Amoxicillin resistance in Helicobacter pylori isolated from Thai patients

Abstract

Amoxicillin is an important component of combination therapies for Helicobacter pylori eradication and there is an increasing rate of amoxicillin resistance in many countries. This study investigates the prevalence of amoxicillin resistance in H. pylori isolated from Thai patients and determines the molecular mechanism of amoxicillin resistance in H. pylori by detecting mutations in the pbp1 gene. The minimal inhibitory concentration (MIC) of amoxicillin was examined by the E-test. Mutations in the pbp1 gene were analyzed by PCR and DNA sequencing. Fifty H. pylori isolates were obtained from 357 patients (14%, 50/357) who underwent upper gastrointestinal endoscopy at King Chulalongkorn Memorial Hospital between August 2003 to June 2004. Of the 50 isolates, 49 (98%) were susceptible to amoxicillin and only one isolate was resistant (2%) (MIC 0.75 microgram/ml). MIC[subscript 50] and MIC[subscript 90] were <0.016 and 0.016 microgram/ml, respectively. The DNA sequences of pbp1 were determined by sequencing both amoxicillin-susceptible and amoxicillin-resistant H. pylori isolates. Of the 6 isolates, 5 were susceptible to amoxicillin (MICs range = < 0.016-0.25 microgram/ml) and one isolate, HP-1144, was resistant to amoxicillin (MIC = 0.75 microgram/ml). When compared with amino acid sequence of PBP1 of H. pylori 26695 from GenBank, the result from this study showed forty-two different amino acid substitutions in both amoxicillin-sensitive and resistant isolates at positions 13, 16, 17, 35, 45, 79, 114, 120, 125, 148, 242, 243, 322, 324, 332, 352, 374, 392, 406, 408, 414, 432, 469, 479, 504, 508, 509, 515, 534, 535, 543, 547, 556, 589, 593, 595, 611, 648, 649, 653, 654, 656 and one amino acid deletion at position 400. Fifeen amino acid changes were shared among resistant and susceptible strains. All mutations in the resistant isolate, HP-1144, were found in susceptible isolates (positions 17, 35, 79, 125, 148, 324, 479, 504, 508, 509, 515, 535, 543, 648 and 649). Interestingly, our results showed that mutations in PBP1 at position 406, 414, 515, 535, 543, 556, 593, 648, 649, and 656 which other investigators reported to be linked to amoxicillin resistance were present in our amoxicillin susceptible isolates. HP-1144 was tested for production of beta-lactamase using the chromogenic cephalosporin method and beta-lactamase activity was not detected. Therefore, mutation in PBP1 and production of beta-lactamase may not be associated with amoxicillin resistance in HP-1144. Other mechanisms must be involved in amoxicillin resistance such as decreased membrane permeability, alterations in OMPs and efflux pump.