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.

Competition of various beta-lactam antibiotics for the major penicillin-binding proteins of Helicobacter pylori: antibacterial activity and effects on bacterial morphology.

The penicillin-binding proteins (PBPs) of helical (log-phase) Helicobacter pylori ATCC 43579 were identified by using biotinylated ampicillin. The major PBPs had apparent molecular masses of 47, 60, 63, and 66 kDa; an additional minor PBP of 95 to 100 kDa was also detected. The relative affinities of various beta-lactams for these PBPs were tested by competitive-binding assays. Only PBP63 appeared to be significantly bound to each of the competing antibiotics, whereas PBP66 strongly bound mezlocillin, oxacillin, amoxicillin, and ceftriaxone. Whereas most of the beta-lactams significantly bound two or more PBPs, aztreonam specifically targeted PBP63. The influence of sub-MICs of these beta-lactams on the morphologies of log-phase H. pylori was observed at both the phase-contrast and transmission electron microscopy levels. Each of the eight beta-lactams examined induced blebbing and sphere formation, whereas aztreonam was the only antibiotic studied which induced pronounced filamentation in H. pylori. Finally, studies comparing the PBPs of helical (log-phase) cultures with those of coccoid (7-, 14-, and 21-day-old) cultures of H. pylori revealed that the major PBPs at 60 and 63 kDa seen in the helical form were almost undetectable in the coccoid forms, whereas PBP66 remained the major PBP in the coccoid forms, although somewhat reduced in level compared to the helical form. PBP47 was present in both forms at approximately equal concentrations. These studies thus identified the major PBPs in both helical and coccoid forms of H. pylori and compared the relative affinities of seven different beta-lactams for the PBPs in the helical forms and their effects on bacterial morphology.