Evaluation of CRISPR-like sequences and spoT gene among Helicobacter pylori isolates and determination of their association with antibiotic resistance and biofilm formation

Abstract

Most Helicobacter pylori (H. pylori) isolates carry the spoT gene which significantly involves in biofilm formation. In addition, part of clustered regularly interspaced short palindromic repeats (CRISPR)-like sequences in biofilm formation and antibiotic resistance in this bacterium hasn't been investigated yet. Therefore, this research aimed to study the association of the spoT gene and CRISPR-like sequences with antibiotic resistance and biofilm formation in H. pylori isolates. Also, our study aimed to assess the role of the spoT gene in biofilm formation and antibiotic resistance and its expression by the PNAs (Peptide Nucleic Acid). Methods: Thirty-six isolates of H. pylori were subjected to study the presence of spoT gene and CRISPR-like sequences by PCR method and their correlation with antibiotic resistance and biofilm formation. The antibiotic resistance profile of isolates was investigated by the microtiter-plate method against amoxicillin, tetracycline, metronidazole, and clarithromycin. Biofilm formation of isolates was analyzed by microtiter-plate-based-method. The effect of PNA was evaluated against antibiotic resistance and biofilm formation and expression level of the spoT gene by Real-time PCR. Results: Out of 33 spoT- positive isolates, 26 isolates were able to form biofilm and none of the 3 spoT- negative isolates were able to form biofilm. The frequency of resistance to amoxicillin, tetracycline, metronidazole, and clarithromycin in spoT+ H. pylori was, 12 (36%), 19 (57.5%), 19 (57.5%) and 22 (66.66%), respectively. Out of 23 CRISPR-like positive isolates, 19 isolates were able to form biofilm and 7 of 13 CRISPR-like negative isolates could form biofilm. The frequency of resistance to amoxicillin, tetracycline, metronidazole, and clarithromycin in CRISPR-like positive H. pylori were 11 (48%), 18 (78%), 18 (78%), and 23 (100%), respectively. MIC of amoxicillin, tetracycline, metronidazole and clarithromycin without PNA was 16, 2, >32 and 0.0625, and with PNA was 4, 2, >32 and 0.25. OD of the tested isolate with PNA was 0.41 and without PNA was 0.434. The expression ratio of the spoT gene in strains exposed to PNA was reduced 20% in comparison to strains, which were not exposed to PNA.