Effects Of Moringa Oleifera Leaves Extracts On Biofilms Of Vibrio Parahaemolyticus Isolated From Seafood

Biofilm consists of a bacterial colony embedded in a matrix of extracellular polymeric substances(EPS)which protects the microbes from adverse environmental conditions and results in infection.Vibrio parahaemolyticus,a common halophilic gram-negative bacterium,mainly distributed in sea water and fish,shrimp,crab,shellfish and other marine products.It can cause a series of diseases such as acute gastroenteritis,wound infection and septicaemia.When V.parahaemolyticus forms a biofilm,this mechanism can enhance its resistance to adverse environment,thus reducing the efficacy of antibiotics and increasing the risk of human infection.Moringa oleifera has obvious antibacterial activity and is listed as a new food resource by the Ministry of Health.It has been attracted much attention in the field of food research.Therefore,it is urgently needed to develop a safe,efficient,and natural anti-biofilm material and Moringa oleifera is a good choice.In this study,V.parahaemolyticus was isolated and identified from seafoods in Guangzhou,and the antibiotic resistance of V.parahaemolyticus was evaluated.We investigated the suitable extraction conditions of the antibacterial substances from Moringa oleifera leaves,and then analyzed the antibacterial spectrum.The antibacterial activity and the MICs for V.parahaemolyticus isolates were determined,and the inhibition and eradication effects of the extract on the biofilm of V.parahaemolyticus were investigated.The biofilm formation and metabolic activity of the biofilm were assessed using crystal violet assay(CV)and XTT reduction assay respectively.The treated and control biofilms were observed by fluorescence microscopy and confocal laser scanning microscope(CLSM).The main contents and results of this study are as following:Isolation and identification of V.parahaemolyticus from seafoods in Guangzhou.Forty-two samples of seafood were collected,and 8 isolated strains were identified by themethod of national standard GB 4789.7-2013 and 16 S rDNA sequencing.The isolated strains were mainly from fish and shrimp,only one from clams.The sensitivities of the isolated strains and ATCC 17802 standard strain to 20 antibiotics were evaluated using Kirby-Bauer method.All isolated strains were found to be susceptible to 8 antibiotics such as polymyxin B,ceftazidime,gentamicin,doxycycline,chloramphenicol,erythromycin,ciprofloxacin,and nelidixic acid;resistant to penicillin and vancomycin,and showed different degrees of resistance to other antibiotics.Moreover,all strains exhibited more significant multi-drug resistance compared with ATCC 17802,and their multi-drug resistance was positively correlated with its biofilm formation ability.Extraction of the antibacterial substances from Moringa oleifera leaves and investigation of the antibacterial effects.It was found that the method of water extraction was better to inhibit Vibrio parahaemolyticus under the conditions of ratio of material to liquid at 1:50,extraction time in 50 min,ultrasonic power at 200 W and extraction temperature at 50?,and the inhibition zone reached 24 mm,MIC was 3.13mg/mL.The contents of polyphenols and flavonoids in in the extracts were determined.The inhibitory abilities of the extracts to common pathogenic bacteria were significant,and there was no antibacteral effect on lactic acid bacteria and fungi by analyzing the antimicrobial spectrum.The results showed that the MICs of the extract against 8 isolated strains were from 6.25mg/mL to 12.5mg/mL.The inhibition and eradication abilities of the extract on V.parahaemolyticus biofilms.The antibiofilm effects were determined by CV method.The results showed that the inhibitory effects of the extract on biofilm formation and metabolic activity were significant,and eradication effect on the mature biofilm was also obvious.The inhibitory and eradication effects were different among the isolated strains.One strain was selected and studied in detail.The treated biofilms were observed by fluorescence microscope.The effect of the extract on the biofilm was further studied via observing the changes of biofilms structure by CLSM.The red dead cells in the biofilm treated with the extract were more than those in the control biofilm,and the green living cells were less and more dispersed in the biofilm.Different concentrations of the extract had different effects.The results of CLSM alsoshowed that there were more dead cells and less exopolysaccharides in treated biofilms.The two results are consistent with the above experimental results.It is reveal that inhibiting,killing bacteria and destroying the EPS are part of the mechanism of inhibition and eradication of Vibrio parahaemolyticus biofilms.In summary,the extract of Moringa oleifera leaf had antibacterial effect.It also possessed the activities of inhibiting and eradicating V.parahaemolyticus biofilms.