing was performed by standard agar diffusion methods according to CLSI. Subsequently, MICs were determined by E test and standard agar dilution method. Molecular typing of the clinical strains was performed using multiplex PCR for the detection of vacA and cagA genotypes. Metronidazole resistance was characterized by molecular methods for the detection of rdxA gene mutations. THE INTERNATIONAL ARABIC JOURNAL OF ANTIMICROBIAL AGENTS


Introduction
Recently, the medical community has been increasingly interested in Helicobacter pylori (H pylori) related diseases as several studies have been reporting a major role of H. pylori in chronic gastritis and peptic ulcer worldwide [1,2]. Colonization of the gastric mucosa with H. pylori constitutes a major risk factor in the pathogenesis of gastric cancer and gastric mucosa-associated lymphoid tissue (MALToma) [3][4][5].
H. pylori is equipped with an armamentarium of biological factors that enable this microaerophilic bacterium to survive in the harsh conditions present in human stomach. For instance, all strains of H. pylori possess a unique stable urease enzyme which is used to neutralize the stomach acidity and provide a suitable pH for the growth of this bacterium. In addition, the presence of numerous binding proteins and adhesion molecules ensure optimal colonization of H. pylori in the gastric mucosa [6]. Among the H. pylori virulence factors, cytotoxin A (CagA) and vacuolating cytotoxin A (VacA) are the most studied since the production of these toxins increases the risk for gastric atrophy, intestinal metaplasia and gastric carcinoma [7,8]. However, genetic variation within the genes encoding these virulence factors contributes to differences in the pathogenicity of strains [9]. It has been reported that a round 70% of the H. pylori strains isolated in the western countries possess the cagA gene while higher prevalence of cagA-positive strains is Results: Among 72 symptomatic patients, 13(23%) patients showed positive H. pylori infection by both rapid urease test and culture. The antibiotic susceptibility profile showed that all of the isolates were sensitive to amoxicillin. Resistance to, clarithromycin, ciprofloxacin and levofloxacin were observed in 15%, 23% and 8% of the isolates respectively while 92% of the strains were resistant to metronidazole (MIC ≥ 32ug/ml). Metronidazole resistance due to mutations in rdxA gene was only observed in one strain (8%) suggesting other resistance mechanisms. Correlation between antibiotic resistance and virulence factors was statistically not significant (p > 0.05).
reported in East Asian countries [10,11]. On the other hand, vacA is present in approximately all H. pylori strains however, allelic variation in three main genetic regions encoding and regulating this protein has been observed [12,13]. Depending on the type and combinations of vacA different alleles, distinct abilities of vacA to induce vacuolation in epithelial cells can occur [14]. Associations between infection with strains containing certain vacA alleles and increased risk for progression of gastric precancerous lesions and for gastric carcinoma were also reported [15,16].
The eradication of H. pylori and attenuating its associated virulence factors can contribute to improve clinical conditions of the patients infected with this bacterium including accelerating peptic ulcer healing and minimizing the recurrence of gastric cancer. Current approaches for effective eradication of H. pylori include the use of at least two antibiotics and a proton pump inhibitor (triple and more recently quadruple therapy [17,18]. Triple therapy, including two antibiotics, amoxicillin and clarithromycin, and a proton pump inhibitor given for a week has been recommended as the treatment of choice [19]. Other treatments have also been proposed, including metronidazole, as well as tetracycline, fluoroquinolones, and rifamycins. However, due to the emergence of antibiotic resistance among H. pylori clinical strains particularly against clarithromycin, and to a lesser extent metronidazole, higher rates of treatment failure of triple therapy were reported [21,22]. Global studies in Europe reported a prevalence of H. pylori resistance to clarithromycin among adults and children of approximately 20%. In contrast, higher rates of resistance against metronidazole were reported in Europe and USA [23,24].
Only few data regarding the resistance of H. pylori clinical strains to the conventional antimicrobial agents used in the clinical practice were reported which might be due to the technical difficulties to isolate and perform the standard methods for antimicrobial susceptibility testing on H. pylori. Our aim is to study the prevalence of H. pylori resistance to these various antibiotics especially in light of the resistance mechanism using both microbiological and molecular methods in addition to characterize the virulence factors and their possible association with antibiotic resistance among H. pylori strains.

Materials and methods
Patients and specimens. Antral and corpus mucosal gastric biopsies from stomach of 72 Jordanian patients attending the gastrointestinal unit of the Jordan University Hospital presented with any of the following clinical manifestations active gastritis, peptic ulcer or gastric cancer were obtained. Informed written consent was obtained from all patients participating in this study after ethical approval has been provided. Biopsies were transferred into 4 ml aliquots containing 1% Proteose Peptone (Oxoid, UK) for transport purposes.
Isolation and identification of H. pylori. For optimal recovery of clinical strains of H. pylori, all biopsy tissues were homogenized using a tissue homogenizer (IKA, Germany) and aliquots of 100 µl of the homogenate of each sample were inoculated into Columbia blood agar base (Oxoid, UK) containing 7 % laked horse blood and Helicobacter pylori selective supplement (Dent, Oxoid, UK) [25]. All of the plates were incubated at 37°C under microaerophilic conditions using CampyGen atmosphere generating system (Oxoid, UK) in anaerobic Jars for 5-7 days. Growth of H. pylori was confirmed according to colony morphology, Gram staining, microaerophilic growth at 37°C, oxidase, catalase and urease and subsequently by standard PCR of 16S rDNA test [26]. H. pylori cultures were stored at -70°C in Trypticase soy broth (Oxoid, UK) containing 10% v/v fetal calf serum (PAA, Austria) and 15% glycerol.
Antimicrobial susceptibility testing and MIC measurements. Bacterial suspensions were prepared to the 2 McFarland's standard and subse- quently uniformly spread on a solid growth medium in a Petri dish. Antibiotic discs including amoxicillin (25 µg), metronidazole (5 µg), clarithromycin (15 µg), ciprofloxacin (5 µg), and levofloxacin (5 µg) (Mast, USA) were used. Plates were incubated for 5-7 days under appropriate cultivation conditions. Antibacterial activity was determined by the production of an inhibition zone around the disc. The minimal inhibitory concentrations (MICs) of each antimicrobial agent were determined by both E-test (Mast, USA) and by the two fold agar dilution method according to CLSI recommendations [27]. Antimicrobial agents were provided as powder from local pharmaceutical manufacturers and dissolved in distilled water for stock solutions. Next, two fold serial dilutions of each antimicrobial agent incorporated to molten Columbia blood agar plates supplemented with 7% (v/v) horse blood were prepared. Spots of H. pylori (1x 10 5 CFU) were applied on the surface of each plate and the growth of visible colonies was determined after 5-7 days of incubation at 37°C under microaerophilic conditions. MIC was recorded as the lowest concentration that inhibited visible growth of H. pylori in the agar dilution method and by measuring the ellipse edge intersects the strip in E-test. Triplicates of each antimicrobial agent were performed and the average of the results was taken. The resistance breakpoints of antibiotics were defined for amoxicillin ≥ 0.5 µg/ml, metronidazole > 8.0 µg/ml, ciprofloxacin and levofloxacin > 1.0 µg/ ml and clarithromycin ≥ 1.0 µg/ml.
Detection of cagA and Vac A alleles. Detection of CagA gene, and VacA allele variation was performed as described by Atherone et al., 1995 and Zheng et al., 2000 with slight modifications [28,29] (Table 1). In brief, multiplex PCR was performed with the primers of concentrations of 0.2 µM each and 1X ready to use master mix (New England biolab, UK) with adjusted concentration of MgCl 2 of 2.5 µM in a final reaction volume of 50 µl. The amplification was performed with the following parameters; 95°C for 10 min followed by 40 cycles of 30s at 95°C, 45s at 58°C and 45s at 62°C. Positive and negative controls were used in all experiments and the PCR amplified products were analyzed by 2% agarose gel electrophoresis.
Detection of rdx Agene deletion in Metronidazole resistant H. pylori. Detection of NADPH nitroreductase was performed by conventional mutagenic-based PCR with the following conditions and parameter; reactions were carried out in 25 μl mixtures containing 12.5 μl PCR master mix (New England biolab, UK), 9.5 μl sterile deionized water, 1 μl template DNA (100 ng) and 1 μl of each oligonucleotide primers (0.4 μM). Initial denaturation at 94°C for 5 mins followed by 30 cycles of denaturation at 94°C for 45s, annealing for 1min at 55°C, extension at 72°C for 30s. The expected fragment was 800 bp if the gene is normal and 650 bp if the gene is mutated. PCR amplified products were analyzed by 1.5% agarose gel electrophoresis [30].

Statistical analysis
The results were statistically analyzed using the Chi square or Fisher's exact test. The level of significance was set at p < 0.05.

Results
Prevalence of H. pylori and patients characteristics. A total of 72 patients presented with gastroduedenal diseases were included in this Study. 13 cases (23%) presented positive culture for H. pylori, with one of the patients (8%) presenting growth only in the biopsy of the gastric corpus, five patients (38%) in the biopsy of the antrum, while 7 patients (54%) were positive for H. pylori growth in both biopsy cultures. In general, statistically significant differences were not reported for the prevalence of H. pylori as a function of gender, age groups and endoscopic findings (p > 0.05) (  AMX= amoxicillin, CLR= clarithromycin, CIP= ciprofloxacin, LEV= levofloxacin, MTZ= metronidazole. R indicates resistant, S indicates sensitive thermore, the presence of H. pylori in patients who used antibiotics and or NSAID was not statistically significant. Although the rate of H. pylori colonization increased with the use of PPI as reported in this work, no statistical significance was reported. In regards to the clinical manifestations, H. pylori colonization was significantly higher in patients presented with heartburn and dyspepsia (p < 0.05).
Antimicrobial susceptibility profile of H. pylori. All of the tested strains were sensitive to amoxicillin with values of MICs range of 0.015-0.03 µg/ml. Resistance to ciprofloxacin and levofloxacin was observed in 23% and 8% respectively ( Figure  1). MIC values of ciprofloxacin resistance in H. pylori were 4-16 µg/ml ( Table 3). 15% of the strains were resistant to clarithromycin with MIC value of 8 µg/ml. Resistance to metronidazole was dominating with 92% of the strains showed moderate to high levels of resistance with MIC range of 32-128 µg/ml.
Characterization of cagA and vacA genotypes and their relation to antibiotic resistance. Molecular analysis of different alleles of the vacA gene showed that one sample (8%) were found to have both m1 and m2 alleles with notable moderate multi-drug resistant behavior against the tested antibiotics ( Table 4). The majority of the tested H. pylori strains possess the m2 allele (78%). No statistical significant association between the vacA allelic genotype and resistance to antibiotics or the clinical manifestation could be established (p > 0.05).
cagA gene was detected in 46% of the tested strains with no correlation to antibiotic resistance and clinical manifestations.

Discussion
H. pylori has been increasingly considered as one of the most common bacterial infections affecting all ages worldwide. In Jordan, there is a limited number of epidemiological reports with a remarkable lack of information regarding the susceptibility of the clinical strains to the conventional antimicrobial agents used for the treatment of H. pylori infections [31]. One reason for this shortage of information is the technical difficulties of isolating this microaerophilic bacterium and the sensitivity of the methods used for this purpose [32].
This study has been conducted to characterize the susceptibility of the clinical strains of H. pylori to the conventional antimicrobial agents used in the region with particular concern for metronidazole and clarithromycin since there is an increasing number of recent reports about the emergence of multi-drug resistant H. pylori strains in the developing countries [33,34].
Our study reported a very high rate of metronidazole resistance among the clinical isolates of H. pylori in Jordan compared to countries from Europe and Asia [35][36][37]. It could be suggested that the misuse of metronidazole, which is one of the common prescribed drugs by the general practitioners particularly in adults, would be a major factor for this high rate although the differences in gender and age were not statistically significant. In addition, the chemical nature of this antimicrobial agent plays a significant role in selecting the resistant strains. One interesting finding of our work is the low occurrence in deletion mutations in rdxA gene among H. pylori metronidazole resistant strains which is similar to data from Japan and supports the studies which questioned the mutational inactivation of