Effects Of Polystyrene Microplastics On The Growth, Film-forming Properties And Virulence Of Vibrio Parahaemolyticu

The randomly discarded plastics are gradually decomposed into small particles of<5mm in diameter through a series of effects（e.g.light,weathering,etc.）,forming microplastics.Microplastics,because of their large surface area,can easily become the colonization site of pathogenic bacteria.Vibrio parahaemolytics（VP）is a saline-loving gram-negative pathogenic bacteria widely distributed in seawater and seafood,which is easily adsorbed on the surface of microplastics when exposed to seawater,thus forming a biofilm structure.This undoubtedly increases the risk of infection for seafood consumers.However,there are few studies on the interaction between microplastics and pathogenic bacteria and potential health risks,and the synergistic biological effects are still in their infancy.In this paper,we selected polystyrenemicroplastics（PS-MP_S）,a commonly detected microplastic in the aqueous environment,as the material,and adopted a co-culture of VP and PS-MP_Sto analyze the effects on VP growth,biofilm formation ability and related membrane wall characteristics at different concentrations of PS-MP_S（1μg/L-1000μg/L）,and the combined toxicity of PS-MP_Sand VP was evaluated using the model organism Caenorhabditis elegans.The main studies and the results obtained are as follows.（1）Effect of polystyrene microplastics on the growth and film-forming characteristics of Vibrio parahaemolyticusThe method of co-cultivation of VP and PS-MP_Sis adopted.Firstly,the absorbance of VP at OD600_nmwas measured by micro UV spectrophotometer and the growth curve of VP was obtained,and the growth of VP was found to be promoted by PS-MP_S.It was found that the biofilm formation was positively correlated with the concentration of PS-MP_S;further analysis of the factors related to the biofilm formation of VP showed that the self-coagulation ability and hydrophobicity of VP were also positively correlated with the concentration of PS-MP_S,while the motility showed no correlation with the concentration of PS-MP_S.This indicates that PS-MP_Shas a"concentration-effect"relationship in promoting the growth of VP and biofilm formation.That is,"the growth,hydrophobicity,self-coagulation ability and biofilm formation of VP increased with the increase of PS-MP_Sconcentration.（2）Determination and analysis of the main substances in the extracellular polymer of Vibrio parahaemolyticusTo further understand the role of extracellular polymers in biofilm formation,the microstructure of extracellular polymers（EPS）in biofilm was observed by scanning electron microscopy（SEM）and the content of extracellular polysaccharides and extracellular proteins,which are the main substances in extracellular polymers,were measured.The results showed that VP in PS-MP_Sat high concentrations（100μg/L-1000μg/L）resulted in significant aggregation of bacteria into clusters with rougher surface morphology and the bacterium was encapsulated by extracellular polymers.The analysis of the secreted extracellular polymers revealed that the extracellular polysaccharide was the main substance of PS-MP_Sthat caused the change of the extracellular polymer of VP,and the extracellular polysaccharide acted as a molecular gel in the biofilm structure formed by VP,which contributed to the formation of a more dense biofilm structure.Fluorescence quantitative PCR results showed that the expression of aph A,cps A and cps Q were significantly increased,while the expression of Luxs was significantly decreased,indicating that multiple genes were involved in regulating the formation of extracellular polymers outside the biofilm of Vibrio parahaemolyticus.（3）Synergistic toxicity of polystyrene microplastics and Vibrio parahaemolyticus to Caenorhabditis elegans.In order to understand the effect of PS-MP_Son VP virulence,the model organism Caenorhabditis elegans was used to establish the synergic effect model of PS-MP_Sand VP.The acute exposure toxicity of C.elegans under PS-MP_Sand VP alone was compared with that under the coexistence of the two conditions.The combined toxic effects of both were measured by measuring the biological endpoints of C.elegans:motility behaviour,longevity experiments,colony forming units（CFU）,ROS reactive oxygen levels and lipofuscin accumulation.The expression of antimicrobial genes and oxidative stress-related genes in C.elegans was determined using fluorescent quantitative PCR.The results showed that PS-MP_Senhanced the bacterial toxicity of VP,which led to the decrease of movement frequency and the shortening of life span of nematodes,the increase of intestinal ROS level,intestinal spontaneous fluorescence（lipofuscin）and the accumulation of CFU in vivo,and the toxicity showed a concentration dependent trend.When PS-MP_Sreached high concentration（100μg/L-1000μg/L）,the amplification effect was statistically significant.Fluorescence quantitative PCR results showed that VP alone exposure and VP+（PS-MP_S）combined exposure could induce the expression levels of antimicrobial genes（lys-7,lys-8,spp-1,clec-60,clec-87）and oxidative stress genes（sod-1,sod-2）in C.elegans.Induce the innate immune response of C.elegans to pathogenic bacteria and change the molecular basis of oxidative stress in C.elegans.In summary,PS-MP_Spromotes VP growth and biofilm formation by regulating changes in the expression of aph A,cps A,cps Q and Luxs genes,resulting in the formation of a more dense biofilm structure,increased hydrophobicity,self-coagulation and increased secretion of extracellular polysaccharides.In terms of effects on C.elegans,VP displayed synergistic toxicity with PS-MP_S,mainly leading to a reduction in the frequency of C.elegans motor behaviors and reduced lifespan as well as increased intestinal ROS levels,accumulation of lipofuscin and CFUs,eliciting a C.elegans innate immune response to pathogenic bacteria and modifying the molecular basis of oxidative stress in C.elegans.