Cell-free supernatants of Bacillus inhibit the biofilms ofPseudomonas lundensiscultured with and withoutAcinetobacterjohnsonii

Abstract:

[objective]Pseudomonas as one of the dominant spoilage bacteria highly form biofilms in chilled meat products and processing environment when contaminating single or mixed with other species.This study aims to investigate the antibiofilm properties of the cell-free supernatants (CFSs) of three Bacillus species isolated from fermented food and rice seeds on Pseudomonas lundensis(PL) or and Acinetobacter johnsonii(AJ) as mono-or dual-species.[Methods] Biofilm biomass,extracellular polymeric substances (EPSs),and biofilm structure were measured by crystal violet staining,spectrophotometry,confocal laser scanning microscopy (CLSM),respectively,as well as transcription of biofilm-related genes determined by qPCR.[Results]The CFSs of Bacillus amyloliquefaciens ZG08,B.velezensis B5,and B.subtilis YB11 inhibited the biofilm formation of PL and AJ without affecting their growth.The treatment with 50% CFSs of ZG08 and B5 decreased the cell viability of two biofilms by 12.73%–21.04%,which was higher than that of YB11(0.15%–4.38%).The inhibition rates of 50% CFSs of the three strains were 59.75%–79.59% against the PL biofilm and 63.62%–78.57% against the biofilm of PL+AJ,in which the CFS of YB11 had weaker activity.The content of exopolysaccharides and exoprotein in the two biofilms treated with these CFSs were reduced by 53.77%–73.30% and 54.84%–62.38%,respectively.The treatment with the three CFSs also reduced the adhesive cells,loosened biofilm structures,and thinned their thickness by 57.63%–74.49% and 60.43%–64.64%,respectively.Moreover,the CFSs of ZG08 and B5 effectively eradicated by 41.77%–69.79% against the mature biofilms of PL and PL+AJ,compared to weak activity of YB11.In addition,the antibiofilm activities of the three CFSs were stable under four enzyme digestion and heating conditions.Compared with the control,the CFSs of ZG08 and B5 significantly down-regulated the expression of six biofilm-related genes,lapA,alg44,pelG,luxR,wspR,and rpoS.[Conclusion] The CFSs of ZG08 and B5 have strong antibiofilm activities against PL and AJ as mono-or dual-species.