Study On Resistance Characters And Genes Associated With Biofilm Formation In Imipenem-resistant Pseudomonas Aeruginosa

Objective:To investigate the antibiotic resistance of imipenem-resistant Pseudomonas aeruginosa of clinical isolates in Tianjin.To evalution the ability of biofilm formation of Pseudomonas aeruginosa of clinical isolates,a laboratory method was established.To probe the correlation between the ability of biofilm formation of Pseudomonas aeruginosa isolates and expression of gene involved in the synthesis of polysaccharides,and to study the role of erythromycin in biofilm formation of Pseudomonas aeruginosa.It will be helpful for clinical doctors to reasonably use antibiotics.Methods:Sixty isolates of imipenem-resistant Pseudomonas aeruginosa were collected from three hospitals in Tianjin from January to December in 2006, antimicrobial susceptibility test was done by microdilution test.Sixty isolates of Pseudomonas aeruginosa were tested for their biofilm formation with a modifed microtiter test,four patterns were identified in the 60 isolates of Pseudomonas aeruginosa.We selected two strains compared with control strains PAO1.One has higher ability of biofilms formation,the other has lower ability.Congo red binding assay was used for analysis of polysaccharides product.Real-time PCR was used to quantify mRNA to analyze their mRNA level during planktonic growth and biofilm formation on 6-well flat-bottomed plastic tissue culture plate.Results:Most of Pseudomonas aernginosa of clinical isolates come from sputum. MIC Antibiotic susceptibility tests revealed high percentage of resistance to most antibiotics in the 60 clinical isolates of P.aeruginosa included in this study,Amikacin was the most sensitive antibiotics,which resistant rate was 30%,the resistant rate of Aztreonam was 50%,while Meropenem,Cefepime,Ceftriaxone,Ceftazidime, Cefotaxime,Cefoperazone/Sulbactam,Gentamicin,piperacillin were higher than 60%. Four patterns were identified in the 60 isolates of Pseudomonas aeruginosa by their different ability of biofilms formation.Most of the Pseudomonas aeruginosa of clinical isolates tested formed strong biofilm.The relative expression levels of pslA, algD and pelA in PAO1 were the highest at 24h and then the relative expression levels of pslA and pelA depressed gradually.Those were the lowest at sixth day at near planktonic levels.But the relative expression levels of algD had no significant variation in the procedure of biofilms formation.We selected 10 strains at random for further experiment.The results indicated that the relative expression levels of pslA of sessile growth bacteria(24h) were higher than that of planktonic(6h) in 10 strains tested.2mg/l erythromycin acted with Pseudomonas aeruginosa for 24h.Then the numbers of colonies were counted and relative expressions of pslA mRNA were evaluated in biofilms.These results indicated that the numbers of bacteria with erythromycin in biofilm not so many as that of without erythromycin.There were no statistical differences between two groups in expressions of pslA mRNA.Conclusions:MIC Antibiotic susceptibility tests revealed high percentage of resistance to most antibiotics in the 60 clinical isolates of IRPA included in this study. Most of Pseudomonas aeruginosa of clinical isolates tested formed strong biofilm. The relative expression levels of pslA,algD and pelA in PAO1 were the highest at 24h.Analysis of the relative expression levels indicated that the level of transcripts of pslA was significantly higher than that of algD and pelA in the procedure of biofilms formation.Our datas show that transcripts of the pslA involve in biofilm formation of Pseudomonas aeruginosa.There is an important value for biofilm formation of IRPA. Subinhibitory erythromycin can inhibit the formation of biofilm by Pseudomonas aeruginosa,but it has no effect on the expression of pslA.