| L. monocytogenes is a particularly serious public health concern. Invasive infection by L. monocytogenes causes the disease listeriosis. The manifestations of listeriosis include esepticemia, meningitis and corneal etc. The fatality rate from listeriosis infections in some counties is estimated to be as high as 28%. Organic acids are widely used in meat industry to control pathogens, specifically, lactic and acetic acids are employed as washes to decontaminate meat surfaces of bacteria, and their salts are added to processed meats to prevent outgrowth of bacteria. Levulinic acid has been investigated recently for similar applications. Virulence in L. monocytogenes has been linked to acid and bile resistance, because each of these factors contributes to pathogen survival in the gastrointestinal tract. Several prior studies have shown exposure to mild acid conditions (pH<5.5) may induce resistance to acid or bile in L. monocytogenes, but little is understood regarding the response of L. monocytogenes to the weakly acidic conditions that characterize ready-to-eat or enhanced meats. Because widespread use of organic acids in the meat industry may have unintended consequences in L. monocytogenes virulence, it is important to develop a greater understanding of the effects of organic acid salts on the physiology of this important pathogen.Thus, this research investigated the intrinsic resistance to acid and bile of four human-pathogenic strains L. monocytogenes, as well as the effect of organic acid habituation at pH 6.0 on induced acid or bile resistance in these strains. Furthermore, the potential mechanisms of the enhanced acid resistance induced by organic acid were conducted from the altheration of membrane fatty acids profiles and intracellular metabolites. The main results are followed:1. Growth curves of those four strains of listeria monocytogenes:The growth trends of each strain were similar in media at pH 6.0 with or without the suppliment of organic acid (including 4.75 mM L-lactic acid, levulinic acid or acetic acid). All strains entry into the log phase and stationary phase at the same time, and the max colony number were 8.9-9.2 log10 CFU/ml. The growth rates were different between strains, of which strain C1-056 entried stationary pahse earlier two hours than other strains.2. Intrisic responses of those four strians to acid and bile:Compared with log phase, strains grown in stationary pahse could be resistant with sevever acidic or bilious conditions. The differences of acid or bile resistance between stains were more evident in log phase than those in stationary phase. As for effect of the the serotype on the acid/bile resistance, serotype 4b were more resistance to pH3.0 than serotype 1/2a, but the bile resistances were stain specific.3. Induced responses of those four strians to acid and bile by pre-exposure in weak acid conditons:Growth in the HCl control broth medium induced increased survival against acid challenge with three of the four strains as compared to the baseline control. More importantly, habituation of L. monocytogenes in TSB at pH 6.0 that contained an added organic acid induced greater survival against acid challenge as compared to HCl control for all strains except L. monocytogenes C1-056. The ability of induced acid resistance for organic acids were in order: acetic acid> levulinic acid> L-lactic acid. L. monocytogenes has relatively high intrinsic resistance to bile, but we found habituation in the HCl control medium significantly (P<0.05) decreased survival of all strains against the 0.2% bile challenge as compared to the baseline control. However, cells habituated in TSB at pH 6.0 with added organic acids generally survived the bile challenge at a rate that was comparable to the pH 7.4 baseline control.4. Membrane fatty acids alteration of those four strians by pre-exposure in weak acid conditons:by Eleven kinds of fatty acids (C14～C18) were detected in the cell membranes of each strain, including 6 kinds of branched chain fatty acids,3 kinds of saturated straight chain fatty acids and 2 kinds of unsaturated straight chain fatty acids, of which the anteiso fatty acids were dominent. The content of the fatty acid were in order ai-15> ai-17>i-15> i17> C16:0, and the first two kinds of fatty acids was greater than 50%. Afther the acid habituation, the content of ai-15 and C16:0 decreased in most of strains, while the contents of i-15, C16:0, ai-17 and i-17 increased. Ratios of membrane anteiso/iso and C15/C17 fatty acids, which are key factors influencing membrane fluidity, were significantly lower (P<0.05) in cells grown at pH 6.0 versus pH 7.4, which suggested a decrease in membrane fluidity in response to acidic pH. However, the presence or absence of organic acids did not induce additional changes in the membrane composition of cells grown at pH 6.0. These results suggest that previously observed differences in cell resistance to acid and bile after habituation to different organic acids were not based on acid-specific changes to membrane fluidity. The correlation analysis showed that the suvival in pH 3.0 after acid habituation were correlated to the ratio of C15/C17, and the higher ratios resulted a lower acid resistance.5. Intracelluar metabolites alteration of those four strians by pre-exposure in weak acid conditons:Four groups of small-volatile substances were detected via GC-MS, including 8 kinds organic acids,9 kinds of carbohydrate and phosphates,21 kinds of amino acids,16 kinds of fatty acids and its derivatives. Of which the content of the sum of amino acids was the highest, followed by fatty acids, carbohydrate and phosphates, and organic acids. And glycerol was the highest content among all the single components. After the acid habituation, the content of 2,4-dihydroxybutanoic acid decreased in four strains, while the percentage of frutose decreased in three of four strains (R2-499,C 1-056 and N1-227), and glucose also decreased in two strains (R2-499 and N1-227), compared with those in baseline control. The contents of asparitic aicd in organic acid treatments (strains N1-227 and N3-013) were lower than those in HCl control. The alteration of the contents of lysine, valline, tryptophan and histidine were strains and treatment-specific. It indicated that the inhanced acid resistance were related the decrease of 2,4-dihydroxy butyric acid, fructose and glucose. The differences of the induced acid resistance between organic acids and HCl control maybe caused by the alteration of the asparagine acids. Furthermore, the content of lactic aicd in L-lactic acid treated cells of four strains were higher than those in other treamments, and also the content of levulinic acid in levulinic acid treated cells of two strains (R2-499 and C1-056) were higher than the content in other treated cells, which suggested the induced acid resistance by organic acid in L. monocytogenes were related to the accumulation of intracellular acid anions. |
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