Study On The Regulation Mechanism On Cell Programmed Death Systems Of Staphylococcus Aureus In Polymicrobial Biofilms

In China,under the effective control of food quality and safety,although the rate of substandard food is still low-level,food safety caused by microorganisms is still one of the three major food safety issue.Staphylococcus aureus is a typical foodborne pathogen with strong biofilm forming ability and high pathogenicity.At present,due to the endless emergence of multi-drug resistant S.aureus in animal husbandry,alternative antimicrobial methods have been concerned,and one of the potential methods is to regulate the inherent programmed cell death system of bacteria.In recent years,it has been discovered that the natural stress pressure formed by Pseudomonas aeruginosa could significantly regulate the PCD system of S.aureus in the polymicrobial biofilm,and at the same time significantly inhibit the growth of S.aureus and even kill it completely.Therefore,for the inherent PCD system of S.aureus,study on the regulation of P.aeruginosa on the cell PCD system and lethal mechanism of S.aureus in polymicrobial biofilm,which can provide a theoretical basis for preventing and removing S.aureus contamination in food.1.Constructing different growth characteristics of colony polymicrobial biofilms model between P.aeruginosa and S.aureus,and the RNA-seq sequencing of the polymicrobial biofilm at different periods were performed.Transcriptome results shows that during the formation of polymicrobial biofilms,two P.aeruginosa(ATCC27853 and HQ)significantly regulates the transcription of S.aureus 10071 in several aspects,including energy metabolism,amino acid metabolism,nucleic acid metabolism,metal ion uptake and programmed cell death system and stress response.At the same time,the energy metabolism and iron uptake of two P.aeruginosa have also be significantly differentially expressed.As for energy metabolism,two P.aeruginosa inhibits the aerobic respiration of S.aureus,and P.aeruginosa ATCC27853 has the strongest inhibitory effect.S.aureus mainly conducts anaerobic respiration and anaerobic fermentation,accumulating ammonia and weak acids.In addition to aerobic respiration,two P.aeruginosa also obtains energy by metabolizing lactic acid secreted from S.aureus.As for iron uptake,in the polymicrobial biofilm,P.aeruginosa ATCC27853 will share iron ions with S.aureus,while P.aeruginosa HQ competes iron ions with S.aureus.The biofilm formation of S.aureus was promoted by iron-limitation promotes,which contribute to S.aureus coexist with P.aeruginosa HQ for a long-term co-culture(5 d).For the PCD systems of S.aureus,P.aeruginosa ATCC27853 inhibits cell autolysis by inducing antiholin protein to influent the structure of the biofilm and activate cid C-mediated cell death during the biofilm formation.Although P.aeruginosa HQ activated cid C-mediated cell death,it did not induce antiholin protein.2.Constructing different metabolism models(aerobic respiration,anaerobic respiration and anaerobic fermentation)applied for co-cultivation of P.aeruginosa and S.aureus.The results show that under different metabolic states(aerobic respiration,anaerobic respiration and anaerobic fermentation),none of P.aeruginosa strains inhibited the growth of S.aureus 10071 early stage,but all the P.aeruginosa induce S.aureus 10071 cells death in the stationary phase.Under aerobic respiration,after 24 hours of culturing,P.aeruginosa PAO1 and ATCC27853 can reduce the cultivable number of S.aureus 10071 to 0,while P.aeruginosa HQ can coexist with S.aureus 10071 for 5 days.Under anaerobic respiration and anaerobic fermentation,S.aureus 10071 can co-exist with three strains of P.aeruginosa(PAO1,ATCC27853,HQ)to grow for 5 days.Besides,the growth curves of two S.aureus strains(110749 and 121931)in coculture with two P.aeruginosa(ATCC27853 and HQ)are similar to S.aureus 10071.In all coexist groups,SCVs of S.aureus 10071 were accounted for more than 80% of the total culturable cells.At the same time,SCVs of S.aureus 10071 induced by the three strains of P.aeruginosa was restored to the normal cell colony after being cultured in fresh medium for 6 h.3.Apart from inhibiting the exponential growth of S.aureus 10071,the supernatants of all P.aeruginosa with aerobic and anaerobic respiration can induce S.aureus cells death in stationary phase.According to the decline range,the effect of the supernatant of P.aeruginosa in each metabolic state is as follows: aerobic respiration > anaerobic respiration > anaerobic fermentation.As for S.aureus,the transition of S.aureus 10071 from aerobic respiration to anaerobic respiration or fermentation growth in the supernatant of P.aeruginosa is one of the ways to improve its survival rate.Comparing with alkaline(p H = 9.0)condition,the high-level cells death of aerobic-respiration S.aureus 10071 was induced by aerobic-respiration supernatant of all P.aeruginosa under neutral(p H = 7.0)conditions,but under these range of p H(p H = 7.0-9.0)the growth of anaerobic-respiration or-fermentation S.aureus 10071 had been no influenced by aerobic-respiration supernatant of all P.aeruginosa.It is preliminarily inferred that all the aerobic respiration supernatant of P.aeruginosa can activate the cid Cmediated PCD system by inhibiting the aerobic respiration of S.aureus 10071,which leads to cell death and accumulation of weak acid.Comparing with anaerobic respiration or fermentation,P.aeruginosa ATCC27853 could significantly induce the up-regulation of the two PCD systems of S.aureus 10071(cid C and lrg AB)in aerobic co-culture,while P.aeruginosa HQ only activates the two PCD systems of S.aureus 10071 in the early stage of aerobicrespiration culture(24 h).This further confirms the molecular-regulation mechanism of P.aeruginosa ATCC27853 on the PCD systems of S.aureus 10071.