Abstract:

Objective To discuss the effect of quorum sensing-related gene expression on biofilm formation and drug resistance in clinically multidrug-resistant Pseudomonas aeruginosa， and to clarify the mechanism of enhacing drug resistance. Methods A total of 77 strains of Pseudomonas aeruginosa were collected. Based on drug resistance， the strains were divided into multidrug-resistant group and sensitive group. The optimal biofilm formation conditions were determined using the microtiter plate method； biofilm formations of the stains in both groups was observed under an optical microscope； crystal violet staining was used to semiquantitatively detect biofilm formation ability of P. aeruginosa in both groups； microbroth dilution method was used to determine the minimal inhibitory concentration （MIC） values of the quorum sensing inhibitor （C-30） against Pseudomonas aeruginosa in both groups； RNA was extracted from two groups using a commercial kit， while RNA from planktonic state and biofilm state of multidrug-resistant strains was extracted using modified TRIzol method； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the mRNA expression levels of quorum sensing-related genes （lasR/I， RhlR/I， PqsR/A） of the stains in multidrug-resistant group and sensitive group， as well as before and after adding the quorum sensing inhibitor C-30. Results Among 77 strains of Pseudomonas aeruginosa， 56 were multidrug-resistant （multidrug-resistant group） and 21 were fully sensitive（sensitive group）. Optimal biofilm formation occurred at a bacterial concentration of 1.5×10⁸ CFU·mL^-1 with 48 h incubation. The biofilm positivity rate was 91%， with strongly positive， moderately positive， weakly positive， and negative biofilms accounting for 16%， 34%， 41%， and 9%， respectively. The biofilm positivity rate in multidrug-resistant strains was 96%， and biofilm formation ability in multidrug-resistant group was higher than that in sensitive group （P<0.05）. When the concentration of C-30 was 8 mg·L^-1 the biofilm formation in most Pseudomonas aeruginosa was inhibited， with enhanced suppression at higher concentrations. The absorbtion （A） value of both planktonic-state and biofilm-state RNA ranged from 1.8 to 2.0. The RT-qPCR results showed that compared with planktonic state， the expression levels of lasR/I， RhlR/I， and PqsR/A mRNA of the stains in biofilm state were significantly increased （P<0.01）. Compared with non-inhibitor group， the expression levels of lasR/I， RhlR/I， and PqsR/A mRNA in biofilm state of inhibitor-treated group were significantly decreased （P<0.01）. Conclusion High expression of quorum sensing-related genes in multidrug-resistant P. aeruginosa promotes biofilm formation， thereby enhancing drug resistance.

Key words: Pseudmonas aeruginosa, Biofilms, Quorum sensing system, Real-time fluorescence quantitative PCR, Drug resistance