Abstract
The quorum sensing (QS) circuit plays a role in the precise regulation of genes controlling virulence factors and biofilm formation in Pseudomonas aeruginosa. QS-controlled biofilm formation by Pseudomonas aeruginosa in clinical settings has remained controversial due to emerging drug resistance; therefore, screening diverse compounds for anti-biofilm or anti-QS activities is important. This study demonstrates the ability of sub-minimum inhibitory concentrations (sub-MICs) of baicalin, an active natural compound extracted from the traditional Chinese medicinal Scutellaria baicalensis, to inhibit the formation of Pseudomonas aeruginosa biofilms and enhance the bactericidal effects of various conventional antibiotics in vitro. In addition, baicalin exerted dose-dependent inhibitory effects on virulence phenotypes (LasA protease, LasB elastase, pyocyanin, rhamnolipid, motilities and exotoxin A) regulated by QS in Pseudomonas aeruginosa. Moreover, the expression levels of QS-regulatory genes, including lasI, lasR, rhlI, rhlR, pqsR and pqsA, were repressed after sub-MIC baicalin treatment, resulting in significant decreases in the QS signaling molecules 3-oxo-C12-HSL and C4-HSL, confirming the ability of baicalin-mediated QS inhibition to alter gene and protein expression. In vivo experiments indicated that baicalin treatment reduces Pseudomonas aeruginosa pathogenicity in Caenorhabditis elegans. Greater worm survival in the baicalin-treated group manifested as an increase in the LT50 from 24 to 96 h. In a mouse peritoneal implant infection model, baicalin treatment enhanced the clearance of Pseudomonas aeruginosa from the implants of mice infected with Pseudomonas aeruginosa compared with the control group. Moreover, the combination of baicalin and antibiotics significantly reduced the numbers of colony-forming units in the implants to a significantly greater degree than antibiotic treatment alone. Pathological and histological analyses revealed mitigation of the inflammatory response and reduced cell infiltration in the peritoneal tissue surrounding the implants after baicalin treatment. Measurement of the cytokine levels in the peritoneal lavage fluid of mice in the baicalin treatment group revealed a decrease in IL-4, an increase in interferon γ (IFN-γ), and a reversed IFN-γ/IL-4 ratio compared with the control group, indicating that baicalin treatment activated the Th1-induced immune response to expedite bacterial load clearance. Based on these results, baicalin might be a potent QS inhibitor and anti-biofilm agent for combating Pseudomonas aeruginosa biofilm-related infections.
Highlights
Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, is one of the major causes of nosocomial infections
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of baicalin against planktonic P. aeruginosa PAO1 are shown in Table 1; baicalin exerted no direct bactericidal effects and no completely bacteriostatic effects on planktonic P. aeruginosa PAO1 cells with both MBC and MIC >1024 μg/mL, at least at the concentration of 1024 μg/mL that we tested
Antibiotic MICs against the quality control strain P. aeruginosa ATCC27853 were obtained in parallel, and all results were within the range recommended by the CLSI
Summary
Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, is one of the major causes of nosocomial infections. P. aeruginosa forms biofilms on damaged tissue or medical implants in addition to living as free planktonic cells within the host [3]. Biofilms comprise more than 80% of all microbial infections associated with catheters, foreign-body implants, urinary tract infections, dental plaque, and gingivitis and commonly produce chronic and persistent infections [4,5,6]. Compared with their free planktonic cell counterparts, bacterial communities embedded in biofilms exhibit distinctive growth, gene expression and cell-cell communication phenotypes [7].
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