Effect And Mechanism Of Signal Molecules On Formation Of Viable But Nonculturable State Of Staphylococcus Aureus

Staphylococcus aureus is a common pathogenic bacterium with wide distribution.When the external environment is bad,Staphylococcus aureus will enter the viable but non-cultable state(VBNC)to resist external pressure.Staphylococcus aureus in VBNC state could not grow on conventional medium,and its morphological structure,resistance,adhesion,virulence gene expression and pathogenicity were changed.When conditions are favorable,Staphylococcus aureus in VBNC state can resuscitate,posing a potential threat to food safety and human health.The quorum sensing system mediated by signal molecules plays an important role in regulating bacterial growth,biofilm formation,cell movement and virulence gene expression.Studying the mechanism of Staphylococcus aureus entering VBNC state from the perspective of signal molecules can provide reference for the targeted elimination of Staphylococcus aureus.Considering the importance of low temperature storage for food and the important role of signal molecules in the regulation of bacterial life,this research studied the effect of quorum sensing signal molecules on the formation of VBNC state of Staphylococcus aureus and the possible mechanism of action.Staphylococcus aureus was induced with pure citric acid and citric acid supplemented with peptide 1,2,3 and 4 at 4?,and the number of culturable bacteria these five groups was recorded every day.The results showed that pure citric acid group,peptide 1 group,peptide 2 group,peptide 3 group and peptide 4 group became unculturable after 29,53,59,31 and 28 days of induction,respectively.Subsequently,Live/Dead Baclight kit and laser confocal microscope were used to observe the number of viable bacteria in unculturable state.The results showed that there were viable bacteria in all the five groups in unculturable state,indicating that they had completely entered the VBNC state.After tryptic soy broth(TSB)method,VBNC state cells in control group,peptide 1 group and peptide 2 group were successfully resuscitated.Transmission electron microscopy was used to observe Staphylococcus aureus under the conditions of logarithmic growth phase,VBNC of control,VBNC of peptide1 group and VBNC of peptide 2 group.Cell walls and membranes no longer fit tightly,and some cells have died.API ZYM kit was used to detect the intracellular enzyme activity in logarithmic phase and VBNC state.The results showed that the intracellular enzyme activity of VBNC bacteria decreased in control group,but increased in peptide1 group and peptide 2 group.This may be one of the reasons why autocrine polypeptides delay the formation of VBNC state in Staphylococcus aureus.The adhesion ability of Staphylococcus aureus in control group,peptide 1 group and peptide 2 group was determined by crystal violet staining.It was found that there was no significant difference between peptide 1 group and control group,while the adhesion ability of peptide 2 group was significantly higher than that of control group and peptide 1 group,which might be because the autocrine polypeptide regulates the adhesion ability of bacteria.The surface Zeta potentials of Staphylococcus aureus in control group,peptide1 group and peptide 2 group were measured by particle size analyzer.It was found that the absolute value of surface Zeta potential of Staphylococcus aureus decreased after the addition of peptide 1 and peptide 2,and the decreasing trend was concentrationdependent,indicating that peptide 1 and peptide 2 could increase the adhesion ability and aggregation degree of Staphylococcus aureus.Cell membrane permeability of Staphylococcus aureus was observed by laser confocal microscope under the same induction time in the control group and the groups with three different concentrations of peptide 1 and peptide 2.The results showed that the bacterial cell membrane permeability increased after the addition of polypeptide 1and polypeptide 2.After the increase of cell membrane permeability,peptide 1 and peptide 2 may enter the cell and interact with the intracellular DNA.Therefore,gel block,UV spectrum and fluorescence spectrum were used to analyze the effect of peptide and DNA in vitro.The results showed that peptide 1 and peptide 2 could bind to DNA,which may be one of the reasons why the two peptides delay the formation of VBNC state.The addition of peptide 1 and peptide 2 delayed the formation of VBNC state of Staphylococcus aureus,which bought time for detection and thus reduced the threat of food safety and human health caused by Staphylococcus aureus in VBNC state.Therefore,this study proposed a new strategy for food safety protection from the perspective of inhibiting the formation of VBNC state.