| Objective: To investigate the positive rate of beta-lactamase and qacâ–³1-sul1 gene by polymerase chain reaction (PCR) method among clinical isolated strains of multi-drug resistant Pseudomonas aeruginosa and Acinetobacter baumannii.The strains'consanguinity were investigated by multi-genes cluster analysis.Methods:1 Sources of strains To collect the strains of Pseudomonas aeruginosa and Acinetobacter baumannii which isolated from patients due to acute exacerbation chronic obstructive pulmonary disease ,acute respiratory distress syndrome,severe pneumonia and neural disease in respiratory intensive care unit(RICU) form Jun.2008 to Dec.2009.2 Antimicrobial susceptibility The Kirby-Bauer method were performed to detect the susceptibility of cefoperazone/sulbactam, piperacillin/tazobactam,ceftazidime , imipenem , meropenem , amikacin , ciprofloxacin , minocycline ,polymyxin B 9 kinds of antimicrobial agents against Pseudomonas aeruginosa and Acinetobacter baumannii.The antibiotics susceptibility test was performed according to the American CLSI 2004 standard.The standard of multi-drug resistant strains were resistant to three or more classes antibiotics.33 strains of multi-drug resistant Pseudomonas aeruginosa and 16 strains of multi-drug resistant Acinetobacter baumannii were isolated from hospitalized patients in RICU.3 PCR templute preparation The template DNA of drug resistant strains were extracted by Proteinase K and they were stored at deep freeze refrigerator.4 Gene analysis Polymerase chain reaction(PCR) was used to detect various beta-lactamase coding genes including blaTEM,blaSHV,blaPER,blaDHA,blaIMP,blaVIM,blaOXA-23,blaOXA-24 and qacEâ–³1-sul1 in multi-drug resistant Pseudomonas aeruginosa and Acinetobacter baumannii.PCR production was observed and stored by gel imaging system.5 DNA sequencing qacEâ–³1-sul1 gene was analyzed by PCR and verified by DNA sequencing and sequence analysis6 Cluster analysis Polygene cluster analysis method was adoptived to analysis the outcome of drug resistance gene.Results:1 Results of susceptibility test The strains of multi-drug resistant Pseudomonas aeruginosa and Acinetobacter baumannii were all susceptible to polymyxin B.The drug resistant rate of multi-drug resistant Pseudomonas aeruginosa to amikacin and meropenem were 45.45%,but to other 6 antimicrobial agents were more than 50%. The drug resistant rate of multi-drug resistant Acinetobacter baumannii to cefoperazone/sulbactam and minocycline were 25.00% and 31.25% respectively, but to other 6 antimicrobial agents were more than 50%.2 Results of drug resistance gene PCR results showed the positive rates of genes blaTEM,blalMP,blaVIM,blaSHV,blaDHA,and qacEâ–³1-sul1 in multi-drug resistant Pseudomonas aeruginosa were 24.24%,18.19%,9.09%,6.06%,15.15% and 84.85% respectively,while genes blaOXA-23,blaOXA-24 and blaPER were all negative.The positive rates of genes blaOXA-23,blaTEM,blaDHA,blaOXA-24,blaPER and qacEâ–³1-sul1 in multi-drug resistant Acinetobacter baumannii were 31.25%,12.5%,12.5%,6.25%,6.25% and 100% respectively,while genes blaSHV,blalMP and blaVIM were all negative.3 Result of DNA sequencing The outcome of sequence analysis was conformity with the GenBank .4 Results of Cluster analysis In Pseudomonas aeruginosa,cluster analysis results showed 1,23,27 were the same clone strains and they all carried the genes of blaVIM and qacEâ–³1-sul1;12,25,26 were the same clone strains and they all carried the genes of blaTEM and qacEâ–³1-sul1; 2,5,6,8,16,19,21,24,29,30,32,33 were the same clone strains and they all carried the gene of qacEâ–³1-sul1.In Acinetobacter baumannii, cluster analysis results showed 3,8,11 were the same clone strains and they all carried the genes blaOXA-23 and qacEâ–³1-sul1;5,6,13,14,15 were the same clone strains and they all carried the gene of qacEâ–³1-sul1. Polygene cluster analysis results discovered that there was a clone transmitted phenomenon.Conclusion:Multi-drug resistant Pseudomonas aeruginosa and Acinetobacter baumannii from RICU have carried various beta-lactamases genes. Multi-drug resistant Pseudomonas aeruginosa mainly carried blaTEM and multi-drug resistant Acinetobacter baumannii mainly carried blaOXA-23.It was evident that class 1 integron was prevalent among multi-drug resistant Pseudomonas aeruginosa and Acinetobacter baumannii. We must pay attention to the clone transmitted phenomenon occurred in our RICU and high positive percentages of qacEâ–³1-sul1 in multi-drug resistant Pseudomonas aeruginosa and Acinetobacter baumannii. |
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