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Emergence of multidrug-resistant (MDR) strains of Helicobacter pylori is a global health concern. This study was aimed to determine the frequency of MDR H. pylori strains in Iran. H. pylori isolates were obtained from cultured gastric biopsy samples on selective culture media after their characterization by PCR and conventional biochemical methods. The minimal inhibitory concentrations of rifampicin, ciprofloxacin, levofloxacin, ampicillin, clarithromycin, erythromycin, metronidazole, and tetracycline were determined for 111 strains that were isolated from 197 dyspeptic patients by the agar dilution method. The primary resistance rates were 61.3% (68/111) for metronidazole, 15.3% (17/111) for ampicillin, and 14.4% (16/111) for rifampicin. Resistance rates for other antimicrobials were as follows: macrolides (erythromycin or clarithromycin) 32.4% (36/111) and quinolones (levofloxacin or ciprofloxacin) 30.6% (34/111). Among the resistant strains, the rates of double and multiple drug resistance phenotypes were 22.6% (19/84) and 34.5% (29/84), respectively. The quadruple drug resistance phenotype encompasses 37.9% of the MDR strains, of which 90% of them was resistant to metronidazole. In conclusion, these results showed a high frequency of MDR phenotypes among the studied H. pylori strains in Iran. The eradication of the H. pylori strains presenting high resistance rates to macrolides, fluoroquinolones, or metronidazole could be achieved by approved tetracycline- or amoxicillin-containing regimens as alternative regimens to standard triple therapy.

The high prevalence of antibiotic resistance in Helicobacter pylori has become a great challenge in Iran. The genetic mutations that contribute to the resistance have yet to be precisely identified. This study aimed to investigate the prevalence of antibiotic resistance and virulence markers in Iranian H. pylori isolates and to analyze if there is any association between resistance and genotype. Antibiotic susceptibility patterns of 68 H. pylori isolates were investigated against metronidazole, clarithromycin, amoxicillin, rifampicin, ciprofloxacin, levofloxacin, and tetracycline by the agar dilution method. The frxA, rdxA, gyrA, gyrB, and 23S rRNA genes of the isolates were sequenced. The virulence genotypes were also determined using PCR. Metronidazole resistance was present in 82.4% of the isolates, followed by clarithromycin (33.8%), ciprofloxacin (33.8%), rifampicin (32.4%), amoxicillin (30.9%), levofloxacin (27.9%), and tetracycline (4.4%). Overall, 75% of the isolates were resistant to at least two antibiotics tested and considered as a multidrug resistance (MDR) phenotype. Most of the metronidazole-resistant isolates carried frameshift mutations in both frxA and rdxA genes, and premature termination occurred in positions Q5Stop and Q50Stop, respectively. Amino acid substitutions M191I, G208E, and V199A were predominantly found in gyrA gene of fluoroquinolone-resistant isolates. A2143G and C2195T mutations of 23S rRNA were found in four clarithromycin-resistant isolates. Interestingly, significant associations were found between resistance to metronidazole (MNZ) and cagA-, sabA-, and dupA-positive genotypes, with p = 0.0002, p = 0.0001, and p = 0.0001, respectively. Furthermore, a significant association was found between oipA “on” status and resistance to amoxicillin (AMX) (p = 0.02). The prevalence of H. pylori antibiotic resistance is high in our region, particularly that of metronidazole, clarithromycin, ciprofloxacin, and MDR. Simultaneous screening of virulence and resistance genotypes can help clinicians to choose the appropriate therapeutic regime against H. pylori infection.

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A b s t r a c tAssociation of the severity of Helicobacter pylori induced diseases with virulence entity of the colonized strains was proven in some studies. Urease has been demonstrated as a potent virulence factor for H. pylori. The main aim of this study was investigation of the relationships of ureB sequence diversity, urease activity and virulence genotypes of different H. pylori strains with histopathological changes of gastric tissue in infected patients suffering from different gastric disorders. Analysis of the virulence genotypes in the isolated strains indicated significant associations between the presence of severe active gastritis and cagA + (P = 0.039) or cagA/iceA1 genotypes (P = 0.026), and intestinal metaplasia and vacA m1 (P = 0.008) or vacA s1/m2 (P = 0.001) genotypes. Our results showed a 2.4-fold increased risk of peptic ulcer (95% CI: 0.483-11.93), compared with gastritis, in the infected patients who had dupA positive strains; however this association was not statistically significant. The results of urease activity showed a significant mean difference between the isolated strains from patients with PUD and NUD (P = 0.034). This activity was relatively higher among patients with intestinal metaplasia. Also a significant association was found between the lack of cagA and increased urease activity among the isolated strains (P = 0.036). While the greatest sequence variation of ureB was detected in a strain from a patient with intestinal metaplasia, the sole determined amino acid change in UreB sequence (Ala201Thr, 30%), showed no influence on urease activity. In conclusion, the supposed role of H. pylori urease to form peptic ulcer and advancing of intestinal metaplasia was postulated in this study. Higher urease activity in the colonizing H. pylori strains that present specific virulence factors was indicated as a risk factor for promotion of histopathological changes of gastric tissue that advance gastric malignancy. K e y w o r d s: Helicobacter pylori, virulence, factor urease activity, histopathological changes

Mixed infections and heteroresistance of Helicobacter pylori contribute to decreased efficacy of treatments. This study aimed to investigate frequency of clarithromycin heteroresistance and its link with mixed infections, medication history, and disease severity. A total of 40 pairs of H. pylori strains were isolated from the antrum and corpus of 97 patients. Susceptibility of the strains to clarithromycin was measured by agar dilution method. Site-specific mutations of 23S rRNA at A2143G, A2142G, and A2142C positions were analyzed by PCR and genomic relatedness of pairs of the strains was determined by random amplified polymorphic DNA (RAPD)-PCR. The results showed a prevalence of 35% (14/40) clarithromycin resistance. Diversity of the antrum and corpus isolates in resistance to clarithromycin was detected among 17.5% (7/40) of the patients. Similarly, diversity in MIC value was also detected in two patients infected with the sensitive strains. Significant difference in frequency of resistance was detected among patients with peptic ulcer disease (PUD) (MIC90 32 μg/mL) and severe gastritis (MIC90 16 μg/mL), compared with those who suffered from non-ulcer dyspepsia (NUD) (MIC90 8 μg/mL) and chronic gastritis (MIC90 0.25 μg/mL). MIC values showed 8-32 folds increased levels in the corpus. A2142G, A2143G, and A2142C mutations were detected in three, two, and two patients, respectively, but not observed in 46% of the resistant strains. RAPD-PCR fingerprints showed identical molecular patterns for the isolates of the corpus and antrum in each patient. In conclusion, microevolution of H. pylori strains during chronic infection, rather than mixed infection, and inappropriate medication appear to be main reasons of treatment failure in adults.

SUMMARY:Infection with different strains of Helocobacter pylori and emergence of new genomic variants during their long-term gastric colonization are assumed to be the main reasons for eradication failure. We used genotyping and arbitrarily primed PCR fingerprinting (RAPD) to detect relatedness and genetic variations among H. pylori single isolates from each patient in Iran. Multiplex-PCR amplification of gene alleles encoding the virulence factors vacA (m/s), cagA, and iceA (A1/A2) and comparison of RAPD patterns of different singles colonies were performed for each individual patient's isolate. Results showed a high frequency of diversity among the H. pylori strains. Nearly 23z of infected patients showed a single type infection. The infection types related, unrelated, and related/unrelated were found among 25.6z, 12.8z, and 38.5z of patients, respectively. Both mixed type infections (77z) and quasispecies development (15.4z) were detected in these patients. Genotype conversion among vacA (41.6z), cagA (41.6z), and iceA (50z) alleles was observed for the strains with identical or related RAPD patterns. Coevolution of different alleles was also detected in a patient infected with strains presenting the same RAPD patterns. Collectively, results of this study revealed the occurrence of quasispecies development, mixed type infections, and changes in virulence properties of H. pylori strains among the studied patients.

Background: Detection of fastidious enteropathogenic bacteria in fecal samples of patients with gastroenteritis is a challenge in clinical microbiological laboratories. Objectives: The aim of this study was to compare the detection limits of the PCR and culture methods for the diagnosis of Campylobacter spp., Yersinia spp., Clostridium perfringens, and Clostridium difficile in human stool samples. Methods: Healthy human stool and sterile phosphate-buffered saline (PBS) samples were separately spiked with 10-fold dilutions of C. jejuni, C. difficile, Y. enterocolitica, and C. perfringens reference strains to obtain final concentrations of 10 1 -10 8 colony forming