English abstract
Introduction: Antibiotic resistance is the leading cause of Helicobacter pylori infection
therapy failure. Statistics on antibiotic resistance and genetic mechanisms of resistance are
lacking in Oman, which is important for choosing the right medications to eradicate H. pylori.
This study aims to investigate the antimicrobial resistance of clarithromycin, amoxicillin,
levofloxacin, metronidazole, and tetracycline phenotypically in H. pylori. Moreover, this
study focuses on the molecular characterization of multi-resistant isolates by Illumina whole
genome sequencing.
Methods: Gastric biopsies were collected through endoscopy from patients with
gastroduodenal symptoms and screened for H. pylori using the rapid urease test; CLO test.
Three hospitals were included in this study: Sultan Qaboos University Hospital (SQUH),
Royal Hospital (RH) and Creativity Medical Centre (CMC). The positive biopsies were
transferred to the microbiology laboratory in a transport media. Phenotypic testing of
antibiotic susceptibility was performed using two methods; broth microdilution and E-test
strips for six antibiotics (clarithromycin (CLA), amoxicillin (AMX), metronidazole (MTZ),
tetracycline (TET), rifampicin (RIF) and levofloxacin (LEV)), The DNA was extracted for
whole-genome sequence (WGS). Data obtained from WGS was analyzed for the genotype
of antimicrobial resistance, virulence factors, Multi-Locus Sequence Typing (MLST), and
phylogenetic relatedness of the strains.
Results: The total number of biopsy samples collected from three different centers according
to inclusion criteria was 169 but the total number which was successfully growing in the
growth culture was only 23. Antibiotic susceptibility testing by using two methods, broth
microdilution and E-test, showed that the highest levels of resistance was in MTZ (93.3%)
and 100% respectively. There was agreement between the two methods in all antibiotics
except metronidazole. For a 95% confidence interval, the limits of agreement for six
antibiotics were as follows: (-42.6,18.4) for CLA; (-9.2,28.4) for AMX; (-94, -8.4) for TET;
(-8.4,12.6) for MTZ; (-6.5,20.7) for RIF; (-.8,1.6) for LEV. None of the isolates were resistant
to rifampicin according to previous study MIC instead of EUCAST guidelines.
WGS data was used to characterize the genotype of resistant isolates by searching for point
mutations in resistance-conferring genes, Among the 20 strains, 33.3% strains showed
resistance to CLA by E test and with mutations in 23S rRNA (A2146G, A2147G, C1707T
and A2144G). Amoxicillin phenotypic resistance in 66.6% of isolates was associated with
point mutations in pbp1, pbp2, and/or pbp3. None of the strains were sensitive to MTZ by
both methods with mutations in rdxA (T31E, R90K, A118T, A67V, C49T, R16C, D59N,
H97T, H97Y, K64N, P106S and S108A) and frxA (Y62D, A16T, A15V, A85V, M126F,
A70G ndV7I). All isolates were genotypically susceptible to tetracycline, although one
isolate was resistant phenotypically. Among the 65.7% RIF-resistant strains, mutations in
ropB were in the positions K2068R, I837V, and Q2079K whereas only 33.3% showed
phenotypic resistance. Strains with mutations in gyrA (N87K, D91G andV172I) were
resistant to LEV (20%). The genotype and phenotype agreement in the six antibiotics tested
was statistically insignificant (p=1). MLST analysis showed that twelve strains belonged to
ST 3120, one isolate belonged to 3104 and seven isolates were novel. The phylogenetic tree
shows that these strains were very closely related, which suggests that the strains circulating
in the Omani population are very similar. The most significant H. pylori virulence genes are
presented in this study along with key findings. The presence of virulence genes can affect
the patients and increase the risk of gastritis, peptic ulcers, inflammation, and gastric cancer
as shown in this study.
Conclusion: In summary, our findings indicate that H. pylori demonstrated a high significant
resistance to metronidazole, followed by clarithromycin, and amoxicillin with contributing
point mutations. Furthermore, antibiotic susceptibility and molecular testing by WGS are
essential to guide appropriate and population-specific antibiotic therapy.
