Amoxicillin resistance with β-lactamase production in Helicobacter pylori

Background Amoxicillin‐resistant Helicobacter pylori with minimal inhibitory concentration (MIC) ≥ 256 mg L^?1 was isolated from a gastritis patient. The aims were to investigate the mechanism of high‐level amoxicillin resistance in H. pylori. Materials and methods The β‐lactamase production was determined by means of nitrocefin sticks and the presence of gene encoding the β‐lactam antibiotic resistance enzyme TEM β‐lactamase was analysed by polymerase chain reaction (PCR), sequencing and dot‐blot hybridization. Sequencing analysis of pbp1A gene was performed and amoxicillin‐susceptible isolate was transformed with pbp1A PCR products from the resistant isolate. The expression of hefC efflux system was analysed using real‐time quantitative PCR. Results Activity of β‐lactamase was detected. Sequence analysis showed that the PCR product derived from H. pylori 3778 was identical to the bla_TEM‐1 (GenBank accession EU726527 ). Dot‐blot hybridization confirmed the presence of β‐lactamase gene bla_TEM‐1. By transformation of PCR product of mutated pbp1A gene from H. pylori 3778 into amoxicillin‐susceptible strain showed that substitutions in Thr₅₅₆→Ser, Lys₆₄₈→Gln, Arg₆₄₉→Lys and Arg₆₅₆→Pro contribute to low‐level amoxicillin resistance. The MIC of amoxicillin for the transformants was 0·75 mg L^?1. Over‐expression of hefC was not found. Conclusions High‐level amoxicillin resistance is associated with β‐lactamase production in H. pylori. Low‐level amoxicillin resistance is linked to a point mutation on pbp1A. Because H. pylori can exchange DNA through natural transformation, spreading of bla_TEM‐1 amoxicillin resistance gene among H. pylori is a potential threat when treating H. pylori infection.