Analysis Of High-level Antibiotic Resistance And The Expression Of TolA,ndvB In Pseudomonas Aeruginosa Biofilms

Objectives:(1) Assessing the capability of biofilm formation by clinical strains of Pseudomonas aeruginosa in vitro, and the change of antibiotic resistance in biofilms.(2) Analyzing the distribution and expression of antibiotic resistance genes, tolA and ndvB, in biofilm-grown and planktonically grown Pseudomonas aeruginosa by quantitative real-time-PCR, to guide a new research direction for clinical therapeutic regimens.Methods:(1) Non-duplicated clinical isolates of Pseudomonas aeruginosa in Xiangya hospital were collected, which were identified by Vitek-two automatic microorganism analytical system. Biofilm formations of60Pseudomonas aeruginosa strains were estimated by the microtiter-plate test. This technique involves fixing the bacterial film with methanol, staining with crystal violet, screening by optical microscope. Polymerase Chain Reaction (PCR) was performed in all isolates for detecting tolA and ndvB genes. The amplified products were sequenced and the sequences were analysed by BLAST.(2) MIC of tobramycin, gentamicin and amikacin against biofilm-grown and planktonically grown Pseudomonas aeruginosa were tested by broth microdilution method, and the MIC fold changes were compared.(3) The expression of tolA and ndvB genes in biofilm-grown and planktonically grown Pseudomonas aeruginosa by quantitative real-time-PCR. Statistical differences among the results obtained by the biofilm-grown and planktonically grown Pseudomonas aeruginosa were examined by the Paird-samples t-test. P values of<0.05were considered significant.Results:(1)58strains of Pseudomonas aeruginosa were succeed in forming biofilms (accounted for97%). All strains showed tolA and ndvB genes positive. All the positve genes were identified by BLAST, and the sequences homology were all above99.0%compared with the known genes in GenBank.(2) Biofilms became1～1024,0～>1024and1～> 256times more resistant to the effects of tobramycin, gentamicin and amikacin than their planktonic counterparts. However, MIC of tobramycin, gentamicin and amikacin against12,20and15strains could not be measured, respectively, because of the serious antibiotic resistance.(3) The biofilm cell showed significantly high expression of ndvB than the planktonic cell (P<0.05). On the contrary, statistical analysis showed no significant difference between the results of tolA expression obtained by the biofilm cell and the planktonic cell (P=0.16).Conclusions:(1) Almost all Pseudomonas aeruginosa can form biofilms, when a surface and an appropriate environment are provided. The tolA and ndvB genes are part of the complete genome sequences of Pseudomonas aeruginosa.(2) Biofilms become several times to several thousand times more resistant to the effects of aminoglycoside than their planktonic counterparts.(3) The highly activated of biofilm-specific gene, ndvB, is common in biofilms, which plays an important role in biofilm bacteria resistance. It may become an anti-biofilm target for the development of anti-infectives. The tolA gene shows no significant difference between the biofilm cell and planktonic cell. The resistant mechanism may be activated in only sevral strains. But it still needs large of samples to confirm the conclusion.