Characterization of amoxicillin resistance mechanisms in the human gastric pathogen Helicobacter pylori

Amoxicillin-resistant Helicobacter pylori have been isolated worldwide and have recently appeared in the United States. To understand the mechanism(s) of amoxicillin resistance in H. pylori, the penicillin binding protein 1 (pbp) gene from an in-vitro selected amoxicillin-resistant (AmoxR) strain (MIC 4-8 mug/ml) was examined. A T438M mutation localized near the SLN penicillin binding domain (PBD) in the transpeptidase region was found. An amoxicillin-sensitive H. pylori strain 700392 (MIC 0.03-0.06 mug/ml) was transformed with this pbp1 gene and a moderately resistant strain (700392T) was isolated (MIC 0.25-0.5 mug/ml). Biotinylated amoxicillin (Bio-Amox) titration assays show that saturation of the PBP1 in 700392T requires 0.6-0.8 mug/ml Bio-Amox compared to 0.05-0.1 mug/ml) needed for parental PBP1. These saturation values are similar to observed MIC levels of these strains, suggesting a possible correlation between degree of amoxicillin binding and MIC.;Porins have been implicated in decreased antibiotic accumulation. Porin genes hopA, hopB, hopC, hopD and hopE in AmoxR were sequenced and compared to their wild type counterparts. While no changes were observed in hopA, hopD and hopE, HopB revealed numerous changes localized between amino acid positions 100 to 160, while HopC possessed a stop codon in position 211. Transformations with these modified genes into 700392 increased MICs 4-fold and 1-fold respectively. [14C] penicillin G accumulation studies show a 25% decrease in accumulated antibiotic in the HopB-transformed strain compared to wild-type, and a 10% decrease in HopC transformed strain. When a double hopB/hopC mutant was introduced into the pbp1 mutant strain, antibiotic accumulation and MIC levels comparable to AmoxR were obtained, suggesting that these 2 porin mutations and the pbp1 mutation comprise all the resistance mechanisms found in AmoxR.;Based on sequence alignments of pbp1 genes from amoxicillin resistant strains, we hypothesized the presence of several mutational hotspots in this gene. To assess this, three mutants were generated, S414R, S543R, and T556S, and the MICs of each H. pylori strain with these PBP1 mutations was 0.25-0.5 mug/ml. Bio-Amox saturation studies of PBP1s from these strains confirmed the hypothesis that there is a direct correlation between amoxicillin saturation of PBP1 and MIC.