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.

Section 1: Construction and screening of a genomic library for the mosquito Aedes aegypti (Diptera, culicidae). A genomic library has been constructed for this important vector of arboviral disease. Total genomic DNA and various classes of RNA from Ae. aegypti were used to screen this library. The results obtained indicate that this species has a short period interspersion pattern of repeated sequences. Transcription of these repeats could not be detected using total cytoplasmic RNA, hnRNA or mRNA as hybridisation probes. Section 2: Sequence organisation of ribosomal DNA in Aedes aegypti. The Aedes aegypti genomic library was used to isolate clones containing the intact ribosomal DNA (rDNA) repeat of this species. This has been restriction mapped and the transcribed regions have been identified. The rDNA repeat is 9.0 Kb in length and is present as approximately 500 head-to-tail tandemly repeated copies. A low level of intraspecies polymorphism of Ae. aegypti rDNA is evident. Two restriction polymorphisms have been identified within the rDNA repeat. Section 3: Analysis of ribosomal DNA variation within Ae. aegypti'and between closely related species. Four variant rDNA clones have been isolated. One of these' may contain the end of a tandem array of ribosomal genes. Another variant contains a duplication of rDNA within the internal transcribed spacer region of the ribosomal repeat. Sequence analysis of this clone has identified regions at the 3' end of the 18S rRNA gene of Ae. aegypti which show very strong homology with the corresponding regions in other species. Some repeated sequences have been identified downstream of the 18S rRNA gene in this clone. Preliminary analysis of the two other rDNA variants indicates that one contains a duplication or insertion of DNA in the 28S rRNA coding region and one contains non-transcribed spacer homologous sequences which are not associated with rRNA coding regions. Section 4: DNA probes for species identification of mosquitoes in the Anopheles gambiae complex. DNA sequences have been isolated which distinguish four of the morphologically identical members of the An. gambiae species complex. Two sequence classes were obtained. Class 1 homologues are highly reiterated in the genomes of An. arabiensis and An. merus, present in low copy number in An. melas and were not detected in An. gambiae s. s. Class 1 sequences are male specific in An. arabiensis. Class 2 homologues are highly reiterated in the genomes of An. merus and An. melas and present in low to middle copy number in An. gambiae s. s, and An. arabiensis. Sex specificity of Class 2 homologues does not occur in the species tested (An. gambiae s. s. and An. arabiensis). Hybridisation of these species specific DNA sequences to mosquitoes squashed directly onto nitrocellulose provides a simplified method of species identification.

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The flavivirus genus is of major concern for world morbidity and mortality and includes viruses causing both encephalitic as well as hemorrhagic diseases. The incidence of Tick-borne encephalitis is increasing in many European countries and several reports have emphasized the expansion of the main vector, Ixodes ricinus. The pattern of vector distribution is also changing in Sweden, which makes it important to set up solid and successful strategies for detection and genetic characterization of novel Swedish TBEV strains. In this study we have generated strategies for detection of broad types of tick-borne flaviviruses in pools of I. ricinus sampled in Sweden. The positive collection on the island of Torö was used to generate a sequence of a complete TBEV genome straight from the arthropod reservoir. This cloned virus was used to construct a self-replicating DNA based sub-genomic TBEV replicon capable of expressing reporter genes. The replicon was used to study the effect of TBEV on neurite outgrowth, which revealed that the MTase domain of NS5 block the formation of the Scribble/Rac1/βPIX protein complex, impairing neurite outgrowth in neuronal growth factor induced PC12 cells. We also demonstrate that TBEV replication is affected by two PDZ binding motifs within NS5 and reveal putative PDZ binding proteins. These interactions might affect cellular pathways and might have a role in flavivirus replication. We also characterize the variable 3´ non-coding region (V3’-NCR) by in silico studies on TBEV. Analysis brings new evidence that V3’-NCR region carries an enhancer element important for different replication/translation dynamics during the viral lifecycle in mammalian and tick cells. We also propose a temperature-sensitive trans-acting riboswitch mechanism; altering the secondary RNA structures of a closed form at lower temperatures and a form open for translation at higher temperatures. This mechanism may explain the low TBEV level observed in sampled ticks. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

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Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1967. / Typescript. Includes bibliographical references.