Analysis Of Acid Adaptation Effects On Salmonella Enterica Serovar Enteritidis

Salmonella is an important pathogenic microorganism that causes many foodborne illnesses worldwide each year.Sublethal environmental stresses can enhance the tolerance of bacteria to the same(direct protection)or a different(cross protection)stress,posing a great threat to mirobial food safety.Development of stress tolerance resulting from acid adaptation in Salmonella has been attracting increasing attention in recent years.The current work determined acid adaptation response in a standard strain,Salmonella enterica serovar Enteritidis(S.Enteritidis)ATCC13076.The main research contents and results are as follows:S.Enteritidis at log-phase and stationary-phase were kept in pH4.5-6.0 for 1-4 h to prepare acid-adapted cells.Log-phase and stationary-phase cultures that were not subjected to mild acidic pHs were used as non-adapted controls.The survival ability of non-adapted and acid-adapted cells in Luria-Bertani(LB)broth acidified to pH 3.0 with hydrochloric acid was then determined.It was found that all treatments induced various levels of acid tolerance response(ATR)that was dependent on pH value,exposure time and growth phase.Moreover,exposure to pH 4.5 for 3 h(log-phase cells)and for 1 h(stationary-phase cells)was the optimal acid adaptation condition that induced the highest degree of ATR in S.Enteritidis.In cross protection test,the effect of acid adaptation(pH 4.5 for 3 h for log-phase cultures;pH 4.5 for 1 h for stationary-phase cultures)on the survival of S.Enteritidis exposed to heat(50 ~oC),cold(4 ~oC and-20~oC),salt(8%NaCl)and ethanol(12%)stress was assessed.The results showed that acid adaptation resulted in tolerance to 50 ~oC and 8%NaCl regardless of the growth phase.However,the tolerance of log-phase and stationary-phase cells to low temperatures(4 ~oC and-20 ~oC)and 12%ethanol was increased and decreased by acid adaptation,respectively.Finally,acid adaptation mechanisms in S.Enteritidis were explored in terms of changes in cell membrane characteristics and gene expression patterns.To this aim,analysis of cell membrane fatty acids,cell membrane permeability,electron micrograph image and expression of stress tolerance genes were determined.The results showed a decrease in the ratio of unsaturated to saturated fatty acids after acid adaptation.However,no significant changes in cell membrane permeability was found and both acid-adapted and non-adapted cells remained intact on the surface.Besides,RT-qPCR analysis revealed that the following genes involved in tolerance to acid(SEN1564A,cfa,fabB),heat(rpoH,uspB and htrA),cold(cspA,cspC,cspE and csdA)and salt(proP,proV and osmW)stress were generally upregulated after acid adaptation.Taken together,acid adaptation resulted in the development of direct protection against acid stress and cross protection against heat,cold,salt and ethanol stress in S.Enteritidis.The induction of stress tolerance was probably due to changes in cell membrane fatty acid composition and the expression of corresponding stress tolerance genes.These findings can enrich the understanding of acid adaptation effect of the important serotype S.Enteritidis and provide theoretical support for the control of Salmonella contamination in food production.