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

Abstract

An amoxicillin-resistant (Amox r ) strain of Helicobacter pylori was selected for by culturing an amoxicillinsensitive (Amox s ) strain in increasingly higher concentrations of amoxicillin, resulting in a 133-fold increase in MIC, from 0.03 to 0.06 g/ml to 4 to 8 g/ml. This resistance was stable upon freezing for at least 6 months and conferred cross-resistance to seven other ␤-lactam antibiotics. ␤-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 ␤-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 ␤-lactam antibiotics in H. pylori can be selected for by prolonged exposure to amoxicillin, resulting in increased resistance to this antibiotic.Helicobacter pylori is the most common cause of gastric and duodenal ulcers and is strongly associated with the development of gastric adenocarcinoma (see references 14 and 32 for reviews). It is estimated that at least a third of the world's population is infected with H. pylori, making it one of the most common infections in humans (14). Successful treatment of H. pylori infections most often employs the use of two or more antibiotics and the addition of either bismuth or a proton pump inhibitor (14,17,18). However, H. pylori resistance to many of the commonly used antibiotics in this triple regimen is rising (19), including resistance to metronidazole (1, 27, 36), clarithromycin (1, 6, 9, 27), rifampin or rifabutin (24), and, recently, amoxicillin (11, 12, 13, 22, 38).Resistance to ␤-lactam antibiotics by gram-negative bacteria is most commonly due to the production of ␤-lactamase, either chromosomally encoded or, more often, plasmid mediated (see reference 30 for a review). Other important mechanisms of resistance include alterations in penicillin-binding proteins (PBPs), decreased permeation of the antibiotic into the bacterial cell, or combinations of these resistance strategies (see reference 28 for a review). Active efflux pumps in gramnegative bacteria which excrete drugs, including multidrug effl...

Medicinal plants in the treatment ofHelicobacter pyloriinfections

“…The organism did not acquire resistance to this bioactive compound at subinhibitory concentrations (0.25 and 0.5 mg/mL) even after 10 passages (Ali et al, 2005). According to other studies, H. pylori strains acquired resistance to amoxycillin and clarithromycin after 10 sequential passages (DeLoney & Schiller, 2000). It should also be mentioned that C. verum can inhibit urease activity and prevent gastric upset (Nabati et al, 2012).…”

Antimicrobial Susceptibility of Helicobacter pylori Strains Isolated in Bangladesh

“…Although 6.6% of the isolates were resistant, none was positive for ␤-lactamase. Amoxicillin resistance develops due to structural alterations in one of the penicillin-binding proteins (4,5,9) or changes in other proteins involved in cell wall synthesis (2,15,26), and the resistant phenotype may be lost due to freezing or storage. All isolates tested in the present study were frozen at least once, and the low prevalence of the resistance phenotype may be due to loss during storage.…”

Production of anti-Helicobacter pylori urease-specific immunoglobulin in egg yolk using an antigenic epitope of H. pylori urease

“…Current triple therapies are effective against Helicobacter pylori infection, but the development of antibiotic resistance and the challenge of re-treatment after initial failure demand that novel approaches be developed (Deloney & Schiller, 2000;Peitz et al, 1998). Vaccines (Kusters, 2001;Del et al, 2001), probiotics (Aiba et al, 1998), natural extracts (Mabe et al, 1999;O'Gara et al, 2000), anti-adhesion compounds and mucosal protective agents (Mysore et al, 1999) are under investigation.…”