High prevalence of antibiotic resistance in Helicobacter pylori isolates from Iran: importance of functional and mutational analysis of resistance genes and virulence genotyping

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 33 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 81.8% of the isolates, followed by clarithromycin (36.4%), ciprofloxacin (36.4%), amoxicillin (30.3%), rifampicin (30.3%), levofloxacin (27.3%) and tetracycline (6.1%). Most of the metronidazole-resistant isolates carried frameshift mutations in both frxA and rdxA genes, and premature termination was 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 isolates. Interestingly, significant associations were demonstrated between intact cagPAI and resistance to rifampicin (P = 0.027), and between susceptibility to amoxicillin and cagPAI intactness (P = 0.016). The prevalence of H. pylori antibiotic resistance is high in our region, particularly that of metronidazole, clarithromycin, ciprofloxacin and multidrug resistance. Occurrence of mutations in resistance genes were involved in the development of resistance, especially in less virulent isolates.


Introduction
Helicobacter pylori (H. pylori) is known as the most common human pathogen infecting more than half of the world's However, the efficacy of eradication treatments has been extremely compromised primarily due to increased resistance 46 to antimicrobial agents in many countries. [5][6][7][8] Today, first-line standard triple therapy is the most widely used eradication treatment for H. pylori infection, which Hence, the focus of the present study was to evaluate the antibiotic susceptibility patterns and underlying resistance mechanisms of H. pylori strains isolated from Iranian patients with different gastric diseases. Furthermore, we determined the presence of genetic mutations that are associated with antibiotic resistance. We also examined the 71 possible association between resistance profiles and a panel of virulence genotypes.

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The Chi-square and Fisher's exact tests were used to determine the statistical significance of differences between 131 categorical variables. A P value of less than 0.05 was considered as statistically significant.

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Characteristics of patients

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As for gyrB subunit, two different amino acid variants including D481E and R484K were detected among 7 isolates.

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The D481E substitution was found to be present in both fluoroquinolone-susceptible and -resistant isolates, whereas

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R484K was exclusively present in resistant isolates. As shown in Table 6 Table 7.

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Association between virulence genotypes and resistance patterns

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The frequency and distribution of strains grouped by virulence genotypes according to each susceptibility pattern is 196 shown in Table 8. High frequencies of cagL-positive and cagA-positive genotypes were found frequently in

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susceptible isolates for all antibiotics tested, with the exception of metronidazole. The cagA ABC motif was also found 198 frequently in susceptible isolates, with the exception of metronidazole and clarithromycin. H. pylori isolates with vacA 199 s1m2 were found more frequently in susceptible isolates. Strains with oipA "on" status and babA, sabA and dupA 200 positivity were also frequently found in susceptible isolates. There were only two isolates that showed resistance 201 against tetracycline and both these strains also were sabA-positive. All ciprofloxacin-resistant isolates also were found 202 to be dupA-positive. There was no association between these virulence factors and antibiotic resistance patterns (P > 203 0.05). Furthermore, H. pylori strains harboring intact or partial cagPAI were variably distributed between susceptible 204 and resistant isolates. Interestingly, significant associations were observed between intact cagPAI and resistance to 205 rifampicin (P = 0.027), and between susceptibility to amoxicillin and cagPAI intactness (P = 0.016).  clarithromycin-based treatment regimens. 12,24,59 However, it has been reported that fluoroquinolone resistance is 240 rapidly expanding around the world. 6,7,53 In a previous study from Iran, the rate of resistance to ciprofloxacin and

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levofloxacin was reported about 27% and 24.3%, respectively. 32 In this study, we found a significant increase of 242 fluoroquinolone resistance, which is of great concern. Nevertheless, studies from Taiwanese

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Among macrolides, clarithromycin is recognized as a major antibiotic for H. pylori eradication therapy because of its

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In contrast, vacA s1m2 genotype was found more frequently in susceptible isolates for other antibiotics examined.

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Likewise, isolates with oipA "on" status and harboring babA, sabA and dupA genotypes were frequently found in

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pylori strains that carry an intact and functional cagPAI are more virulent and frequently associated with severe clinical 308 outcomes than those carrying partial or no cagPAI. 30,38 As far as we know, this is the first study that relates the cagPAI 309 integrity with antibiotic resistance. Our results showed that H. pylori isolates harboring intact or partial cagPAI were 310 variably distributed between susceptible and resistant isolates. However, we found significant associations between 311 intact cagPAI and resistance to rifampicin (P = 0.027), and contrastingly between susceptibility to amoxicillin and 312 cagPAI intactness (P = 0.016). These results are contradictory and did not strongly support the idea that susceptibility 313 to antibiotics is higher in infections caused by more virulent genotypes. Nevertheless, it is likely that infected patients 314 with resistant and hypervirulent strains are at increased risk of progression to more severe clinical outcomes due to 315 failure in H. pylori eradication.

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In conclusion, this study demonstrated that the prevalence of H. pylori antibiotic resistance is worrisome in our country