Characterization of an In Vitro-Selected Amoxicillin-Resistant Strain of Helicobacter pylori

摘要

An amoxicillin-resistant (Amox^r) strain of Helicobacter pylori was selected for by culturing an amoxicillin-sensitive (Amox^s) strain in increasingly higher concentrations of amoxicillin, resulting in a 133-fold increase in MIC, from 0.03 to 0.06 μg/ml to 4 to 8 μg/ml. This resistance was stable upon freezing for at least 6 months and conferred cross-resistance to seven other β-lactam antibiotics. β-Lactamase activity was not detected in this Amox^r strain; however, analysis of the penicillin-binding protein (PBP) profiles generated from isolated bacterial membranes of the Amox^s parental strain and the Amox^r strain revealed a significant decrease in labeling of PBP 1 by biotinylated amoxicillin (bio-Amox) in the Amox^r strain. Comparative binding studies of PBP 1 for several β-lactams demonstrated that PBP 1 in the Amox^r strain had decreased affinity for mezlocillin but not significantly decreased affinity for penicillin G. In addition, PBP profiles prepared from whole bacterial cells showed decreased labeling of PBP 1 and PBP 2 in the Amox^r strain at all bio-Amox concentrations tested, suggesting a diffusional barrier to bio-Amox or a possible antibiotic efflux mechanism. Uptake analysis of ¹⁴C-labeled penicillin G showed a significant decrease in uptake of the labeled antibiotic by the Amox^r strain compared to the Amox^s strain, which was not affected by pretreatment with carbonyl cyanide m-chlorophenylhydrazone, eliminating the possibility of an efflux mechanism in the resistant strain. These results demonstrate that alterations in PBP 1 and in the uptake of β-lactam antibiotics in H. pylori can be selected for by prolonged exposure to amoxicillin, resulting in increased resistance to this antibiotic.