Dissemination, Drug Resistance And Molecular Biology Properties Of The Integrons Among Some Clinical Isolates Of Pseudomonas Aeruginosa

Objective:To investigate the disseminaton of integons carried by Pseudomonas aeruginosa clinical isolates from our hospital and to analyze the correlation between integrons and the multi-drug resistance of Pseudomonas aeruginosa.To study the genotypes of variable region and the biochemical properties of integrons and to disclose its action in the acquisition, transmission or dissemination of antibiotic resistance.Materials and Methods:1. Isolates:Totally 47 strains of Pseudomonas aeruginosa were collected between 1997 and 2003 from infection cases in Jiangsu Province Hospital and randomly chosen for study.2. Method:All the isolates were candidates for screening class 1 and class 3 integrons by using specific primers located on integron-encoded integrase gene, the intI1 gene and intI3 gene, respectively; conjugation experiments was performed between integron positive isolates and PU21 to transfer resistance plasmid; agar dilution method was used to determine MICs of 9 antibiotics against 47 isolates and the transconjugants.The variable regions of class 1 integrons were amplified by using universal primers located on 5' and 3' conserved segments.The PCR amplicons were purified. Then DNA sequencing and blastn program was used to ascertain the genotype of the variable region of class 1 integrons.Every gene cassette in wild-type isolates and transconjugants were amplified by primers designed according to the results of DNA sequencing of variable regions in class 1 integrons, respectively. The DNA sequence of PCR products was determined by direct sequencing.Plasmid DNA was extracted from wild-type isolates and transconjugants by alkaline lysis.Pulsed-field gel electrophoresis method was performed to analyze the homology of integron positive isolates.Results:Of 47 isolates, 29 strains of Pseudomonas aeruginosa were found containing class 1 integron, and no class 3 integron was detected. All class 1 integron isolates were successfully conjugated to the recipient cell. Susceptibility testing revealed that the antibiotic resistance rate of the integron positive strains was higher than that of the integron negative strains. All class 1 integron positive wild-type isolates and the corresponding transconjugants exhibited an overall resistant property to piperacillin, ceftazidime, gentamicin, amikacin, chloramphenicol and streptomycin. Compared with the wild-type Pseudomonas aeruginosa, the transconjugants were all susceptible to piperacillin-tazobactam, ciprofloxacin and imipenem, and MIC₉₀ values of those antimicrobial agents decreased about 8 times, 128 times and 128 times, respectively.A length of 4,300bp variable region was found in all the class 1 integron positive strains by PCR amplification. The result of DNA sequencing revealed that it was comprised of a new array of aac(6')- II -aadA13-cmlA8-oxa-10 gene cassettes, and the Genbank number is EU182575. According to the database in Genbank, aadA13 was previously unreported cassette in Pseudomonas aeruginonsa; chloramphenicol efflux pumping determinant, named as cmlA8 shared 95% nucleotide similarity to cmlA5 gene.All four gene cassettes harbored by class 1 integron in wild-type isolates and transconjugants were detected by PCR amplification, respectively. The direct sequencing results were identical to the results of DNA sequencing of variable region.The length of plasmids DNA extracted from all wild-type isolates and transconjugants were identical to each other and larger than 10Kb.All those class 1 integron positive isolates showed high homology by PFGE pattern analysis after digestion with Spe I .Conclusion:Class 1 integron was wildspread in Pseudomonas aeruginosa clinical isolates from our hospital. Susceptibility testing revealed that the antibiotic resistance rate of the integron positive strains to many antimicrobial agents was significantly higher than that of the integron negative strains. The resistance phenotype and genotype of the variable region harbored by integons suggested that the presence of integron was closely associated with resistance and multi-drug resistance of Pseudomonas aeruginosa. Compared to the wild-type isolates, the correlative transconjugants were susceptible to piperacillin-tazobactam, ciprofloxacin and imipenem.Class 1 integrons disseminated among some clinical isolates of Pseudomonas aeruginosa in our hospital possessed novel array of gene cassettes and located on transferable plasmid. Horizontal or clonal (vertical) transmission might exist between these Class 1 integrons positive isolates.