Infection and Drug Resistance (Nov 2019)
A Newly Discovered Drug Resistance Gene rfaF In Helicobacter pylori
Abstract
Jiansheng Lin,1,2 Xiaoyan Zhang,1,2 Yancheng Wen,1,2 Hao Chen,1,2 Feifei She1,2 1Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350122, People’s Republic of China; 2Fujian Key Laboratory of Tumor Microbiology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350122, People’s Republic of ChinaCorrespondence: Feifei SheKey Laboratory of Ministry of Education For Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350122, People’s Republic of ChinaTel +86 591 22862863Email [email protected]: The purpose of this study was to understand the function of rfaF gene in Helicobacter pylori antibiotic resistance.Methods: The gene homologous recombination method was used for knockout and complementation of H. pylori rfaF gene. Various constructed strains were analysed for drug sensitivity to amoxicillin (AMO), tetracycline (TET), clarithromycin (CLA), metronidazole (MET), levofloxacin (LEV), and chloramphenicol (CHL) by agar plate dilution method. Drug sensitivity was further confirmed using a growth inhibition curve. Ethidium bromide (EB) accumulation experiments were performed to assess cell membrane permeability. PCR and sequence analysis were used to detect the rfaF gene.Results: The minimum inhibitory concentrations (MIC) of TET, CHL, AMO, and CLA in 11,637 rfaF knockout strain (ΔrfaF strain) were 4, 4, 2, and 2 times higher than those in 11,637 wild type (WT) strain, respectively. A multidrug-resistant (MDR) ΔrfaF strain also displayed the same trend; however, the degrees of increase were relatively small. Growth inhibition experiments indicated that the growth of the 11,637 ΔrfaF strain was higher with antibiotics at the MIC of the 11,637 WT strain than that of 11,637 rfaF-complemented strain (ΔrfaF/rfaF strain), whereas the 11,637 WT strain did not exhibit any growth. The 11,637 ΔrfaF strain was significantly reduced compared with the cumulative EB fluorescence intensity of the 11,637 WT and of 11,637ΔrfaF/rfaF strain, and the same trend appeared in the MDR strain. Among the 10 clinical strains, 9 clinical strains were found to have mutations in the conserved sequence of rfaF amino acids.Conclusion: We found a new drug resistance gene, rfaF, in H. pylori, which changes the permeability of cell membrane to confer cross-resistance to AMO, TET, CLA, and CHL and is involved in clinical strain drug resistance. It can be used as a drug target.Keywords: rfaF, amoxicillin, clarithromycin, tetracycline, resistance
