| Summary: | 碩士 === 國立臺灣大學 === 農業化學研究所 === 88 === Biofilms, consisting of microbial cells and their extracellular polymers, are infamous for the recalcitrance to antimicrobial agents. When biofilms are developed, they are at least 500 times more difficult to kill than their suspended counterparts. This fact is a menace in either industry or medical therapy. The explanation of their stubborn resistance can 'be divided into three categories. The first one is the transport limitation of antimicrobial agents. The antimicrobial agents would be neutralized and/or adsorbed by the extracellular polymers. The second explanation is physiological adaptation. The third hypothesis is related to cell-to-cell communication.
Many microbial cells are able to communicate with each other within homogeneous or heterogeneous species and this involves the quorum sensing phenomenon. In many various gram-negative bacteria, quorum sensing can control specific gene expression at a threshold density and regulate distinct physiological process such as bioluminescence of Vibrio fisheri and virulence of Pseudomonas. aeruginosa. Scientists have known that the cell-to-cell communication is carried out via N-acyl-L- homoserine lactones (HSL). The signal molecules are secreted from cell itself and also called autoinducers.
P. aeruginosa commonly distributes in water and soil and is an opportunistic pathgen of humans, causing infections of cystic fibrisis and burns. It also infects plants and insects. The production of virulence factors In P. aeruginosa was controlled by las and rhl quorum sensing systems that contains a transcriptional activator (LasR/ RhlR) and an autoinducer synthase (LasI/RhlI). At low cell density, autoinducers are synthesized at basal quantity and accumulated to medium. LasI directs the synthesis of 3-oxo-C12-HSL. When cells grows to threshold density, 3-oxo-C12-HSL will bind to LasR to regulate the production of several virulence factors such as LasA protease, elastase, exotoxin A and exoenzyme S. RhlI directs the synthesis of C4-HSL binding to RhlR to regulate the biosynthesis of rhamnolipid, haemolysin, RpoS and elastase.
In this study, wildtype P. aeruginosa and lasI and rhlI gene null mutant strains and double mutant strain were used to cultivate biofilms. The formation and resistance between these species were investigated. The results show that the viable cell density for the wildtype of P. aeruginosa biofilms reached (4.96±0.47)×109/cm2 after 72 hours of cultivation, while those for mutants were (8.78±1.34)×108/cm2 (rhlI mutant), (3.36±0.50)×108/cm2 (lasI mutant) and (5.37±1.14)×108/cm2 (lasI, rhlI mutant), respectively. The total polysaccharides content for the wildtype biofilms was (55.39±3.22) μg/cm2, and those for mutants were (5.55±0.11) μg/cm2 (rhlI mutant), (2.32±0.42) μg/cm2 (lasI mutant) and (4.95±1.39) μg/cm2 (lasI, rhlI mutant), respectively. Under fluorescence microscope, the wildtype biofilms displayed about 150μm thickness, while mutants ranged from 20 to 50 μm. When biofilms were treated with 100μg/ml kanamycin for two hours, the surviving fraction of wildtype was above 0.7 and that in mutants was 0.46±0.03 (rhlI mutant), 0.088±0.008 (lasI mutant) and 0.035±0.005 (lasI, rhlI mutant), respectively. These results indicate that the quorum sensing regulation mechanism might play an important role in biofilm development and the resistance to antibiotics.
|
|---|
