Nojoumi, Seyed Ali; (1997) Inducible responses to acidity and to thermal stress in gram-negative bacteria. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis discusses experiments on acid sensitivity and thermal stress in some Gram-negative bacteria. Microorganisms can be exposed to a wide variety of unfavourable environmental conditions including acid pH (e.g. in nature: rivers, estuaries; in foods: preservatives and in the human body: gastric juice) and heat. Therefore they may possess adaptive systems to overcome the damaging effects of these stresses. In this study it was shown that all the strains involved, were killed by lethal acid pHs in nutrient broth (NB) but they tended to habituate when they were grown at sub-lethal pH (e.g. pH 5) acquiring resistance which allowed them to survive when exposed to acid, whereas the non-habituated cells which were grown at the optimal pH (7.0) were considerably more sensitive to acidity. It was also found that prolongation of exposure time leads to higher resistance as compared to non-habituated cells. The process of habituation took place in river water (both for stationary and exponential cultures) as well as broth and resulted in more acid resistance in habituated cells. Unlike NB in which habituation occurs rather quickly, this takes slightly longer to happen in water. Pathogenic species would not normally resist acid foods (owing to presence of low pH and organic acids) but it has been proposed that prior to entry into food they become habituated during the food processing treatment, therefore they may survive the normally lethal acidity in the final product. In addition to NB and water, slight acid resistance was shown to occur in six Gram-negative strains used when treated with four acid foods (i.e. vinegar, yogurt, mayonnaise and salad cream). Growth of neutrophilic bacteria including E.coli K12 is poorer and slower at pH 5.0 than pH 7.0. Growth of Salmonella enteritidis was stimulated at pH 5.0 (adjusted with HCl) when 10 mM potassium phosphate was added to the culture; whereas its addition to pH 7.0 grown cells had no stimulatory effect. Such a stimulatory effect of phosphate on growth at pH 5.0 was also observed for Acinetobacter calcoaceticus, Proteus mirabilis, Vibrio parahaemolyticus, Citrobacter koseri, Citrobacter freundii, Klebsiella aerogenes,, Enterobacter gergoviae, Enterobacter cloacae, Serratia marcescens, and Salmonella typhimurium but not for Aeromonas formicans and Klebsiella edwardsii. The strain E.coli K10 (that lacks PIT system) was subjected to six organic acids (i.e. acetic, benzoic, sorbic, citric, propionic and transcinnamic acids) and found to be inhibited by the weak acids. When phosphate was added to the cultures it failed to stimulate the bacterial growth resulting in full inhibition in higher concentrations of organic acids. The basis for the different effects of phosphate in the presence and absence of organic acids was discussed. Following an induction by heat a rapid, transient response resulting in the synthesis of heat shock proteins occurs in E.coli. Induction of these proteins that may bring about thermotolerance might also lead to acid resistance. Temperature upshift conferred resistance to acid in E.coli 1829 ColV, I-K94 (which is more sensitive to acid than its ColV-free isogenic strain). In another experiment the effect of acid on heat- induced E.coli K12 strain (NM81) which is mutant in nhaA gene was tested and it was found to be less resistant to acid than its wild type strain. The strains of E.coli 1829 ColV, I-K94, 1829 himA, 1829 hns, 1829 fur, 1829 relA and 1829 nhaA grown at 25°C were shifted to 44°C and then challenged with acid to study the effect of regulatory components on heat induced cells. As a result, it was shown that H-NS protein is involved in resistance to acid in heat induced cells, since the 1829 hns cells were the most sensitive ones at 44°C. Sensitivity of heat induced cells of 1829 ColV, I-K94 to acid when treated with various inhibitors during induction were studied. Accordingly, addition of Nalidixic acid appeared to reduce induction of the resistance to acid in heat induced cells.

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