Reasearch On The Resistance Of Plasmid Mediated Shigella Spp. Resistance

Bacterial resistance to antibiotics means that bacteria can get the instinct of survival in the drugs that originally can kill or weaken them. There are two ways of the source of the bacteria resistance:genetic mutation and the acquisition of resistance genes. The main reason for antibiotic resistance of bacteria widely spreading is that the bacteria can get external resistance genes through mobile genetic elments, including plasmids, transposons, integrons and Insertion sequence common region(ISCR), etc.Plasmid is an independent, self-replicating genetic element, which can carriy resistance genes, transposons, integrons and ISCRs to transfer between different bacteria promoting the spread of drug resistance. Then we can see plasmids play a key role in the diffusion process of the drug-resistant genes. Therefore, to study the structure of the plasmid DNA and to analyze the relationship beween the plasmids carrying the functional genes which is related to drug-resistance and bacterial resistance is significant for the further study of molecular mechanism of bacterial resistance.In order to study the horizontal transfer of genes systematically, our research group had built a multi-drug resistant shigella spp. ZT by conjugation(Donor bacteria was E. coli E667resistant to CF, SMZ, NOR, GM,isolated from hospital; Recipient bacterium was Shigella flexneri Z23sensitive to above four antibiotics.), and filtered out some sequences by suppression subtractive hybridization experiments. Then the results of dot-blot hybridization had indicated that these sequences were associated with antibiotic resistance by cloning conversion, located on the plasmids donor strains E.667and tansfer strains ZT. However we still couldn’t make the conclusion the plasmid containing these sequences related drug-resistance was a resistance plasmid and mediated conjugation beween E667and Z23by itself, which is worthy of our consideration. That is to say we still need further study to identify the role of the plasmid which contains these drug-resistant seqeuence during the process of the horizontal transfer and the retionship between the plasmid and drug-resistance. So we tried to gain transformant of the plasmid which contains those sequences related to antibiotic resistance through the transformation experiment, then analyzed the relationship beween the plasmid and the drug-resistance, got the drug-resistant genes of the plasmid and mobile gentic elments related antibiotic resistance, which helped us make out the role of the plasmid for the drug-resistance tansfer and provide the basic information for the study of the bacteria resistance molecular mechanisms.Methods1The acquisition of transformants:By improving plasmid transformation method, reducing rotation speeds and tempreture and prolonging time of preincubation. plasmid stored in the library which was extracted from conjugal transfer shigella spp. ZT.(Donor bacteria was multiresistant E. coli E667isolated from hospital; Recipient bacterium was Shigella flexneri Z23sensitive to antibiotics.)was transformed into competent Ecoli cells DH-5α, and then the bacteria solution was coated on the LB solid culture medium Which contained one single type of antibiotic, including MIC and1/2MIC of cephalothin(CF), sulfonamide(SMZ), norfloxacin(NOR), gentamicin(GM), cultured18h-24h at37℃. If there were bacterial colonies growing on the LB solid culture medium, we would use colony PCR to amplify the drug resistance related sequence T2G6to screen the positive clones.2Plasmid extraction:The positive clones were picked to LB liquid medium plus antibiotic NOR and shaken overnight at37℃, and then plamids were extracted by using a small amount of plasmids extraction kit which was bought from Axygen biotech company to obtain the plasmids, which were analyzed by0.5%agarose gel electrophoresis with original plasmid.3Antimicobial resistance test:We used antimicrobial susceptibility testing standards of disk diffusion method and U. S. National Institutes for Clinical Laboratory Standards Committee (NCCLS) guidelines as a reference to identify the susceptibility of antibiotics of the transformants by the Kin-Bauer disc agar diffusion. The antibiotic susceptibility paper included:cephalothin(CF), cefazolin(CFZ), cefuroxime sodium(CXM), cefotaxime/clavulanic acid (CAZL), ceftazidime(CAZ), cefepime(FEP), cefotaxime(CTX), cefotaxime/clavulanic acid (CTXL), ceftizoxime(ZOX), cefoperazone(CFP), chloramphenicol(CHL), Azotreonam(AZT), sulfonamide(SMZ), norfloxacin(NOR), amoxicillin(AMX), ampicillin(AMP), tetracycline(TET), streptomycin(STR), gentamicin(GM), methylthiouracil(TMP), Imipenem(IPM)and so on. ATCC25922was the quality control bacteria.4Resistance genes screening:Basing on the results of antimicobial resistance test, we slected some resistance genes which may locate on the plasmid, including cephalosporin resistance genes like blaTEM, blaSHV, blaCTX-M, Ampc, quinolone resistance genes like qnrA, qnrB, qnrC, qnrD, qnrS, qepA, aac(6’)-Ib-cr, Sulfa-drug resistance gene sull, chloramphenicol acetyltransferase gene cat.5Mobile genetic elements related to drug-resistance screening:According to the related literatures, we got the primers of insertion sequences like ISEcpl, IS26, IS903, ISCR1, integrase genes intll, intI2, intI3and conjugal plasmid gentic markers traA and trbC. The genes were screened by PCR and the sequeces were submitted to BLAST.Results1The acquisition of transform ants:After the screening of positive clones with the method of colony PCR, the transformants were gained from NOR(1/2MIC) LB solid medium while there were no positive clones growing on the rest of the antibiotic mediums. The results of plasmid agarose gel electrophoresis showed that the plasmid of the transformant was the same with the original plasmid.2Identification of antibiotic susceptibility:Competent E. coli cells DH-5α were sensitive to all antibiotics; transformants were resistant to CAZ, AZT and intermediarily resistant to CTX, ZOX while they were sensitive to other antibiotics; compared with Competent E. coli cells, antibacterial rings of CTX, CFP, ZOX, FEP, NOR, CIP, LVF, SMZ, TMP and CHL decreased more than5mm to transformants while that of CFZ, CXM, AMP, AMX, GM, STR, TET, IPM changed less than5mm. ESBLs confirmatory experiments showed that the transformant was non ESBLs producing strain.3Resistance genes screening:It was found that resistance genes TEM-116, sull and cat located on the plasmid, however we didn’t not detect the quinolone resistance-related genes.4Resistance-associated mobile gentic elements screening:The insertion sequences were not detected on the plasmid while we found intll and trbC were located on the plasmid.Conclusions1The plamid which was proved to be a wide-host conjugal plasmid mediated the transfer of the resistance genes beween Wild E. coli667and Shigella spp..2The plasmid was a multiresistant plasmid which mediated the bacteria resistance to cephalosporin antibiotics and monobactam antibiotics, and was related to the development of resistance to quinolones, chloramphenicol and sulfonamide antibiotics.3The resistance genes and resistance-associated genetic elements couldn’t fully explain the resistance phenotypes, so the mechanism of action of the plasmid deserves further research.