| Biofilm is a form of survival of most of the bacteria in the nature,which often consisted of multiple different species of bacteria.Once biofilm is formed,it will pose a serious threat to the quality and safety of food.Pseudomonas fluorescens and Shewanella baltica,with biofilm forming ability,are dominant spoilage bacteria in cultured fishes during the chilled storage.Previous studies showed that quorum sensing(QS)system has involved in the formation of biofilm.P.fluorescens was found to secrete AHLs,but S.baltica failed.In the present study,the biofilm formation,spoilage phenotypes and AHLs activity of five P.fluorescens isolates were comparatively characterized.A PF strain with strong ability of biofilm formation and high AHLs activity was screened out.Effect of culture temperature at 30℃,15℃ and 4 ℃ on the mono-and dual-species biofilm formation and adhesion properties of Pseudomonas fluorescens PF07 and Shewanella baltica SB 11 was evaluated.The interspecies interaction on dual-species biofilm formation were investigated.A mutant knocked out luxl gene was obtained,biofilm and adhesion between wild strain andmutant were compared,as well as the effect of AHLs on interspecies interaction.The main results are as follows:In the present study,the biofilm formation and AHLs activity of five P.fluorescens isolates were comparatively characterized.The biofilm forming ability and adhesion of the isolates were measured by using crystal violet staining,phenol sulfuric acid assay,bead vortexing method and fluorescence microscopy.Swimming motility,protease activity,siderophore and AHLs activity were performed.The results showed that five P.fluorescens isolates grew fast in LB media at 28 ℃,and formed thick pellicle at the air liquid(A-L)interface for 24 h.All of them produced rapidly biofilm on microtiter plates,and PF01、PF06、PF07 and PF10 reach the maxmium amount of biomass at 12 h,however,PFuk4 arrived at 18 h.The isolates produced continuously exopolysaccharides with the extension of time and arrived to the maxmium amount at 18-24 h.Five isolates adhered to stainless steel quickly,possessed high swimming motility and protease activity,as well as production of siderophore.Only PF07 and PFuk4 were detected to short chain AHLs activity,which could be associated with higher ability of biofilm formation,adhesion,swimming and protease.P.fluorescens PF07 was screened for further study,which had high ability of biofilm formation and high AHLs activity.P.fluorescens PF07 and S.baltica SB 11 from Pseudosciaena crocea were co-cultured,and the effect of temperature on mono-and dual-biofilm formation was evaluated.Biofilm formation and adhesion of two spoilage bacteria were performed by using crystal violet(CV)staining,and visualization using scanning electron microscopy(SEM)and confocal laser scanning microscopy(CLSM).The results showed that PF07 and SB 11 grew rapidly at three different temperature,however,the planktonic cell density of PF07 in the co-culture decreased remarkably at low temperature.Two isolates reached the maximum biofilm biomass after cultured 12 h at 30 ℃,24 h at 15 ℃ and 72 h at 4℃.Meanwhile,PF07 had quicker adhesion and dispersion as well as higher biofilm biomass than SB 11 at initial phase.PF07 and SB 11 formed sticky and fragile pellicle,respectively,and exhibited different spatial architecture of biofilm.Compared to single PF07,the biofilm-forming steps were prolonged in the dual-species biofilm,biofilm formation and cell adhesion at the developing phase decreased at three different temperature.Cultured at 4 ℃ for 48 h,the biofilm biomass of single PF07 and mixed-species were 2.15 and 0.82(OD590),respectively.The CLSM results revealed that the biofilm thickness of SB11,PF07 and mixed-species were 46.8 um,41.4 um and 31.0 um respectively.All the pellicle,biofilm biomass and adhesion revealed that biofilm formation of mono-and dual-species at 4 ℃ or 15 ℃ were significantly higher than at 30 ℃.The maturing biofilm biomass at low temperature was high 3 times than at 30 ℃.Furthermore,high C4-HSL levels were detected in the PF07 culture,whereas,AHLs activities in co-culture significantly decreased,suggesting that AHLs mediated quorum sensing could affect the interspecies interaction of dual-species biofilm.P.fluorescens PF07 luxl gene and AHLs activity in mutant strain constructed was verified by PCR amplification,sequencing and C.violaceum CV026 biosensor strain and HPLC-MS/MS.The growth,adhesion,biofilm formation,EPS and swimming motility were compared between wild PF07 and mutant ΔluxI at 30℃ and 4℃.Compared to wild PF07,AHLs activity in the mutant decreased 80%.The growth of PF07 and mutant ΔlusI had no significant difference,however,the cell adhesion inΔlusI sharply reduced,and the cell density were 1 Logio CFU/mL inferior to PF07.CLSM results revealed that biofilm biomass in the ΔIuxI decreased 72.5%compared to PF07,and biofilm thickness of PF07 and AluxI was 52.5 um and 13.9 um respectively.Similarly,EPS amount in ΔluxI decreased 21.2%compared to PF07,After 24 h incubation at 30℃,the diffusion diameter of PF07 and ΔluxI were 73.3 and 60.9 mm,respectively,indicating the difference of swimming between PF07 andΔluxI at the early phase.In addition,exogenous addition of C4-HSL in the mutant significantly enhanced the adhesion,biofilm biomass and EPS production,which was hinted that luxl gene could be involving in biofilm formation and swimming ability.The dual-species biofilm formed by SB11 and PF07 or ΔluxI exhibited significant difference,especially at 4℃.Compared to single PF07,the biofilm biomass of PF07 and SB 11 decreased 54.8%for 4 d at 4℃,while,no great effect onΔluxI co-cultured with SB 11 at inital period.After 5 d incubation,the biofilm biomass of....decreased 14.3%.The CLSM results revealed that,the biofilm biomass of PF07 co-cultured with SB 11 decreased 48.0%,while AluxI increased 28%.Furthermore,exogenous addition of PF07 CFS resulted in the decrease of 32.2%in SB11 biofilm biomass,however,the CFS of ΔluxI had no effect on SB 11 biofilm.This study revealed that various isolates of P.fluorescens exhibited the different ability of biofilm formation and AHLs activity.Two spoilage bacteria,PF07 and SB 11,interacted in a competitive manner during the co-culture.Low culture temperatures greatly stimulated biofilm formation and adhesion of psychrotrophic bacteria,which would threaten the preservation and processing of aquatic products.In addition,it was indicated that AHLs signal could be involved in regulation of biofilm formation and interspecies interaction. |
