Effects Of Quorum Sensing Inhibitors On Lactobacillus Fermentum Biofilm Formation

As a kind of beneficial microorganism in the human gut,Lactic acid bacteria usually takes the form of biofilms to improve their resistance to external adverse environments.Study shows that the formation of lactic acid bacteria biofilm can be regulated by quorum sensing,but whether Autoinducer-2(AI-2),as a quorum sensing signal molecule,participates in the formation of lactic acid bacteria biofilm remains to be studied.In this study,Lactobacillus fermentum 2-1 with high-yield signal molecule AI-2 screened by previous group was used as the research object to explore the effects of three quorum sensing inhibitors(QSIs)on the activity of signal molecule AI-2,biofilm formation,cell morphology and lux S gene expression of the strain.At the same time,the effects of acid and bile salt stress on strain growth,signal molecules and biofilm were studied.With most biofilm forming,the effect of QSIs on strain signal molecules and biofilm was explored.Finally,the whole genome sequencing of L.fermentium 2-1 was carried out to locate the potential genes related to biofilm formation and determine the effect of QSIs on its relative expression.The main results are as follows :(1)D-galactose,D-ribose and furanone were selected as QSIs,which can inhibit the activity of L.fermentium 2-1 signaling molecule AI-2 and the formation of biofilm in a certain concentration range,while its effect on the growth of the strain was not obvious.As a result,the optimal concentration of the inhibitor was 100 μmol/L D-galactose,200μmol/L D-ribose and 200 μmol/L furanone.The results of scanning electron microscopy and fluorescence microscopy showed that none of QSIs had significant effect on the cell morphology in the planktonic and biofilm state of the strain at the optimal final inhibitory concentration,but the adhesion of the strain was reduced.The expression level of lux S,a key quorum sensing gene,was down-regulated and the time of gene expression peak was pushed back.(2)With different p H values and bile salt concentrations,the growth,signal molecule activity and biofilm formation of the strain were affected.When p H value was 2.5 and bile salt concentration was 0.1%,the strain no longer grew.When p H value was 3.5 and bile salt concentration was 0.03%,most biofilm of the strain formed.At the same time,the signal molecule AI-2 activity of the strain was greatly affected by p H value and bile salt concentration,Which may decrease the activity and delay the peak.When the p H value was 3.5 and the bile salt concentration was 0.03%,the addition of three kinds of QSIs also had no significant effect on the growth of the strain,but could inhibit the biofilm formation ability and the activity of signal molecule AI-2.(3)After sequencing and assembling the genome of L.fermentium 2-1,the total length of the sequence was 2101587 bp and the average GC content was 51.75%.A total of 2095 coding genes were obtained,while 58 t RNAs and five sets of 16s-23s-5s ribosomal RNA operons were predicted.Locating to the potential genes gap A,pgm B,tuf,rib BA,rib F,rpo C,rpo D and rpo E related to biofilm formation,each gene was highly expressed in the Logarithmic growth period of strain growth.With the decrease of culture time,the expression of each gene was down-regulated through adding QSIs in the logarithmic phase and the early stationary phase of the strain.