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Antimicrobial Agents and Chemotherapy, December 2000, p. 3368-3373, Vol. 44, No. 12
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Cindy R. DeLoney and Neal L. Schiller*

Division of Biomedical Sciences, University of California, Riverside, Riverside, California 92521

Received 31 January 2000/Returned for modification 25 April 2000/Accepted 5 September 2000

An amoxicillin-resistant (Amoxr) strain of Helicobacter pylori was selected for by culturing an amoxicillin-sensitive (Amoxs) 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 beta -lactam antibiotics. beta -Lactamase activity was not detected in this Amoxr strain; however, analysis of the penicillin-binding protein (PBP) profiles generated from isolated bacterial membranes of the Amoxs parental strain and the Amoxr strain revealed a significant decrease in labeling of PBP 1 by biotinylated amoxicillin (bio-Amox) in the Amoxr strain. Comparative binding studies of PBP 1 for several beta -lactams demonstrated that PBP 1 in the Amoxr 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 Amoxr 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 Amoxr strain compared to the Amoxs 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.

* Corresponding author. Mailing address: Division of Biomedical Sciences, University of California, Riverside, Riverside, CA 92521. Phone: (909) 787-4569. Fax: (909) 787-5504. E-mail: neal.schiller{at}ucr.edu.

Antimicrobial Agents and Chemotherapy, December 2000, p. 3368-3373, Vol. 44, No. 12
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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