The Molecular Characteristics Of AmpC β-lactamase Gene, Virulence Gene Detection And Out Membrane Proteins Nanlysis In Escherichia Coli Isolates From Ducks

It was currently recognized that bacterial mechanisms of resistance to beta-lactam drugsincluded the following four kinds:①changes of penicillin-binding protein,②g enerationofβ-lactamase enzymes,③outer membranebarrier function,④bacterial active efflux mechanism.Of resistance produced by β-lactamase enzymes, resistant AmpC β-lactamase (AmpC enzyme)and extensive spectrum β-lactamase enzyme (ELBLs) was the most common. Gram-negativebacilli produce both chromosome-mediated AmpC enzymes and plasmid-mediated AmpCenzymes. AmpC enzymes are a class of enzyme which are capable of hydrolyzing cephalosporinand cannot be inhibited by β-lactam inhibitors. Chromosome-mediated AmpC were originallydiscovered mainly from Enterobacter, C.freundii and Providencia, etc. Later it was found thatmost of the plasmid-mediated AmpC enzymes were from the chromosomal-mediated AmpCenzymes, resistant genes can transfer between chromosomes and plasmids in various formsincluding transposon, insertion sequence and integron, cause widely disseminated clinicalresistance, which made greater harm than chromosomal-encoded AmpC enzymes. There havebeen a lot of reports about the E. coli plasmid-mediated AmpC enzymes, but no detection andmolecular features research of duck Escherichia coli AmpC enzyme blaDHA-1gene was reported.This paper collected fifty three strains of clinical duck Escherichia coli, broth dilutionmethod was applied for the determination of resistance phenotype, and high AmpC strains werescreened using cefoxitin and3D test. The AmpC gene of AmpC enzyme positive strains weredetected using PCR. Whether the AmpC enzyme genes could spread by plasmid conjugation wasconfirmed by plasmid conjugation test. The gene-environment of the AmpC enzyme genes wasanalyzed by cloning experiments. Strains were analyzed by plasmid incompatibility group,phylogenetic typing, and ERIC-PCR technology to research the cloning evolutionary relationshipamong strains from different regions. The virulence genes were amplified by PCR to analysis therelationship among virulence genes, multidrug-resistant phenotype and phylogenetic type. Inaddition, SDS-PAGE was used to analyze the outer membrane protein, and common E. coli outermembrane protein gene (OmpA, OmpC and OmpF) were amplified by PCR to investigate the relationship deletion between outer membrane protein deletion and multidrug-resistant. Wehope that we can provide a scientific basis for the clinical use of drugs and prevention andtreatment of multiple drugs use; provide a research data basis for the development of new drugsand looking for targets for antibacterial drugs.Eleven suspected positive AmpC were screened from fifty three clinical duck Escherichiacoli by cefoxitin. Six high AmpC strains were obtained from3D test, of which two were detectedto contain blaDHA-1gene and two containing CMY-2gene by PCR amplification. MIC test showedthat the majority of the bacteria were multi-drug-resistant strains, all the fifty three ducks E. coliresistant to florfenicol and the rate of cefoxitin-resistance reached58.5%, resistant rates totetracycline, doxycycline, grace enoxacin, kanamycin, cefotaxime, ciprofloxacin, cephalosporinsspecial piperidine, cephalosporins special piperidine/sulbactam, cefotaxime/sulbactam,cefepime and amikacin reached96.2%,88.7%,83.0%,75.5%,73.6%,69.8%,26.4%,24.5%,24.5%,15.1%and13.2%, respectively. Three zygotes were obtained by the membrane bondingtest, while only one bacterium was detected containing blaDHA-1gene using PCR. Plasmidconjugation experiments showed that blaDHA-1gene was located on a conjugative plasmid.Plasmid incompatibility group analysis was carried for zygote plasmid, and results showed thatthe plasmid did not belong to any one of the common E. coli plasmids. According to thephylogenetic typing method, multiplex PCR were applied to the eleven E. coli. Results showedthat we got four A-class strains, the proportion of which was36.4%; and seven B1class of strains,the ratio was63.6%, no high virulence strain in the eleven strains were detected.PCR technology showed that cefoxitin resistance genes including iron ions conditioningsystem (irp2) gene in the high pathogenicity island (HPI), P-type fimbriae structural gene (papA),the serum tolerance gene (iss, cvaC) and adhesion pilus genes (felA, csgA) of the11strains. Theresults showed that the rate of irp2, Papa, iss cvaC of csgA in clinical isolates was54.5%,90.9%,18.2%,9.1%,90.9%.Plague streptozotocin receptor genes (fyuA), type I pili protein gene (fimC),adhesion pilus genes felA,were not detected. ERIC-PCR results showed that the strains ofdifferent sources may belong to the same clone type; strains of the same source may belong todifferent clone origins.A bacteria recombinant plasmid was obtained from enzyme digest and transformation,sequence analysis of the recombinant plasmid showed a fragment14583bp long. Throughcomparison and analysis of the recombinant plasmid fragment,we found that the plasmid carriedboth blaDHA-1and qnrB4gene, this sequence also contained a partial sequence of the insertedsequence ISCR1,transporter protein (SAPC SapB SAPA), inducible protein CinA,phage shockprotein (pspF, pspA, pspB, pspC, pspD) and AmpC enzymes transcriptional regulator ampR gene. It can be drawn from the above results that blaDHA-1and qnrB4gene were located in the sametransferrable plasmid, and that these two resistance genes can be co-transferred andco-propagated.SDS-PAGE electrophoresis was applied to analyze the outer membrane proteins of the fiftythree clinical duck E. coli, and we found that the number of strains with deletion of OmpA,OmpC and OmpF of Omp (A+C),Omp (A+F), Omp (C+F), Omp(A+C+F) was28,35,9,15,3,6,4; ratio of which was52.8%,66.0%,17.0%,28.3%,5.7%,11.3%and7.5%.,respectively.Three pairs of primers, OmpA (F, R), OmpC (F, R), OmpF (F, R) were designed and PCR methodwas used for genetic testing of fifty three bacteria. Results showed that the number of strainsmissing the OmpA, OmpC, OmpF, Omp (A+C),Omp (A+F), Omp (C+F),Omp (A+C+F)was28,44,7,26,4,7,4; respectively; the proportion was52.8%,83.0%,13.2%,49.1%,7.5%,13.2%and7.5%, respectively. Contrast SDS-PAGE and PCR assay results, we found that thegene and protein deficiencies difference were almost similar to each other except the OmpC geneand protein.