Amoxicillin resistance with beta-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 beta-lactamase production was determined by means of nitrocefin sticks and the presence of gene encoding the beta-lactam antibiotic resistance enzyme TEM beta-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 beta-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 beta-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 Thr(556)-> Ser, Lys(648)-> Gln, Arg(649)-> Lys and Arg(656)-> Pro contribute to low-level amoxicillin resistance. The MIC of amoxicillin for the transformants was 0 center dot 75 mg L-1. Over-expression of hefC was not found. Conclusions High-level amoxicillin resistance is associated with beta-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.