Detection And Sequence Analysis Of Aminoglycoside And Quinolones Resistance Genes In Riemerella Anatipestfer

The Riemerella anatipestfer (RA) is the causative agent of a contagious septicaemic diseaseespecially in ducklings, gooses, turkeys and other birds. The disease accounts for seriouseconomic losses to the duck industry due to high morbidity and mortality. At present, thebacterial strain with antibiotic resistance to many kinds of antibiotics includingaminoglycoside and quinolone antibiotics, and even multiple drug-resistant strains haveappeared due to the unreasonable use of antibiotics in prevention and treatment of the disease.In this study, the antibiotic resistance to aminoglycoside and quinolone antibiotics and theresistance gene in38RA isolates were tested, and it can provide a reference for clinicalmedicine.1. The research of the resistance phenotype to aminoglycoside and quinoloneantibiotics among RA strainsIn this study, a total of38RA isolates were analyze the aminoglycoside and quinolonessusceptibility tested by K-B (Kirby-Bauer) method of WHO and evaluated the MICs togentamicin, amikacin, ciprofloxacin and norfloxacin by the microdilution method accordingto Clinical and Laboratory Standards Institute (CLSI) guidelines. The results displayed thatthe resistant incidence rates were exhibited to aminoglycoside antibiotics includinggentamicin (76.3%), streptomycin (71.1%), amikacin (55.3%), neomycin (50.0%) andkanamycin (39.5%); and to quinolone antibiotics including norfloxacin (55.3%), ciprofloxacin(52.6%), pefloxacin (44.7%) and ofloxacin (28.9%). There are33RA isolates (86.8%)produce the antibiotic resistance to two or more drugs, and2RA isolates (5.3%) produce theantibiotic resistance to all the above drugs. The MIC’s result indicated that the38RA isolatesdisplayd the resistance to gentamicin (63.2%), amikacin (50.0%), norfloxacin (60.5%) andciprofloxacin (55.3%). The above of the results showed that the38RA isolates displayedantibiotic resistance to many kinds of aminoglycoside and quinolone antibiotics, and thephenomenon of the multiple drug-resistant are very seriously.2. Testing and sequence analysis the aminoglycoside-resistant gene in38RA isolates In this research, the modifying enzyme genes (ant(3’’)-Ia, aac(6’)-Ib and aph(3’)-IIa) andthe16S rRNA methylase genes (armA, rmtA and rmtB) which mediated resistance toaminoglycosides were examined by PCR. The result showed that the detection rate ofant(3’’)-Ia, aac(6’)-Ib and aph(3’)-IIa were31.6%(12/38),89.5%(34/38) and94.7%(36/38),respectively. The nucleotide identities of the ant(3’’)-Ia, aac(6’)-Ib and aph(3’)-IIa sequencesbetween RA and E.coli were96.1%-100%,98.4-100%and98.9%-100%, and nucleotideidentities of ant(3’’)-Ia and aac(6’)-Ib gene between RA and Salmonella were94.3%-100%and98.4-100%, respectively. The16S rRNA methylase gene, which mediated the high levelresistance to aminoglycosides in the38RA isolates, were not detected.3. Testing and sequence analysis the quinolones-resistant gene in plasmid in38RAisolatesIn this research, the qnr genes (qnrA, qnrB, qnrC, qnrD and qnrS), QepA gene andoqxAB gene which in plasmid mediated resistance to quinolones, were detected by PCRrespectively. The result showed that only detected the qnrS gene in the quinolones resistancegenes. The detection rate of qnrS gene was28.9%(11/38). The nucleotide identities of qnrSgene between RA and E.coli or Salmonella were91.8%-99.8%or91.6%-98.8%, respectively.The study also found that there were4(11.8%) RA isolates contained aac(6’)-Ib-cr genewhen the sequence of aac(6’)-Ib gene which mediated the resistance to aminoglycosides in34positive RA isolates were analysised. The amino acid sequence of aac(6’)-Ⅰb-cr gene whichmediated low level resistance to quinolones with the Trp102→Arg and Asp179→Tyr.