Drug-resistant Analysis Of Laboratory Animals Pseudomonas Aeruginosa Isolates In Beijing Area And Comparison Of PFGE And MLVA Typing Method To These Isolates

Objective:To understand the drug resistance, distribution of genotypes and homology of Pseudomonasaeruginosa isolates derived from laboratory animals in Beijing area. And to carry outepidemiological analysis and compare the advantages and disadvantages of both pulsed-fieldgel electrophoresis(PFGE) and multilocus variable number tandem-repeat analysis (MLVA)typing method. To ensure laboratory animals microbial quality control and related animalexperiments carried out succsessfully.Methods: (1) P. aeruginosa isolates, collected at17experimental animal origins between2007to2011,were identified by the API bacterial identification system and16sRNA sequencing of thebiochemical identification of the strains;(2) According to the Clinical and Laboratory Standards Institute (CLSI) standard method, weselected eight antimicrobial agents of seven Groups, including cephems, penicillins,β-lactam, aminoglycosides, fluoroquinolones, carbapenems, lipopeptides. six to eight kindsof antibiotics, to conduct drug sensitive test for134P. aeruginosa by agar dilution method;The imipenem-resistant mechanism analyzed by IMP-EDTA test and outer membraneprotein OprD encoding gene sequencing.(3)134isolates were digested by Spe I restriction enzyme to conduct an epidemiologicevaluation and molecular typing using pulsed-field gel electrophoresis(PFGE), PFGEpatterns were processed and cluster analyzed by Bionumerics software.(4) The134isolates were amplified by13VNTR loci using capillary electrophoresis andanalyzed Bionumerics software, to calculate the various strains for each locus the number ofrepeat sequences, clustering analysis of the VNTR polymorphism by Bionumerics software.The cluster results compared with the PFGE.Results:(1) The results of API20E biochemical identification demonstrated that134strains wereP.aeruginosa doubtlessly isolated from the2988experimental animals and55drinking watersamples of135suspected P.aeruginosa isolates. The strain PA73was identified asAchromobacter xylosoxidans. All the rest of tested strains produced21species biochemicalnumerical profile, and the first identification results are P.aeruginosa. Biochemical profileofof CMCC10110was220200463. The strains identification ID%were among98.9%to99.9%, but strains PA001, PA002and PA060were25.2%,25.2%,57.7%and36.9%respectively,, the T index were0.5to1.134isolates were confirmed134P.aeruginosa.(2) The134P. aeruginosa isolates of laboratory animals drug sensitive test by the agardilution method and susceptibility testing, the results of resistance to CTX, IMP, Coli GEN,CIP five antibiotics, including resistance to CTX was11.9; IMP resistance was1.5%; rightColi resistance rate was2.2%; on the GEN resistance rate of41%; CIP resistance was1.5%;PIP, PIP-of TAZ and CAZ are sensitive. The existence of CTX-Coli-GEN-CIP (n=1) andmulti-drug resistant strains of the CTX-Coli-CIP (n=1) and CTX-GEN (n=11) double resistance. All resistant strains were isolated from the Beijing Fengtai District. Analysisshowed that the outer membrane protein OprD gene mutation leading to the main reason forresistance to imipenem mechanisms of the two imipenem-resistant strains.(3)134strains were cut out15to20DNA fragments, the size between20Kb to1200Kb. Thetotal isolates were divided into22clusters (A-N) and108patterns by Bionumerics software,the Simpson’s index of diversity D=0.955. The main cluster J, M, N and U, the proportionwere39/134(29.1%),15/134(11.2%),22/134(16.4%) and13/134(9.7%)respectively. Mostof the isolates were independent infection, but these strains had a high genetic similarity indifferent source.(4)134strains were divided into32clusters (1-32),51genotypes (MP1to51), the resolutioncoefficient D=0.763. The respective proportions of12and21clusters of MLVA were56.7and14.2%. In all of134strains, two stains had a high homology with Libya and Spainrespectively through comparing with MLVA database (minisatellites.u-psud.fr/MLVAnet/).(5) PFGE and MLVA have the same typability of drug-resistant strains of IMP, Coli, CIP, andGEN, the ability of PFGE genotyping of CTX resistant strains slightly better than MLVA.Conclusion:P. aeruginosa of experimental resistant to imipenem, cefotaxime, gentamicin, ciprofloxacin,and colistin strains in Beijing. gentamicin and cefotaxime resistance higher. The mechanismsof imipenem-resistant strains is due to the mutation of the outer membrane protein OprDgene. PFGE and MLVA method can effectively type test strains. The results of two methodtype the resistant strains are basically consistent. There are high genetic similarity P.aeruginosa in different source. In same source and at different time, the stains isolated haddifferent patterns. This result indicate presence of different sources of infection or mutationin those isolates. The reason of drug-resistant is possiblely due to mutation of P. aeruginosaitself. MLVA was better for detecting genetic relatedness, nevertheless PFGE was morediscriminatory than MLVA for determining genetic differences in P. aeruginosa.