Supervisors info:
Παναγούλα Κόλλια, Αναπληρώτρια Καθηγήτρια Μοριακής Γενετικής Ανθρώπου, ΕΚΠΑ
Διονύσιος Σγούρας, Διευθυντής Ερευνών, Εργαστήριο Ιατρικής Μικροβιολογίας, Ελληνικό Ινστιτούτο Παστέρ
Ισιδώρα Σ. Παπασιδέρη, Καθηγήτρια Κυτταρικής & Αναπτυξιακής Βιολογίας, ΕΚΠΑ
Summary:
Helicobacter pylori infection is the etiologic factor for the development of digestive diseases, such as chronic active gastritis and peptic ulcer and remains a primary risk factor for the development of gastric adenocarcinoma. For this reason, its successful eradication remains a priority for the WHO, especially due to the rising levels of observed antibiotic resistance, which affects therapeutic efficacy. According to a multicenter study conducted by the Hellenic Pasteur Institute in Greece between 2012-2015, primary resistance levels to clarithromycin, metronidazole and levofloxacin were determined to be 27,2%, 32,1% and 8,2% respectively. In the present study, a Whole-genome sequencing (WGS) approach was applied and bioinformatic analysis was performed to investigate the genetic basis of antimicrobial resistance mechanisms. We analyzed 38 H. pylori clinical strains isolated from adult symptomatic patients, in which phenotypic antibiotic susceptibility had been previously characterized using the E-test®. Premature stop codon and frame-shift mutations, as, point mutations (silent mutations included) were observed in genes related to clarithromycin (23S rRNA), metronidazole (rdxA, frxA, rpsU) and levofloxacin (gyrA, gyrB) resistance with reference to 26695 strain. We observed mutations frequently associated with antibiotic resistance uniquely in resistant strains, but we also identified some previously reported as resistance-associated mutations, in both resistant and susceptible strains. Moreover, a number of Single Nucleotide Polymorphisms (SNPs) was identified, however, further analysis is required in order to examine their impact on H. pylori eradication. H. pylori strains are characterized by genetic diversity which is greater than within most other bacteria and this is something that should be taken into account when studying the role of the SNPs. The association between the genetic variants of the aforementioned genes and the phenotype of antibiotic resistance were analyzed using Fisher's exact test. Whole-genome sequencing technology is a practical state-of-the-art tool for analyzing genomic variations associated with antibiotic resistance in H. pylori, that way contributing significantly to the study of resistance mechanisms. Despite the benefits of the technology, which allows detailed characterization of pathogens, providing a large amount of information including strain type, virulence, antibiotic resistance there are barriers to implementing WGS in the determination of antibiotic susceptibility of H. pylori in clinical microbiology practice. The most common arguments mainly concern the long turnaround time, which needs to be lowered in order to have a significant clinical impact on decision making. It should be noted that there is need not just for shortening of turnaround time but making it no longer than the traditional methods’ duration. In order WGS to be integrated into clinical workflows, another barrier should be overcome and that is the correlation of phenotypic susceptibility and resistance to the WGS genotype. We cannot assume that a specific strain is phenotypically resistant or susceptible to an antimicrobial drug just by revealing the presence of gene mutations related to antibiotic resistance, but we should clearly link new findings to a diagnosis and treatment choice. Finally, regarding the pipelines (data analysis workflow) used to extract sequence data, it is important that the established software is user-friendly and the information extracted to be accurate and clinically applicable.
Keywords:
Helicobacter pylori, antimicrobial resistance, Whole Genome Sequencing, genomic variations
