Analysis Of Resistance To Florfenicol In Swine E.coli And Research Of The FloR Gene

The disease of Escherichia coli from swines is an important disease which often occurs inintensive pig farms.It occurs in the form of yellow or white seour of newbom piglets and pigletedema disease. Nowdays, there are no ideal vaccines which are used to prevent this disease, be-cause there are too many serological types. Antibiotic plays an important role in prevention andtreatment of these disaeses.In recent years, flofenicol is widly used to cure these diseases causedby Escherichia coli. But with the increase of incorrectly use, the sensibility of this antibiotic is b-ecoming lower or lost. With the appearance and dissemination multidrug resistant bacteria, pre-vetation of animal disease is influced.And it threats to the safety of zoogenic food and the healt-h of people.1、Determining MIC,MPC,MSW and narrowing MSW in vitroMinimal Inhibitory Concentration （MIC）, Mutant Prevention Concentration (MPC) andMutant Selection Window（MSW）were determined by agar double dilution method andspreading1011cfu/mL colony on agar plates at different drug concentrations for florfenicol. TheMSWs of S19，S22，S23whose MSWs for florfenicol are both relatively larger were determinedby florfenicol combined with doxycycline.The resistant rates of35strains swine Enterococcifrom Jilin province to florfenicol were42.86%；The combination index of florfenicol anddoxycycline for S19, S22, S23were0.75,0.09,0.4375, showing additive or synergistic effects.For florfenicol, MPC became from256μg/mL,128μg/mL, and460.8μg/mL to8μg/mL,8μg/mLand16μg/mL. Mutation select window became closed or narrower. The results showed that:florfenicol combined with doxycycline could narrow or close swine Enterococci resistantmutations select window.2、Detection and location of the floR gene alonge with conjugation ofplasmidPolymerase chain reaction (PCR) was done with the template of cell. All bacterial plasmidsand genomes were all extracted as templates to achieve PCR reaction.Then plasmid conjugationexperiments were achived. We came to the conclusions: we did not get floR gene from genome.For all the strains whose MIC≥32μg/mL,we got the aim sequences whose sizes were all1031bp. Its homology to the sequence reported is more than98%. The floR genes can be passedthrough a combination of plasmids.Its transmission frequency is5.1×10-7.3、 Detection of floR gene from gene cassettes in intergronsBy PCR, gel electrophoresis, plastic recycling, ligation, transformation, restriction verifica-tion, sequencing and other tests,we can get to the following conclusions: in these Escherichia coli from swines, theⅠtype integrase positive rate was94%; theⅠtype integron positive rate was13.9%; theⅡtype integrase positive rate was27.8%; theⅡtype integron positive rate was25%;The homology of theⅠtype intergron in the experimentsis is99%to Escherichia coli class1integron dihydrofolate reductase (dfrA12), hypothetical protein, and adenyltransferase (aadA2)genes reported and100%to Escherichia coli strain ZM-6plasmid classⅠintegron dihydrofolatereductase (dfrA1) and aminoglycoside adenyltransferase (aadA1) genes reported.It is a pity thatwe have no detected the gene of floR from gene cassettes in intergrons.In conclusion, Escherichia coli from swines have some resisitance to flofenicol,the resultsof MPC and MSW show that, Escherichia coli is very easy to have resistance to flofenicol andhard to recover. Florfenicol combined with doxycycline could reduce or close swine Esc-herichia coli resistant mutations select windows.The detection of floR genes show that in thesestrains, floR genes are all located in plasmids.And they can transfer by plasmids conjugati on.wehave no found floR gene in intergron.