The Antibacterial Effect Of Antibiotics Combined With Proton Pump Inhibitors And The Relationship Of Hefa Efflux Gene Expression And Multiple Drug Resistance Of Helicobacter Pylori

Objectives:To observe the combined antibacterial effect of antibiotic combined with proton pump inhibitors in Helicobacter pylori(Hp);To analyze the relation-ship of outer-membrane protein hefA gene expression and multiple drug resistance of Guiyang Helicobacter pylori.Methods:1.Clarithromycin,amoxicillin and levofoxacin sensitivity of Hp clinical isolates was detected by break point method.The sensitive strains(n=5)and resistant strains(n=5)of clarithromycin,amoxicillin,levofoxacin were selected,the minimum inhibitory concentration of three antibiotics and two proton pump inhibitors(PPI)including omeprazole and rabeprazole before and after combination were detected by checkboard method.Fractional inhibitory concentration index was calculated by the formula to determine the effect of amtibacterial drug combined with;the FICI of three antibiotics combined with omeprazole and rabeprazole were compared,statistical analysis was carried out by t test.2.The genomic DNA of multi-drug resistant strains(n=10),sensitive strains(n=6)and control srtrain ssl were extracted,hefA gene fragment were amplified by PCR,the PCR product was subjected to agarose gel electrophoresis,and then the PCR products were sequenced.Sequences were input into NCBI for BLAST comparison.The RNA of these bacteria was extracted by TRIzol reagents,and gene of 16sRNA was the reference gene.Real-time quantitative PCR of hefA gene and reference gene was carried on the reverse transcripted cDNA.CT value of hefA gene and reference gene were detected and,the differences of hefA gene expression in multi-drug resistant strains and sensitive strains were judged by comparing2-??Ct.Results:1.There were 15(21.7%)multi-drug resistant strains among the 69 Hp strains.2.The FICI of all combined antibacterial tests were less than 0.75 both in sensitive strains and resistant strains group.The FICI of antibacterial test of rabeprazole combined with levofloxacin against sensitive strains was smaller than levofloxacin combined with omeprazole(P=0.035<0.05,t test),There was no significant difference of the FICI of omeprazole or rabeprazole combined with amoxicillin or clarithromycin against sensitive strains(P=0.545and0.156>0.05,t test);Against resistant strains,the FICI of amoxicillin combined with rabeprazole was smaller than amoxicillin combined with omeprazole(P=0.012<0.05,t test),FICI of omeprazole or rabeprazole combination with levofloxacin or clarithromycin had no significant difference(p=0.789and0.461>0.05,t test)3.140bp fragments of hefA gene could be amplified from all strains.In the Real-time PCR,2-??CT Twas 0.89510.540 in sensitive strains,and 2-??CT was 3.387±1.597 in MDR strains,The expression of MDR strains was significantly higher than the sensitive strains(P=0.001<0.05,t test).Conclusions:1.Omeprazole or Rabeprazole combinated with Clarithromycin,levofloxacin and amoxicillincan respectively have synergistic antibacterial effect.2.The combined antibacterial effect against sensitive strains of rabeprazole and levofloxacin was higher than levofloxacin combined with omeprazole;Against resistant strains,the combined antibacterial effect in resist-ant strains of rabeprazole combined with amoxicillin was higher than amoxicillin combined with omeprazole.3.When antibiotics combined with PPI,some resistant strains were sensitive to amoxicillin,levofloxacin and clarithromycin.4.Higher expression of hefA gene could be relative to multiple drug resistance of Helicobacter pylori isolates of Guiyang.