Quorum Quenching Effect Of Lactobacillus Crustorum ZHG 2-1 On Pseudomonas Aeruginosa

Aquatic products are one of the important food sources for human survival.With the rapid development of the aquaculture industry,aquaculture diseases caused by microorganisms are becoming increasingly prominent.Pseudomonas aeruginosa is an important food-borne pathogen and a spoilage bacterium.One of the most important factors leading to such diseases is antibiotic resistance to antibiotics.In addition to health consequences,food spoilage caused by foodborne pathogens can cause considerable economic losses to consumers and producers.The pathogenic effect of P.aeruginosa is closely related to quorum sensing(QS).Lactic acid bacteria(LAB)are a safe and effective biological preservative.Studies on lactic acid bacteria as quorum quenching(QQ)bacteria have been rare at home and abroad.In this paper,LAB with N-acylated homoserine lactones(AHLs)degradation activity were screened from the natural environment,and it was identified with 16S r DNA sequencing identification were performed.Gas chromatography-mass spectrometry(GC-MS)was used to analyze the degradation effect of active LAB on AHLs signal molecules common to several Gram-negative bacteria.In addition,the effects of ZHG 2-1 crude extract(CE)treatment on QS-regulated virulence factors such as extracellular polysaccharides,pyocyanin,chitinase,and bacterial movement were studied;The effects of ZHS 2-1 CE treatment on QS-related genes las I/R,rhl I/R expression were analyzed by quantitative real-time polymerase chain reaction(q RT-PCR),and the effects of ZHG 2-1 CE on the virulence of macrophages in mice infected with P.aeruginosa were analyzed by flow cytometry.Finally,96-well plate method combined with scanning electron microscopy(SEM)was used to analyze the inhibitory effect of ZHG 2-1 CE,azithromycin,and the combination of the two on the formation of P.aeruginosa biofilms and the removal of pre-formed biofilm.The main results are as follows:1.Using 96-well plate method and agar diffusion method to screen 156 strains of LAB isolated from traditional fermented foods and plant roots and other ecological environments.The results showed that strain ZHG 2-1 derived from Fuxin Zhangwu fermented cucumber had the strongest degradation activity close to100%.The strain ZHG 2-1 was identified as Lactobacillus crustorum by physiological and biochemical reactions and 16S r DNA analysis.GC-MS results showed that ZHG 2-1 CE can significantly degrade C₄-HSL and 3-oxo-C₁₂-HSL in P.aeruginosa.The minimum inhibitory concentration(MIC)of ZHG 2-1 CE on P.aeruginosa was 4.0 mg/m L.The three sub-MIC of ZHG 2-1 CE do not affect the growth of P.aeruginosa.And it has a degradation effect on the purple bacteriocin produced by the Chromobacterium violaceum CV026.2.The effects of strain ZHG 2-1 CE on the QS virulence factors and motility of P.aeruginosa were evaluated under three sub-MICs.The results showed that the ZHG 2-1 CE at 1.0,2.0 and 3.0 mg/m L were effective against P.aeruginosa protease,chitinase,pyocyanin,rhamnolipid,alginate and extracellular polysaccharides.The inhibition rates were 28.37%-70.26%,29.63%-63.34%,42.16%-71.84%,25.36%-59.47%,24.25%-59.47,39.83%-79.54%.q RT-PCR analysis showed that the QS genes las I/R and rhl I/R expression were down-regulated and down-regulated by nearly 50%.ZHG 2-1 has a strong inhibitory effect on flagella-mediated swamming movement and pili-mediated swimming movement.All of the above effects have shown dose dependence.In addition,the results of flow cytometry analysis showed that 3.0 mg/m L strain ZHG 2-1 CE could reduce the virulence of P.aeruginosa on mouse macrophage Ana-1 cells.3.The 96-well plate method was used to determine the inhibitory effect of ZHG2-1 CE,azithromycin,and their combination on the formation of P.aeruginosa biofilms and the removal of pre-formed biofilms.The results showed that the ZHG2-1 CE could significantly increase the sensitivity of azithromycin to biofilms,and the inhibition rate at three sub-MICs was 58.4-90.3%.SEM images showed that the combination of ZHG 2-1 CE and azithromycin can inhibit the formation of biofilms and reduce the thickness of pre-formed biofilms.The cell viability of the treated biofilms further confirms the above results.Besides,it had no effect on the survival of planktonic cells when used alone or in combination.