Characterization of an In vitro-selected amoxicillin-resistant strain of Helicobacter pylori.
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 microg/ml to 4 to 8 microg/ml. This resistance was stable upon freezing for at least 6 months and conferred cross-resistance to seven other beta-lactam antibiotics. beta-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 beta-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 (14)C-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 beta-lactam antibiotics in H. pylori can be selected for by prolonged exposure to amoxicillin, resulting in increased resistance to this antibiotic.
DeLoney
C R
CR
Division of Biomedical Sciences, University of California, Riverside, Riverside, California 92521, USA.
Schiller
N L
NL
D000658:Amoxicillin / Q000494:pharmacology*; D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000378:metabolism; D003429:Cross Reactions; D018432:Drug Resistance, Multiple / Q000502:physiology*; D016480:Helicobacter pylori / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006602:Hexosyltransferases; D006801:Humans; D008802:Mezlocillin / Q000494:pharmacology; D002267:Muramoylpentapeptide Carboxypeptidase / Q000378:metabolism; D010400:Penicillin G / Q000494:pharmacology; D010403:Penicillin Resistance / Q000502:physiology*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D018440:beta-Lactam Resistance / Q000502:physiology; D001618:beta-Lactamases / Q000378:metabolism
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D000658:Amoxicillin; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases; D008802:Mezlocillin; D010400:Penicillin G