| Background and purpose:Helicobacter pylori(H.pylori), a gram-negative bacterium, has been recognizedto cause life-long infection of the gastric mucosa in billions of people worldwide, andis a major cause of peptic ulcers and also a key risk factor for gastric cancer andgastric MALT lymphoma, Once H.pylori is established in the gastric mucosa, infectedindividuals usually carry it for life unless treated. With the widespread use ofantibiotics in recent years, the problem of drug-resistant of H.pylori has becomeincreasingly serious; Furthemore, multidrug resistant (MDR) strains that aresimultaneously resistant to three or more antibiotics have been reported. How toovercome antibiotic resistance of H. pylori and to improve the eradication rate of H.pylori has become a worldwide problem. Efflux pump，which pumps toxic substrateout and exists widely in bacteria，is considered as the main mechanism of drugresistance,especially in multidrug resistant. Five families of multidrug efflux pumpsare recognized in bacteria which contribute to resistance against importantdrugs.among this efflux pumps: The resistancenodulation-division (RND) family(also named AcrAB-TolC efflux pump), which was originally discovered inGram-negative bacteria, have a crucial role in the generation of pathogenicity anddrug resistance in Gram-negative bacteria.In order to elucidate what kind of role the AcrAB-TolC efflux pump plays inmulti-drug resistance of H.pylori, we made an investigation to see if thereare discrepancies in the expression of the gene that codes for the protein whichrelated to AcrAB-TolC efflux pump between MDR strains and drug-sensitive strains.In addition, we observed whether mutations existed in the resistance genes which areimplicated in antibiotic resistance in multi-drug resistance strains. Furthermore, wehad made an investigation to see whether efflux pump inhibitors (EPIs) can influencethe antibiotic susceptibilities of H. pylori MDR strains.Method:1. Screening of H. pylori strains: From653H. pylori clinical isolates, we had screened10H. pylori MDR strains(drug-resistant group) and10susceptible strainswhich are sensitive to9kinds of commonly used antibiotic (drug-intensive group),sensitivity of these H.pylori strains to9kinds of antibiotics was remeasured byusing E-test and K-B methods.2. The detection of mRNA expression of protein-coding genes related toAcrAB-TolC efflux pump: Using q-PCR method to detect the expression of hefA、hefB、hefC、hefD、hefE、hefF、hefG、hefH、hefI、hef1489、hef1488; Making ananalysis of the correlation between the expression rate of protein-coding genesrelated to AcrAB-TolC efflux pump and the sensitivity of H. pylori to antibiotics bythe SPSS17.0software.3. The detection of resistance genes which are implicated in antibiotic(Metronidazole, Clarithromycin, Levofloxacin) resistance: It’s recognized that InH.pylori, mutations in the rdxA and frxA genes are implicated in metronidazoleresistance;23SrRNA and gyrA genes are respectively implicated in clarithromycinand evofloxacin resistance. We have made a detailed analysis of these genes afterdetecting them, Mainly including:①Extracting of genome DNA of H. pylori;②Finding out the gene sequences of rdxA、frxA、23SrRNAand gyrA through GenBank,then designing the differential primers about those resistance genes by Using thesoftware Primer Premier5.0. And we would obtain those appropriate targetedfragments by PCR amplification.③Gene sequencing and Bioinformatics analysis:Sequencing those PCR products,then doing a bioinformatics analysis of the sequencewith the software Primer Premier5.0andAlignX.4. Influence of efflux pump inhibitors on the multidrug resistance ofHelicobacter pylori: Adding several kinds of efflux pump inhibitors to the agar, thendetermining MICs of the antibiotics. The concentrations of efflux pump inhibitors asfollows: phenyl-arginine—βnaphthylamide(20mg/L), Carbonyl Cyanide m-Chlorophenylhydrazone(10mg/L), omeprazole(10mg/L), rabeprazole(10mg/L),pantoprazole (10mg/L), lansoprazole(10mg/L), esomeprazole(10mg/L).4-fold or4times more fold increase in MIC of antibiotics for the MDR strains was consideredsignificant.Results: 1. Expression of protein-coding genes related to AcrAB-TolC efflux pump：There was no high expression of protein-coding genes related to AcrAB-TolC effluxpump in all the susceptibal strains, nevertheless,7of the MDR strains presentedobviously high expression of those genes, and there was no significant difference wasobserved in expression of those protein-coding genes between susceptible and MDRstrains (P>0.05)①Expression of hefA in drug-intensive group was0.96±0.71, and3.35±9.25in drug-resistant group. R3stains was above29, all other strains werebelow3;②Expression of hefB in drug-intensive group was1.13±0.73, and4.20±7.22in drug-resistant group. R3and R5stains were above10, all other strainswere below3;③Expression of hefC in drug-intensive group was1.41±0.93, and5.73±13.64in drug-resistant group. R3stains was above44, all other strains werebelow4;④Expression of hefD in drug-intensive group was0.86±0.85, and3.83±7.30in drug-resistant group. R3and R9stains were above15, all other strains were below3;⑤Expression of hefE in drug-intensive group was9.73±8.79, and9.73±8.79indrug-resistant group. R1、 R3and R9stains were above16, all other strains werebelow4;⑥Expression of hefF in drug-intensive group was0.76±0.68, and5.30±9.42in drug-resistant group. R9and R10stains were above22, all other strainswere below4;⑦Expression of hefG in drug-intensive group was0.78±0.48, and6.61±13.84in drug-resistant group. R3、R8and R10stains were above6, all otherstrains were below2;⑧Expression of hefH in drug-intensive group was1.23±0.66,and4.38±7.40in drug-resistant group. R4and R10stains were above12, all otherstrains were below3;⑨Expression of hefI in drug-intensive group was1.12±0.80,and0.85±0.58in drug-resistant group. All the strains were below3.⑩Expression ofHP1489in drug-intensive group was1.31±0.49, and1.74±0.64indrug-resistant group. All the strains were below5； Expression of HP1488indrug-intensive group was1.92±0.61, and2.23±0.70in drug-resistant group. All thestrains were below5。2. The relationship between the expression rate of protein-coding genes relatedto AcrAB-TolC efflux pump and the sensitivity of H. pylori to antibiotics: The resultshowed us that there was no obvious correlation between the expression rate ofprotein-coding genes related to AcrAB-TolC efflux pump and the sensitivity of H. pylori to antibiotics.3. The detection of three kinds of resistance genes:①8MDR strains whichresistant to clarithromycin had mutations in the23SrRNA gene, the mutational sitesare all at2143point;②All the MDR strains had mutations in the gyrA gene, thedominating mutational sites was261point, some at271and272point;③All theMDR strains had mutations in the rdxA and frxA genes, the mutational sites weresporadic.4. Influence of efflux pump inhibitors on the multidrug resistance ofHelicobacter pylori:①After adding PAβN to the agar, MIC of Clarithromycindecreased at least4-fold in4MDR strains;②After adding CCCP to the agar, MIC ofMetronidazole decreased at least4-fold in4MDR strains, and Clarithromycindecreased at least4-fold in2MDR strains;③The MIC of antibiotics didn’t changeat all after adding5kinds of proton pump inhibitors.Conclusion:1. There is a high expression rate of protein-coding genes related to AcrAB-TolCefflux pump in several MDR stains, and low expression in all the sensitive strains.Furthermore, a part of the efflux pump inhibitors can selectively decrease the MICvalue of antibiotics. These indicate that the AcrAB-TolC efflux pump plays animportant role in multi-drug resistance of H. pylori.2. Not every MDR stains shows high expression rate of protein-coding genesrelated to AcrAB-TolC efflux pump, and the high expression of genes have nothing todo with the MIC, it implicate that multi-drug resistance of H. pylori are notcompletely caused by the AcrAB-TolC efflux pump system.3. The situation of mutations in resistant genes are consistent with thesensitivity of H. pylori to antibiotics, it implicate that multi-drug resistance of H.pylori is the result of a combination of the AcrAB-TolC efflux pump system and theresistant genes. |
PDF Full Text Request