The Mechanism Of Acid Tolerance Response In The Listeria Monocytogenes Based On Changes Of Intracellular Concentration Of Lactic Acid Anions And PH Value

The Gram-positive bacterium Listeria monocytogenes is a ubiquitous pathogen that could infect livestock and man.It has 25~30%fatality rate with septicaemia,meningitis,gastroenteritis and abortion.Since it was found to be responsible for several outbreaks of food-borne diseases in the eighties,studies on this bacterium have intensified.Lactic acid is commonly used for control the growth of pathogens in meat industry.However,recent studies had indicate that lactic acid could induce bacteria adapting acidic environment.Acid Tolerance Response(ATR)is a strong acid resistant reaction.It can be induced by sublethal acidic environments.Acid-adapted L.monocytogenes cells shows the increased tolerance even with other stressors,which also shows a higher virulence.In recent years,most studies have tried to find out the antimicrobial mechanism of organic acid,lacking of reports for L.monocytogenes Acid Tolerance Response.But,only few studies have focused the L.monocytogenes'Acid Tolerance Response with tracing the intracellular pH and the concentration of intracellular acid content.Taken into account all the previous study,this experiment mainly forced on the relationship between Acid Tolerance Response and the intracellular pH value and lactic anions content of L.monocytogenes.Therefore,the present study evaluated the adaptive responses to mineral acid(HCl)or lactic acid(3mM,4.75mM and 15mM)at three different pH levels(5.5,6.0,6.5)of L.monocytogenes.with pH 7.4 was set as blank group.Three strains were selected in the study which were 4b and 1/2a type of Listeria monocytogenes and Listeria innocua.The intracellular pH and concentration of lactic anions in cells were measured,and the mechanism of was discussed.The main results were as following:1.The growth curve of L.monocytogenesThree strains of Listerias were incubated in 37°C with shaking for 24h.The different growth phases were monitored.All the organisms reached mid-exponential phase at 8h.Listerias grew the best in BHIM cultures without acid stress,while organisms were inhibited obviously in pH 5.5 lactic acid(15mM).Listerias at mid-exponential phase were collected as samples to determine pH_i value and lactic acid concentration in cells in the following study.2.Induced responses of those three strains to acid in 13 culture conditionsCompared to blank control group and the HCl control group,three strains of Listerias in lactic acid treatment group(pH5.5 and 6.0)were significantly increased while toleranced to severe acid stress.Those of 4b and 1/2a type of L.monocytogenes in pH 5.5 acquired distinct acid resistance compared to that in pH 6.0.The acid tolerance ability of Listeria innocua in different pH was lower than other two strains.Minimal acid resistance was observed when Listerias was cultured in pH 6.5 of organic acid.The result suggest that the low-pH conditions probably have the potential for L.monocytogenes selection with virulence(4b and 1/2a).3.The intracellular pH of the three strains of Listerias cultured in 13 culture conditionsThe three strains of Listerias were incubated in 13 different acid culture conditions which could resist pH_i in the range of 7.4~8.2.The pH_i of 4b type of L.monocytogenes was resisted in the range of 7.4~7.7,and the pH_i of 1/2a type of L.monocytogenes was resisted in the range of 7.7~7.9,and the pH_i of Listeria innocua resisted in the range of 7.6~8.2,respectively.The data suggested that nontoxic Listeria innocua had least stability of pH_i in acid conditions.Comparing to L.monocytogenes incubated in pH 7.4,those organisms cultured in p H 5.5 and 6.0 lactic acid acquired significant lower pH_i.There was no differences in pH_i between organisms cultured in pH 6.5 and organisms in blank group.This phenomenon was in accordance with Acid Tolerance Response in the study.4.The concentration of lactate ion inside 1/2a type of L.monocytogenes under 13culture conditionsChanges of acid lactate ion inside cells of 1/2a type of L.monocytogenes were determined.The cells cultured in blank group with the highest intracellular lactic acid,which was 2.47?g/m L.Compared to organism cultured in other conditions,it acquired significant high lactate ion in cells.There were no differences in the concentration of lactate ion in cells between organism cultured in HCl and lactic acid.