芽孢杆菌无细胞上清液对致腐隆德假单胞菌单/混合生物被膜的抑制活性

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

摘要:

【目的】假单胞菌(Pseudomonas)是冷鲜肉制品及加工环境中的优势腐败菌之一，在单一或混合培养下均具有较强的生物被膜形成能力。本研究评价食品来源的3株芽孢杆菌无细胞上清液(cell-free supernatants,CFSs)对隆德假单胞菌(Pseudomonas lundensis,PL)单培养及与约氏不动杆菌(Acinetobacter johnsonii,AJ)混合培养下的抗生物被膜作用。【方法】采用结晶紫染色、分光光度法、激光共聚焦显微镜(confocal laser scanning microscopy,CLSM)观察、qPCR等方法分别测定生物被膜细胞活力、生物量、胞外聚合物、被膜结构及其相关基因转录变化。【结果】解淀粉芽孢杆菌ZG08、贝莱斯芽孢杆菌B5、枯草芽孢杆菌YB11的CFSs能有效抑制2种腐败菌的生物被膜形成，并且不影响其生长。经50% CFSs处理后，ZG08和B5处理组的单种或混合生物被膜的细胞活性下降12.73%−21.04%，显著高于YB11处理组(0.15%−4.38%)。3种50% CFSs处理后，隆德假单胞菌单种和混合生物被膜的抑制率分别为59.75%−79.59%和63.62%−78.57%，其中YB11的CFS抑制效果较弱。同时，CFSs处理显著降低胞外基质分泌，单/混合生物被膜的胞外多糖和胞外蛋白含量分别下降53.77%−73.30%和54.84%−62.38%，处理后的被膜变得疏松，分散贴壁黏附，被膜厚度减少57.63%−74.49%和60.43%−64.64%。此外，与YB11无清除活性相比，ZG08和B5的CFS对PL和PL+AJ成熟生物被膜的清除率为41.77%−69.79%。经稳定性评价，3种CFSs在4种酶消化和加热处理后仍保持稳定的拮抗活性。与对照相比，ZG08和B5的CFSs显著下调单/混合假单胞菌的6个生物被膜相关基因lapA、alg44、pelG、luxR、wspR和rpoS。【结论】ZG08和B5的CFS对单种或混合培养下隆德假单胞菌和约氏不动杆菌均有较好的抗生物被膜活性。

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.