Amoxicillin resistance with beta-lactamase production in Helicobacter pylori.
BACKGROUND
Amoxicillin-resistant Helicobacter pylori with minimal inhibitory concentration (MIC) >or= 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.


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 0.75 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.
Tseng
Y-S
YS
Department of Microbiology, Kaohsiung Medical University, Kaohsiung, Taiwan.
Wu
D-C
DC
Chang
C-Y
CY
Kuo
C-H
CH
Yang
Y-C
YC
Jan
C-M
CM
Su
Y-C
YC
Kuo
F-C
FC
Chang
L-L
LL
D000658:Amoxicillin / Q000494:pharmacology*; D004352:Drug Resistance, Microbial / Q000187:drug effects* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects* / Q000235:genetics; D016481:Helicobacter Infections / Q000188:drug therapy* / Q000235:genetics; D016480:Helicobacter pylori / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008826:Microbial Sensitivity Tests; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000187:drug effects* / Q000378:metabolism
D000658:Amoxicillin; D001618:beta-Lactamases