Mechanism On The Development Of Stress Tolerance In Salmonella Enterica Serovar Enteritidis In Response To Ethanol Adaptation

Salmonella is a foodborne pathogen that poses a great threat to food safety.There are currently more than 2610 Salmonella serovars,of which Salmonella enterica serovar Enteritidis?S.Enteritidis?is the largest single cause of Salmonella infection.Chemical disinfection is a commonly used approach to control Salmonella in food industries.However,some strains of Salmonella are able to survive in food processing environments in spite of intensive cleaning and sanitation treatments.In fact,sublethal concentrations of disinfectants are commonly present in environmental niches and on equipment surfaces due to their instability,inappropriate application and dilution in the environment.Adaptation to sublethal concentrations of disinfectants can induce bacterial tolerance to homologous?direct tolerance?or heterologous?cross tolerance?stressing agents.This may counteract the effectiveness of currently employed food control measures,thus compromising food safety.In this context,it is of paramount significance to uncover why Salmonella mounts stress tolerance after exposure to chemical disinfectants in order to provide some guidance for the appropriate use of disinfectants and for the development of new intervention measures for this pathogen.Ethanol is commonly employed as a chemical disinfectant,a food preservative as well as a food processing aid in food industries.Furthermore,ethanol is frequently found in fruits,fruit juices,alcoholic drinks as well as fermented foods.Adaptation to sublethal concentrations of ethanol has been reported to induce direct tolerance and cross tolerance in foodborne pathogens such as Vibrio parahemolyticus and Cronobacter sakazakii.Nevertheless,there is no available information on the development of stress tolerance in Salmonella after ethanol adaptation.To address this gap,the influence of ethanol adaptation on stress tolerance and cell membrane/wall characteristics of S.Enteritidis was determined in the current work.The correlation between stress tolerance phenotypes and gene expression patterns in ethanol-adapted S.Enteritidis was then assessed.Finally,global proteomic analysis and mutagenic analysis were conducted to reveal the strategies utilized by S.Enteritidis to acquire direct tolerance to ethanol and cross tolerance to freezing stress following ethanol adaptation.The major findings regarding the above-mentioned aspects are as follows.1.Influence of ethanol adaptation on the development of direct tolerance and cross tolerance in S.Enteritidis.The tolerance of ethanol-adapted S.Enteritidis to food processing-related stresses was determined by plate count method,disc diffusion test or minimum inhibitory concentration and minimum bactericidal concentration assays in this chapter.The main results are as follows.?i?Adaptation in 2.5-10%ethanol significantly induced tolerance to 15%ethanol in S.Enteritidis and the maximum ethanol tolerance was observed when the ethanol concentration used for adaptation was increased to 5%.?ii?The stability of ethanol adaptation was dependent on temperature in the broth assay.Ethanol adaptation was stable in Luria-Bertani?LB?broth at 4°C for up to 48 h but was completely reversed within 60 min at 25°C or after 40 min at 37°C.Furthermore,ethanol adaptation stability was retained in chicken juice and was enhanced by pork juice.?iii?S.Enteritidis acquired cross tolerance to freezing temperature?-20°C?,hydrogen peroxide and malic acid after ethanol adaptation.However,it failed to develop cross tolerance to low temperature?4°C?,high temperature?50°C?,sodium chloride,potassium chloride,sodium hydroxide,potassium hydroxide,acetic acid,hydrochloric acid,lactic acid,ascorbic acid,citric acid,tetracycline,doxycycline,ceftriaxone,ampicillin,aztreonam,chloramphenicol,norfloxacin,ciprofloxacin,trimethoprim/sulphamethoxazole,amikacin,gentamicin,ceftazidime and cephalothin.?iv?The cell number of S.Enteritidis in orange juice was not significantly affected by ethanol adaptation.However,an increased survival was noted with ethanol-adapted S.Enteritidis compared to the non-adapted control in apple juice stored at 25°C,but not at 4°C.Furthermore,variations in cell membrane/wall characteristics of S.Enteritidis were assessed in terms of cell membrane permeability,cell membrane fluidity,cell surface morphology,cell surface hydrophobicity and cell auto-aggregation.It was found that ethanol adaptation significantly decreased cell surface hydrophobicity and increased cell auto-aggregation rate of S.Enteritidis as determined by spectrophotometry.However,cell surface morphology and cell membrane fluidity did not significantly change in response to ethanol adaptation.2.Gene expression characterization of ethanol adaptation in S.Enteritidis.The expression profiles of 81 stress tolerance-related genes were determined following ethanol adaptation by reverse transcription quantitative real-time PCR?RT-qPCR?.The results are as follows.?i?Two acid tolerance-related genes?rpoS encoding?^S and SEN1564A encoding an acid shock protein?were upregulated after exposure to ethanol.?ii?The expression of heat tolerance-related genes?rpoH,dnaK and dnaJ?did not significantly alter after ethanol adaptation.?iii?The cspA gene encoding the major cold shock protein was downregulated in response to ethanol adaptation.However,other four cold shock genes?cspB,cspC,cspD and cspE?were not significantly differentially expressed.?iv?Out of the eight assessed genes?katG,sodA,katE,tpx,hslO,bcp,SEN0385 and oxyR?related to oxidative stress tolerance,only katG encoding catalase showed differential expression after ethanol adaptation.?v?Ethanol adaptation did not significantly change the expression levels of the genes?tolA,mreB,mreC,mreD,mrdB and yjeE?involved in cell wall/membrane integrity.3.Proteomic analysis of direct tolerance to ethanol challenge and cross tolerance to freezing stress induced by ethanol adaptation in S.Enteritidis.Molecular mechanism on ethanol-induced stress tolerance in S.Enteritidis was further explored in terms of direct tolerance to ethanol challenge and cross tolerance to freezing stress.The iTRAQ technique was utilized to uncover the proteomic response of this bacterium to ethanol adaptation and subsequent freezing treatment.The expression pattern of differential proteins was verified at the mRNA level by RT-qPCR analysis.The regulatory networks regarding the above-mentioned stress tolerance phenotypes were then established based on the iTRAQ data.The results are as follows.?i?A total of 138 proteins?56 upregulated and 82 downregulated?were differentially expressed after ethanol adaptation.?ii?When S.Enteritidis was subjected to subsequent freezing treatment?-20°C,3 h?,185 differential proteins?88 upregulated and 97 downregulated?were observed in ethanol-adapted cells compared to those in non-adapted controls.?iii?Most of the tested proteins and their corresponding mRNAs displayed a similar expression pattern in response to ethanol adaptation and subsequent freezing treatment,demonstrating an evidence for the reliability of the data derived from proteomic analysis.?iv?Functional annotation revealed that multiple pathways involving metabolism?e.g.,purine metabolism and amino acid metabolism?,ABC transporter,siderophore biosynthesis and uptake,regulator,protein synthesis and virulence coordinately regulated the development of direct tolerance to ethanol and cross tolerance to freezing stress in ethanol-adapted S.Enteritidis.4.Negative regulation of hiuH and proX on the development of direct tolerance to ethanol challenge and cross tolerance to freezing stress induced by ethanol adaptation in S.Enteritidis.Two proteins?HiuH involved in purine metabolism and ProX associated with glycine betaine transport?were screened for functional characterization in this chapter based on protein expression pattern,protein function annotation,stress response network and RT-qPCR analysis.The main results are as follows.?i?Two mutants??hiuH and?proX?and their corresponding complementary strains??hiuH-C and?proX-C?were constructed according to homologous recombination knockout procedures using the cloning vector pMD₁₉-T and the suicide vector pRE112.?ii?Deletion of hiuH or proX did not significantly alter the growth rate of S.Enteritidis in LB broth.Moreover,there was no significant difference in cell surface morphology between the wild type strain and the two mutants after ethanol adaptation and subsequent freezing treatment.?iii?Ethanol-adapted cells of the wild type strain and the two mutants became filamentous in response to subsequent freezing treatment.Hence,filament formation may be a strategy utilized by S.Enteritidis to develop cross tolerance to freezing stress after ethanol adaptation.?iv?The?hiuH and?proX mutants mounted significantly higher tolerance to ethanol challenge and freezing stress compared with the wild type strain after ethanol adaptation.Furthermore,these stress tolerance phenotypes were restored in complementary strains,thereby confirming that proX and hiuH were negatively involved in the acquisition of direct tolerance to ethanol challenge and cross tolerance to freezing stress.In summary,the development of stress tolerance in ethanol-adapted S.Enteritidis was largely dependent on the subsequent stress tested.Generally,variations in stress tolerance phenotypes were accompanied with the differential expression of corresponding stress tolerance-related genes.Especially,ethanol adaptation was able to induce direct tolerance to ethanol challenge and cross tolerance to freezing stress,malic acid and hydrogen peroxide in S.Enteritidis.Complex regulatory pathways associated with metabolism?e.g.,purine metabolism and amino acid metabolism?,ABC transporter,enterobactin biosynthesis and uptake,regulator,protein synthesis,flagellar assembly and virulence were at play during ethanol adaptation and subsequent freezing treatment.Moreover,mutagenic and complementation analyses demonstrated that proX and hiuH were negatively involved in ethanol-induced direct tolerance to ethanol challenge and cross tolerance to freezing stress.Collectively,these results provide some precious insights into the molecular mechanism on the development of stress tolerance resulting from ethanol adaptation in S.Enteritidis as well as a mechanistic basis for the effective control of ethanol-adapted foodborne pathogens with enhanced stress tolerance.