The Preliminary Study Of Helicobacter Pylori Resistance Agaist Levofloxacin And Amoxicillin By Vitro Induction

【Objectives】(1) To compare the propensity of acquired resistance to LEV and AMX, the H.pylori strain ATCC43504 was exposed to LEV and AMX in vitro with the concentration gradually increasing. (2) To study the effects of LEV and AMX concentration on the mutant gene. (3) To Examine the difference of DNA gene between the resistance-strains and susceptive-strains by sequencing.【Methods】(1) The H.pylori strain ATCC43504 was induced to acquisition of resistance against LEV or AMX in vitro by concentration gradually increasing(2n×MIC). After induced successfully, the isolates were transferred for two times on antimicrobial agent-free medium and then followed by redetermination of the final MIC(fMIC). The the success ratio and induction period between the two groups was compared to analyze the propensity of acquired resistance to LEV and AMX when fixed the induction multiple (induced MIC/ basic MIC). (2) LEV resistance-determining region mutations of gyrA and gyrB of resistants were identified by PCR amplification and gene sequencing, so were the AMX resistance-determing region mutations of pbp1 and pbp2. (3) The results of DNA sequencing were analysised by sequence alignment in the Genebank to identify the mutations.【Results】(1) LEV or AMX at the concentration of 2×,4×,6×,8×MIC were added to the H.pylori strain. After 6-9 passages, LEV-resistant strains were induced successfully in all groups of LEV. AMX-resistants were induced successfully at 2×,4×,6×MIC, but the MIC of the strain selected by 8×MIC can not meet the resistance standard. By comparing the induction period: the MIC increased rapidly in the early time of LEV inducton process, but it stalled later, especially when MIC reached 4.0ug/ml; during the AMX induction process, when MIC increased to 1.0 ug/ml, it did not continue to increase or even decline. (2) The resistans selected at low concentration(2×MIC) of LEV and AMX were non-target mutants. There are six changes of gyrA and two changes of gyrB for the resistants that selected at high concentration(4×MIC) of LEV, such as: Asp87→His, Gly81→Asp, Asp678→Glu, Leu536→Pro, Tyr628→leu, Arg752→Arg; and Lys447→Glu, Tyr 628→Leu; Gly320→Val, Phe423→Leu, Ala79→His, Thr541→Leu, Asn593→Thr pbp1 mutations and Asn296→Ala, His494→Ser, Ser541→Val pbp2 mutantions were selected at high concentration(4×MIC)of AMX.【Conclusions】(1) From all the outcomes above, the resistance to LEV and AMX H.pylori can be selected, while LEV-resistants were more likely to be selected. (2) The different concentration of LEV and AMX have significant effect on resistants mutations: the LEV and AMX target site mutations were not detected in resistants while at low concentration(2×MIC); both of the LEV and AMX resistants have target gene changes while at high concentration(4×MIC). (3) the results of sequencing indicated that: gyrA and gyrB mutations were involved in LEV resistance; AMX resitants aslo exist pbp1 and pbp2 mutations.