Supervisors info:
Π. Κόλλια, Αναπλ. Καθηγήτρια (επιβλέπουσα)
Β. Αλεπόρου, Αναπλ. Καθηγήτρια
Δ. Σγούρας, Ερευνητής Β΄ PASTEUR
Summary:
Helicobacter pylori (H. pylori) is a Gram-negative bacterium that infects the human gastric mucosa with a prevalence of about 50% worldwide. As a rule, the infection manifests as chronic active gastritis, usually asymptomatic, while in 10% of the cases it presents the etiologic factor for the development of duodenal or peptic ulceration. Worldwide the infection is a primary risk factor for the development of non-cardia gastric adenocarcinoma and gastric MALT lymphoma. The usual first-line treatment for the eradication of H. pylori includes two antibiotics, such as clarithromycin and amoxicillin, and a PPI proton pump inhibitor. The second-line schemes include the use of fluoroquinolones, such as levofloxacin. H. pylori can develop resistance against the antibiotics used to treat the infection, not by usual mechanisms of plasmid transfer, but through the occurrence of point mutations in the bacterial genome. The percent of H. pylori resistance to levofloxacin are alarmingly rising, making it imperative to detect the resistance of the bacteria to levofloxacin, especially after failed eradication efforts. In daily practice, the detection of resistance to both clarithromycin and levofloxacin is based on phenotypic antibiotic methods, which require the isolation of the H. pylori strain from the gastric biopsy, which is not always feasible. Molecular detection of point mutations to assess bacterial susceptibility can be an alternative approach. Levofloxacin resistance has been shown to correlate mainly, but not exclusively, with mutations in the gyrA gene resulting in modifications mainly to aa residue 87 (N to K) or aa residue 91 (D to G, D to N or D to Y). The purpose of this study was to characterize the mutations in bacterial genome, responsible for the resistance to levofloxacin in the Greek population of symptomatic patients. Initially, H. pylori clinical strains were isolated from symptomatic patient biopsies and their susceptibility was determined phenotypically using the E-test. A collection of susceptible, as well as, resistant clinical H. pylori strains to levofloxacin and other antibiotics used in eradication schemes, was prepared and genomic DNA isolated. Due to a lack of published studies on prevalent mutations in levofloxacin-resistant H. pylori strains in the Greek population, primers were designed to amplify the gyrA gene regions under study, followed by sequencing. After plotting the molecular and phenotypic effects against levofloxacin resistance, a real-time PCR was designed to determine levofloxacin resistance.
