The Molecular Mechanisms Of Viable But Nonculturable Staphylococcus Aureus By Nonthermal Plasma (NTP)

Nonthermal plasma(NTP),an emerging nonthermal technology,has the advantages of the efficient inactivation activity on a broad spectrum of microorganisms and environmentally friendly.Therefore,NTP has been considered as a hot topic in the field of food sterilization.NTP has an excellent antimicrobial capacity and can overcome some shortcomings of traditional sterilization techniques.However,based on the previous studies and the preliminary research results from our lab,NTP might cause incomplete inactivation of microorganisms.The plasma-generated reactive species,such as reactive oxygen species(ROS),can induce microorganisms into a viable but nonculturable(VBNC)state.VBNC microorganisms fail to grow,reproduce,and form the visible colonies on the medium,which renders them with the ability to escape from the culture-dependent detection and leads to false negative results.VBNC microorganisms still possess a certain level of metabolic activity.Under suitable conditions,they can resuscitate and regrow on the medium.Therefore,VBNC microorganisms have been regarded as the invisible sources of hazard.In view of the potential risks in the formation of VBNC microorganisms by NTP,this thesis employed foodborne Staphylococcus aureus as the research model.The mechanism underlying NTP induced VBNC S.aureus and its perniciousness were systematically investigated in this study.This research lays a theoretical foundation for the optimization of NTP process and the efficient inhibition of the formation of VBNC microorganisms to assure the food safety.The major content and results are summarized as follows:(1)Induction of VBNC Staphylococcus aureus by nonthermal plasmaPropidium monoazide combined with quantitative polymerase chain reaction was established(PMA-qPCR)to determine the total viable counts of S.aureus.The optimized concentration of PMA was 50 μM,and the limitation of detection value of PMA-qPCR was 220 CFU/mL.PMA-qPCR combined with the plate count method was used to monitor the changes in the counts of S.aureus in various states during the storage at 4℃ after the NTP treatment with various applied energies.The results indicated that the total viable counts of S.aureus were insignificantly unchanged during the period of storage.The culturable counts of S.aureus were gradually decreased,and the VBNC counts were increased accordingly.When the applied NTP energies were increased from 8.1 to 24.3 kJ,the time required for S.aureus to completely enter the VBNC state was shortened from 80 to 16 h.The maximum possible number(MPN)method was used to evaluate the effect of various factors on the resuscitation ability of VBNC S.aureus.The results showed that the lag time before resuscitation of VBNC S.aureus and the levels of successfully resuscitated cells were inversely proportional to the incubation temperature.When the resuscitation temperature was enhaced from 15℃to 37℃,the lag time before resuscitation of VBNC S.aureus was decreased from 66 h to 10 h,with the decrease in the levels of successfully resuscitated cells from 3.46 log10 MPN/mL to 2.01 log10 MPN/mL.The addition of chemical compounds(glucose,sodium pyruvate and Tween 80)had insignificant impact on the resuscitation.S.aureus in various period times after entering the VBNC state poccessed different abilities to recover into the culturable state.S.aureus with longer period time in the VBNC state had lower resuscitated capacity,required longer lag time before resuscitation and achieved lower level of resuscitated cells.(2)Biological characteristics of VBNC Staphylococcus aureusThe methods,such as luciferin-luciferase,BCECF-AM fluorescence probes,etc.,were applied for the investigation of the physiological characteristics of NTP-induced VBNC S.aureus.The results showed that compared with the cultivable S.aureus,the intracellular ATP level and pH of VBNC S.aureus were significantly reduced.The activities of 7 metabolic enzymes(lipase C14,leucine arylaminase,valine aryminase,cystine aryminase,β-galactosidase,α-glucase,β-glucase)were decreased in VBNC S.aureus,but the activities of 5 metabolic enzymes(alkaline phosphatase,acid phosphatase,esterase C4,lipid esterase C8,naphthol-AS-BI-phosphohydrolase)were still retained.Further analysis of membrane fatty acid composition by the gas chromatography indicated that the antesio branched-chain fatty acid content in the cell membrane of VBNC S.aureus was increased from 47.33±0.19 to 54.35±0.30%,while the proportions of saturated fatty acids and long-chain fatty acids(C18:0,C19:0,C20:0)were decreased significantly,indicating an increase in the membrane fluidity of VBNC S.aureus.With the observation and analysis by a transmission electron microscope(TEM),a scanning electron microscope(SEM)and an atomic force microscopy(AFM),it was found that the VBNC S.aureus cells were covered with a layer of polymers,resulting in a rough cell surface.In addition,the bacterial cells were aggregated together.The average cell volumes of VBNC S.aureus appeared to be decreased by around 19.6%compared with those of the culturable S.aureus.(3)The mechanisms underlying the induction of VBNC Staphylococcus aureus by nonthermal plasmaThe changes in the levels of intracellular and extracellular oxidative species before and after NTP treatment,as well as the changes of intracellular ATP concentrations in S.aureus were measured.The results indicated that the oxidative stress induced by NTP was the major contributor to the ATP depletion in S.aureus cells.RNA sequcencing(RNA-seq)technique combined with the annotations of KEGG pathways was employed for the qualitative,quantitative and functional analysis of the differentially expressed genes,in order to reveal the regulation of the metabolism,genetic information processing,and environemtnal information processing pathways in VBNC S.aureus.The oxidative phosphorylation uncoupling agent-carbonyl cyanide mchlorophenyl hydrazone(CCCP)was further applied to artificiallydecrease the intracellar ATP level in order to verify the key role of ATP depletion in NTP-induced VBNC S.aureus.The mechanisms underlying induction of VBNC S.aureus by NTP induced was demonstrated as follows:The oxidative stress from NTP induced S.aureus to initiate a series of oxidative stress response,including the production of the enzymes for detoxifying ROS,the promotion of the Dps protein-mediated DNA protection pathways,and the activation of the methionine-sulfoxide reductase Msr and thioredoxin reductase Trx protein repair systems.The synthesis of proteins associated with stress response,leading to a large loss of intracellular ATP levels.In order to reduce the energy consumption,the energy-dependent processes of peptidoglycan and fatty acid biosynthesis,gene replication,transcription,and translation,as well as signal transduction were significantly inhibited in VBNC S.aureus.Subsequently,the metabolic,growth and reproductive activities were suppressed,and S.aureus was finally induced into the VBNC dormancy state.(4)Stress resistance and pathogenicity of VBNC Staphylococcus aureusThe stress resistance and pathogencity of NTP-induced VBNC S.aureus were systematically investigated.Regarding the stress resistance,the results indicated that the tolerance of VBNC S.aureus towards heat,acid,as well as osmosis showed insignificantly different from their culturable counterparts.The enhanced resistance to the oxidative stress(hydrogen peroxide)was found in VBNC S.aureus and it could be associated with the upregulation in genes involving the oxidative stress response.In addition,the resistance of VBNC S.aureus to ciprofloxacin,chloramphenicol,trimethoprim,and oxacillin was significantly enhanced due to the possible mechansisms,including the upregulated expression of the multidrug efflux pumpsMepA and NorB,the intracellular ATP depletion,etc.Regarding pathogenicity,iTRAQ-coupled HPLC-MS/MS proteomics was used for the quantitative analysis of the virulent factor expressions in VBNC S.aureus.It indicated that most of the virulence factors(e.g.,toxins,exoenzymes)were downregulated by VBNC S.aureus.But the SdrD,ClfB,SasH and SCIN-mediated adhesion and invasion pathways were activated to assure the infective ability of VBNC S.aureus to host cells.What’s more,the expression of pathogen associated molecular patterns(PAMPs),including peptidoglycan,teichoic acid and lipoprotein,was significantly downregulated by VBNC S.aureus,which could avoid the bacteria to be sensed by the hots pattern recognition receptors(PRRs).It was also found that the biosynthesis of capsule was enhanced,which could provid a barrier for the surface molecules of bacterial cells from the recognization by the host cells.