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Published Article / Various foodstuffs have a very low pH and bacteria have been reported to survive such products. Acid substances, such as organic acids are common food preservatives. These substances also lower the pH of processed foods. Decontamination with organic acids which could result in the emergence of acid tolerant food-borne pathogens is causing concern. The objectives of the study were to determine the development of acid tolerance in important food-borne pathogenic bacteria, to investigate evolving changes in the phenotypic characteristics as a result this acid tolerance, and to explore the possibility of repercussions in successful food preservation with organic acids. Bacterial strains were screened for acid-tolerance, by determining viable counts immediately before acid challenge and at various times after challenge. Strains were exposed to increasing concentrations of hydrochloric acid, acidic foodstuff and two organic acids (acetic and citric acid). Protein profiles were generated by SDS-PAGE examined for possible modification(s) as a result of acid tolerance development. Susceptibility to seven organic acids levels were scrutinised to evaluate the probability of a correlation between altered antimicrobial activity and acid tolerance. Salmonella enterica sv. Enteritidis ATCC 13076 and Escherichia coli ATCC 25922 indicated rapid development of acid tolerance, after 36h of acid exposure. In Salmonella enterica sv. Typhimurium ATCC 14028, E. coli 0111 and Pseudomonas aeruginosa ATCC 27853 intermediate intrinsic acid tolerance was obvious. On comparing susceptibility of these pathogens to the organic acids, it was demonstrated that pH played a significant role in inhibitory activity, as it is known that these compounds exhibit optimum antimicrobial activity at a lower pH (pH ≤ 5). Further investigations will be conducted to elucidate the two-way role of pH in foodstuff and the addition of an organic acid, in determining if microorganisms are losing their susceptibility for the preservative as a result of sub-optimal pH levels, or if they become acid-tolerant after surviving exposure to organic acids.

Listeria monocytogenes is a food-borne pathogen able to adapt and survive in a wide range of habitats in addition to being able to overcome host defences. The need to prevent L. monocytogenes entering the food chain and the role that stress plays during the course of an infection, means that the starvation-survival and stress resistance mechanism of this organism are thus of significant interest. The starvation-survival response of L. monocytogenes EGD in a chemically defined medium was induced under glucose- or multiple nutrient-, but not amino acid-limitation. This resulted in 90 to 99.9% loss of viability within 2 days, with viability maintained during prolonged starvation. Surviving cells were reduced in size and developed increased general stress resistance. L. monocytogenes EGD demonstrated densitydependent starvation-survival under multiple nutrient- but not under glucose-limitation. Protein synthesis was required for long-term survival only for the first 8 hours of starvation and survival became independent of cell wall biosynthesis during long-term starvation. Strains bearing mutations in the gene regulators sigB (DESOII) or prfA (DES012) showed a to-fold reduction in starvation-survival compared to EGD after 20 days of glucose limitation. DESOl1 had reduced exponential phase acid stress resistance, but increased H202 resistance. Resistance to H20 2 in exponential phase and long-term starved DES012 cells was over 290- fold and 380-fold greater than in EGD (after 20 minutes and 50 minutes exposure respectively), whilst exponential- and post-exponential-phase acid resistance in the DESOl2 was at least 10-fold greater than in EGD. Both DESOII and DESOl2 also exhibited altered catalase expression. Four transposon insertion mutants (two pairs of siblings) defective in starvation-survival were isolated from a glucose-limitation screen. Both sets of mutations resulted in decreased starvation-survival and altered stress resistance properties. Characterisation of the transposon insertion sites in DES028 and DES029 revealed disruption of a putative ORF encoding for a homologue of YuIB, a DeoR-family transcriptional regulator from Bacillus subtilis. In the isolates DES035 and DES045, the transposon insertion was found to disrupt a putative ORF encoding for a homologue of PhaQ, a protein associated with inclusion bodies of the storage polymer polyhydroxyalkanoic acid in Bacillus megaterium. The roles of these two loci in the starvation-survival response and in stress resistance are discussed.

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