Study On Characteristics Of Drug Susceptibility And Mechanisms Of Quinolones Resistance In Bacillus Cereus

Antibiotic was once commonly used in human history, but with frequentand huge use of it, people had to face up to the negative influence broughtby antibiotic: plenty of drug-resistance especially multi-drug-resistance hasshown up, which makes it harder for people to treat the disease of animal ormankind;drug residues not only restrain the export of Chinese meat, eggs,milk product, but also do harm to the people's health. Animal probiotic hastaken the place of antibiotic. Antibiotic can kill or restrain the animalprobiotic , so consequently animal probiotic can't work as it used to be.This study base on probiotic's resistance to antibiotic as well as itsmechanism, and provide the theoretical foundation for development andapplication of probiotic.We get the bacillus cereus from animal probiotic product through normalbio-experiment. They were identified to be B.cereus and B.subtilis throughmorphology and biochemistry experiments. K-B was used to test thedrug-resistance to 54 kind of antibiotic which belong to seven species. Theresult shows that probiotic-bacillus is most sensitive to FQNs, thenfollowing by β-lactam. We adopt NCCLS M100-S13 scheme to measurethe MIC of 22 strains of bacillus cereus. The standard strain CMCC(B)63301 together with two most sensitive B.cereus of above 22 strains wasartificially induced to create their resistance to CPLX. Drug-resistant modelin vitro was constructed. After being induced, the resistance of three strainsof B.cereus was obviously increased. MIC was increased from 0.0625 to2(mg/mL). Then they turn out to be resistant to CPLX.(judged by thestandard of NCCLS)B.cereus induced by CPLX has differently strengthened its resistance toFQNS except CPLX. It shows that FQNS has crossing drug-resistancebetween each other.We designed prime according to gene sequence of B.cereus announced inGenBank, successfully cloned gyrA gene(804bp) and parC gene(816bp)of standard strain and isolated sensitive B.cereus before being induced in topMD18-T vector. Comparing with the announced sequence in GenBank,their homology is beyond 95 percent. After being induced, gyrA gene sixbasic groups of B.cereus was substituted. As it for L2 strain, they areA223C(Asn→His),A269C(Asp→Ala),A462G. and as it for L18 strain,they are G245C(Arg→Pro),A269C(Asp→Ala),G282C(Gly→Cys).Their point mutation A to C on 228 basic group was same. After being artificiallyinduced, two basic groups of the standard strain was substituted. They areT222C and A269C(Asp→Ala). The point mutation on 269 basic group is samewith two isolated strains. Comparing parC sequence, we found, after beinginduced, there are ten basic group changed. All the three B.cereus strainshave the point mutation G to S on 228 basic group. The point mutation of gyrA andparC may lead to their drug-resistance, and it also may be the result ofmulti-drug-resistance mechanism.In the third experiment, we use energy inhibitor CCCP and SPF toinspect B.cereus's accumulation of FQNs (including drug excretion). so asto find if there is other drug-resistance mechanism ofmluti-drug-excretion.The result show that CCCP did made the intaping ofciprofloxacin much more in 3 strains.This research builds basis for the furtherstudy of B.cereus drug-resistance mechanism.