Intervention Effect And Mechanism Of Azithromycin On Pseudomonas Aeruginosa Biofilm And Virulence Factors

Pseudomonas aeruginosa (P.a) is a common opportunistic pathogen, is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients[1], is an important pathogen infection in bronchiectasis, chronic obstructive pulmonary disease (COPD), is an common pathogen in hospital-acquired infections, such as burn infections, ICU infections. Biofilm (BF) formation, the release of a variety of virulence factors and multiple drug resistance are the main reasons for P.a refractory infections, the resistance mechanism is complex, one of the main reasons is BF formation. The formation of BF and production of virulence factors is under the control of the quorum sensing system (QS). Therefore, inhibition of QS system, thus inhibiting the formation of BF and the release of virulence factors may be the direction subject in the BF infection treatment. This study established BF models in vitro to detect the effect of Azithromycin (AZM) on the formation of BF and bacterial virulence factors, and further tested the QS signal molecule production to reveal the possible mechanism of Azithromycin.Objective: To evaluate the effect of Azithromycin on pseudomonas aeruginosa PAO1 biofilm formation and virulence factors production, and the possible mechanism.Methods: 1) Biofilm formation in vitro: Detect the minimum inhibitory concentration (MIC) of Azithromycin against PAO1 by 2-fold dilution method. Crystal violet staining assay was used for initial adhesion assays. The PAO1 biofilm was established in vitro and observed by scanning electron microscope (SEM). Viable bacterial counts were determined by serial dilution. 2) Virulence factors detect: LasB elastolytic activity was determined by using Elastin-Congo Red. Protease activity was determined by using Azo-casein. Chloroform extraction method was used for pyoverdine assay. The orcinol assay was used to directly assess the amount of rhamnolipids. 3) Determination of the QS signal molecules: Extract the AHLs by ethyl acetate, and determine the production of AHLs by HPLC-MS.Results 1) Scanning electron microscope biofilm and viable bacterial counts of PAO1 adhered to the surface of catheter in PAO1 Azithromycin group were less than the PAO1 control group incubated for 3d and 7d (P<0.05), and the initial adhesion was weaker (P<0.05). 2) The virulence factors production were obviously decreased (P<0.01), LasB elastolytic activity and pyoverdine were even reduced to the same as with the PA-JP3 group (P>0.05), but the protease activity and the rhamnolipids concentration were higher than the PA-JP3 group (P<0.05). 3) The productions of AHLs in AZM group were decreased when compared with the PAO1 control group both in C4-HSL and 3-oxo-C12-HSL, and the QS-deficient strain lasI/rhlI did not produce AHLs.Conclusions Azithromycin can inhibit PAO1 bioflim formation in vitro and virulence factors production by inhibit the QS system of Pseudomonas aeruginosa.