Study On Quorum-sensing System And Pathogenicity Of Pseudomonas Aeruginosa During SOS Response

Background:The acquired infection(HAI)in hospital has become one of the most severe threats to human health.During the separation of pathogenic bacteria,Pseudomonas aeruginosa is one of the most important one.It belongs to gram-negative and conditioned pathogen.Patients with low resistance or invasive operation can significantly increase the risk of infection.Pseudomonas aeruginosa is also one of the most important pathogenic bacteria in the respiratory department and intensive care unit(ICU).Because of its easy transplanting,mutation and complex resistance mechanism and so on,as well as widely use of broad-spectrum antibacterial drugs,immunosuppressive agents and tumor chemotherapy drugs,its antimicrobial resistance and multidrug resistance are significantly enhanced and become a difficult problem to solve in clinical antibacterial therapy.At present,the resistance of Pseudomonas aeruginosa to commonly used antimicrobial agents is gradually increasing.Therefore,it is of great importance to study the pathogenicity and drug resistance mechanism of Pseudomonas aeruginosa,use antibiotics reasonably and develop new antimicrobial agents for effectively treating Pseudomonas aeruginosa infection,improving the prognosis of patients and reducing the mortality rate.At present,the most important means of anti-Pseudomonas aeruginosa infection is still the use of antimicrobial agents.The antibacterial agents for Pseudomonas aeruginosa infection in the clinical treatment mainly include ?-Lactam,aminoglycoside,quinolone,polymyxin and so on.Ciprofloxacin is one of the greatest quinolones for anti-pseudomonas.However,with its wide application in clinic,Pseudomonas aeruginosa's resistance to ciprofloxacin has increased year by year,which has brought great difficulies to clinical treatment.Therefore,it is urgent to find new antibacterial drugs.Linoleic acid is the essential fatty acid in human body,as a kind of active substance and drug carrier,fatty acid has been used in the pharmaceutical industry.The role of ?-3 fatty acid in the treatment of diabetes,cancer,anti-inflammation and immunomodulatory activities encourage people to study its effects on microorganisms.SOS response is also called emergency response,refers to multiple gene-induced DNA repair to maintain its interity when DNA is severely damaged.SOS response is mediated by one of its activation proteins RecA and repressors LexA.The damaged DNA binds to RecA,making the actived LexA auto-cleavage,which is followed by the synthesis of SOS repair enzymes.SOS response is related to the production of biofilm and pyocin and so on.Quorum sensing system is refers to the bacteria monitoring bacterial density in the surrounding environment through the synthesis of a specific signal molecule,after reaching the threshold,the signal molecules start to regulate the expression of related genes in bacteria to control its behavior such as producing toxins or spores,forming biofilm to adapt to environmental changes.Objective:To conform the antibacterial effect of linoleic acid by bacteriostatic test and SOS response and QS system induced by low concentration of ciprofloxacin and linoleic acid with the method of qRT-PCR.To elucidate the changes of QS system and pathogenic ability of Pseudomonas aeruginosa during SOS response,in order to realize the harm caused by the insufficient drug dose,understand the pathogenesis mechanism of Pseudomonas aeruginosa,and assist research and development of antibacterial drugs.Methods:1.Pseudomonas aeruginosa was isolated and cultured from clinical patients,ATCC 27853 was used as quality-control strain to identify our strain by dual PCR.2.The minimum inhibitory concentration(MIC)of ciprofloxain and linoleic acid was determined by broth dilution method and macroscopic observation.3.The mutual effects of sub-MIC ciprofloxacin and linoleic acid on SOS response and QS system at different time points and the connection between SOS response and QS system were analyzed by qRT-PCR.4.The production yield of biofilm,pyocin and extracellular DNA were analyzed during SOS response caused by sub-MIC ciprofloxacin and linoleic acid.Results:1.The MIC of ciprofloxacin is 2 ?g/ml and The MIC of linoleic acid is 1.5 mg/ml checked by broth dilution method and macroscopic observation.2.The results of qRT-PCR showed that RecA was up-regulated by both sub-MIC of ciprofloxacin and linoleic acid at four o'clock and the expression of RecA with sub-MIC of ciprofloxacin was higher than the one with sub-MIC of linoleic acid while RecA was down-regulated by both sub-MIC of ciprofloxacin and linoleic acid at twenty four o'clock.3.The results of qRT-PCR showed that lasI,lasR,rhlI and rhlR was all up-regulated by both sub-MIC of ciprofloxacin and linoleic acid at four o'clock and the expression of lasI,lasR,rhlI and rhlR with sub-MIC of ciprofloxacin was higher than the one with sub-MIC of linoleic acid while lasI,lasR,rhlI and rhlR was down-regulated by both sub-MIC of ciprofloxacin and linoleic acid at twenty four o'clocks.4.The results showed that the production yield of biofilm,pyocin and extracellular DNA with sub-MIC of ciprofloxacin and linoleic acid were increased,and the expression with sub-MIC of ciprofloxacin was higher than another one.Conclusion:1.Below the MIC dose of ciprofloxacin and linoleic acid in a short of time will induce SOS response of Pseudomonas aeruginosa,as time goes on,the bacteria gradually adapt to the adverse environment and start a series of protection mechanisms,so the SOS response was down-regulated.2.The results of qRT-PCR showed that the expression of SOS is consistent with the QS system below MIC dose of ciprofloxacin and linoleic acid.The QS system is enhanced when SOS response is actived while the QS system is declined when SOS response is suppressed.In addition,compared with ciprofloxacin,the sub-MIC of linoleic acid induce lower SOS response and QS system.3.The increased production of biofilm,pyocin and extracellular DNA with sub-MIC of ciprofloxacin and linoleic acid means the pathogenicity will enhance simultaneously and the linoleic acid cause lower pathogenicity than ciprofloxacin.So,the insufficient dose of drug may lead to the enhanced pathogenicity of bacteria,which is not beneficial for bacterial infection treatment.4.Unsaturated fatty acid--linoleic acid has bacteriostatic effect and is expected to be developed as a new antibacterial agent or assistant antibacterial agent.