Listeria monocytogenes (L. monocytogenes) are Gram-positive, facultatively anaerobic and intracellular bacilli, occupying a wide range of ecological niches and are responsible for a number of serious infections in man. Primarily transmitted to humans through contaminated food stocks, L. monocytogenes invade mammalian cells in a phagosome, escaping and growing in the cell cytoplasm. Currently, there is a great deal of information about pathogenesis of L. monocytogenes, however, much less is known about the physiology of the bacteria. In particular, very little is known about the physiology during intracellular growth and even less about host cell physiology and changes in response to infection. The focus of this research was to address these issues using a multidisciplinary approach, utilising multiple biological techniques. The catabolic metabolism of L.monocytogenes was elucidated using mutagenesis and protein purification studies. The results are not completely conclusive; however, it was shown that unlike in Escherichia coli, L.moncytogenes may not be dependent on fermentation enzymes Ldh and Pflb during anaerobic growth. Instead anaerobic respiration is hypothesised, utilising a putative fumarate reductase with fumarate as a terminal electron acceptor. The putative fumarate reductase gene was purified and confirmed to have enzymatic activity.External and internal metabolism of HeLa cells, and the effect of L.monocytogenes infection was elucidated by mass spectrometry. The external metabolomic studies proved inconclusive. The internal metabolomic studies show that a number of key amino acids are being sequestered by L.monocytogenes during the course of an infection. Also, the studies show that a large number of carbon compounds are being sequestered by L.monocytogenes, pointing to a complex carbon metabolism for L.monocytogenes during intracellular growth. A targeted analysis of the nitrogen metabolism of L.monocytogenes has shown that L.monocytogenes may utilise a number of nitrogen compounds with glutamine and glutamate being particularly important. The ability to synthesise glutamine de novo is shown to be essential for normal intracellular growth.

The first aim of this study was to determine the effect of irrigation intervals on the survival of L. monocytogenes on spray irrigated broccoli under field trial conditions, and subsequent survival of the pathogen on broccoli during postharvest processing procedures. The nonpathogenic L. innocua was used as surrogate organism to L. monocytogenes. Broccoli in the field was treated with irrigation water inoculated with L. innocua, during intervals over a period of five weeks and the growth and survival of the organism was monitored weekly. L. innocua numbers remained similar over intervals that received consecutive inoculations and L. innocua numbers decreased by at least 2.3 log cfu/g after inoculation ceased, which showed an inoculation effect and that time had an influence on organism survival. Cessation of irrigation before harvest was found to effectively reduce pathogen contamination levels on the crop, whilst repeated irrigation with contaminated water contributes to maintenance of L. innocua as well as elevated total microbial counts on the broccoli. A lack of correlation between the L. innocua counts and the recorded environmental temperatures in the field, including temperature and relative humidity, suggested that survival is not solely dependent on and influenced by, nor can it be predicted by these parameters. It was found that the presence of high levels of contamination (with, in this case L. innocua) in irrigation water used for vegetable crops, can be associated with an increased microbial population on the crop surface. Secondly, the effect of processing on organism survival post-harvest was assessed. Washing with water caused a 1 log reduction of L. innocua, whilst washing with 200 ppm chlorinated water facilitated a further 1 log reduction. Cooking reduced L. innocua numbers on broccoli by an average of 1.1 log units and aerobic plate counts by between 1 and 2 log units. A combined treatment of washing with chlorine, storage in MAP (5% CO2, 5% O2) for two days at 4°C and final microwave heating resulted in the lowest pathogen numbers, causing a 5.13 log cfu/g log reduction. Therefore, even though chlorine isprocessing, it does not suffice alone to eliminate pathogens (with L. innocua being representative of L. monocytogenes) from vegetables, just as MAP storage is only effective as part of a hurdle procedure. Cooking is essential in destroying L. innocua present on broccoli and to ensure vegetables that are safe for consumption in terms of pathogenic exposure. With this knowledge on the behaviour of L. monocytogenes on broccoli, the risk associated with the application of contaminated irrigation water to fresh produce can be better understood and the hazard managed. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Food Science / unrestricted

Dispersion is a natural part of a biofilm life cycle in many bacterial species. Dispersion occurs when bacteria revert from a stationary, sessile state to a free-swimming, planktonic state and are freed from a biofilm. Bacterial biofilms consist of proteins, polysaccharides, and extracellular DNA that together make up the extracellular polymeric substances. Surrounded by this mucus-like substance, sessile cells can be extremely difficult to eradicate as compared to the planktonic form of Listeria monocytogenes. Biofilms are robust due to increased surface adherence, inhibition of diffusion of harmful compounds, and increased genetic diversity that exists within a biofilm. As a result, traditional biofilm removal methods are often inadequate; and a novel method for the eradication of Listeria monocytogenes biofilms is needed. Here it is shown that two known biofilm dispersal agents, nitric oxide and cis-2-Decenoic acid, do not induce dispersion in Listeria monocytogenes strain LM23. Nitric oxide and cis-2-Decenoic acid do not influence planktonic cell numbers or biofilm biomass. Ten carbohydrates were screened for their influence on biofilm biomass for use in investigation into natural biofilm dispersion in Listeria monocytogenes strain LM23. Carbohydrate source can significantly increase or decrease biofilm biomass as compared to glucose. Natural biofilm dispersion in Listeria monocytogenes remains inconclusive, yet warrants further investigation. Changes in planktonic cells numbers, sessile cell numbers, and biofilm biomass were tracked under static growth conditions, and suggested a possible dispersion event. However, treatment of biofilms with spent media and observation using scanning electron microscopy did not clarify the results obtained. This research deems the nitric oxide donors, molsidomine (N- (ethoxycarbonyl)-3-(4-morpholinyl)-sydnone imine) and MAHMA NONOate (6-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine), as well as cis-2-Decenoic acid as ineffective in inducing biofilm dispersion. It also brings about new research questions into natural biofilm dispersion in Listeria monocytogenes.

The objective of this study was to evaluate the efficacy of buffered vinegar in a marinade solution on inhibiting Listeria monocytogenes growth on cooked broiler breast meat. Broiler breasts were vacuum-tumbled for 30 min in a marinade consisting of dry (0%, 0.4% DV, 0.6%DV, and 0.8%) or liquid vinegar (1.5%), sodium chloride, sodium tripolyphospate, and water. The chicken breasts were then cooked to an internal temperature of 75°C. The breast meat was inoculated with L. monocytogenes, placed into modified atmosphere packaging, and stored at 2°C plus/minus 2 for 0-60 days. L. monocytogenes growth was stable on treatments for up to 30 days. However, from 35 to 60 days, the buffered vinegar treatments had fewer L. monocytogenes counts (P<0.05) than the control treatment. In addition, the 0.8 % DV and 1.5 % LV treatments had fewer than 2.0 log counts of L. monocytogenes after 60 days of storage.

Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis. The infectious process depends upon survival in high bile salt conditions encountered throughout the gastrointestinal tract, including the gallbladder. However, it is not clear how bile salt resistance mechanisms are induced, especially under physiologically relevant conditions. This study sought to determine how L. monocytogenes responds to bile salts under anaerobic conditions. The study found resistance to be strain specific and not dependent upon virulence. Changes in the expressed proteome were analyzed using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. A general response among virulent and avirulent strains found significant alterations in intensity of cell wall associated proteins, DNA repair proteins, protein folding chaperones and oxidative response proteins. Strain viability was correlated with an initial osmotic stress response followed by strain specific proteins associated with biofilm formation in EGDe and a transmembrane efflux pump in F2365.

The purpose of this study was to induce an oxidative-stress adaptation in Listeria monocytogenes Bug600 (serotype 1/2a) and F1057 (serotype 4b) by pre-exposing to sublethal H2O2 and alkali-stress either singly or sequentially. Our findings show that the sequential pre-exposure of cells to pH 9 for 30 min treatment followed by 50 ppm H2O2 for 30 min at 37°C yielded the highest oxidative-stress resistant phenotypes of L. monocytogenes Bug600 and F1057. The sublethal H2O2 and sublethal alkali-stress induced oxidative-stress adaptations were completely reversible within 60 min at 37°C in the absence of such sublethal stress. However, the oxidative-stress adaptation induced at 37°C was stable at 4°C over a 24 h test period in both L. monocytogenes Bug600 and F1057. Future studies will focus on the potential cross-resistance of oxidative-stress adapted L. monocytogenes serotypes 1/2a and 4b to commonly used disinfectants and GRAS antimicrobials.

No description available.

Stormwater runoff is a major environmental concern, particularly in urban environments. Trends in managing stormwater have evolved (and continue to evolve) from a quantity only approach into a sustainable approach, which integrates quantity, quality, the environment, and aesthetics. Best management practices (BMPs) and Low Impact Development (LID) are two well-documented techniques capable of managing to sustainable standards. There are a number of stormwater models available to design professionals today. However, there are few which integrate site-scale BMP/LID analysis in a simplified fashion. The purpose of this study is to determine if there is a demand in the design profession for simplified stormwater modeling tools to help designers make informed decisions about integrating BMP/LID strategies into site plans. A Web-based questionnaire was administered to a group of design professionals to determine their knowledge of BMPs and their technological needs and preferences in meeting stormwater goals and requirements.

Listeria monocytogenes is a deadly, Gram-positive foodborne pathogen that is ubiquitous in the environment. The bacterium expresses a number of virulence and stress adaptation proteins that support its pathogenic capabilities. Two-dimensional gel electrophoresis (2-DE) was used to map L. monocytogenes surface proteins, which play a central role in virulence, and to examine protein expression by L. monocytogenes grown on ready-to-eat meat, an important source of Listeria infections. A novel method for solubilization of surface proteins from L. monocytogenes for 2-DE was developed. Additionally, the unique proteome expressed by L. monocytogenes grown on a meat matrix was uncovered. The developed solubilization method will facilitate efforts to identify and routinely compare surface proteins of Listeria by 2-DE. Furthermore, the 2-DE database of proteins expressed by L. monocytogenes grown on a meat matrix will allow further understanding of the interactions of Listeria with its food environment that influence its ability to cause disease.

Post process application of antimicrobials is one method to control recontamination of Listeria monocytogenes on cooked RTE meat products. This study evaluated the antilisterial effect of externally applied solutions of nisin, buffered solution of sodium citrate supplemented with sodium diacetate (SCSD) and a combined solution of the two antimicrobials on commercial beef frankfurters formulated without antimicrobials. Autoclaved frankfurters were inoculated (104 -105 CFU/g), dipped (5 min, 25 ± 2°C) in treatments consisting of 2000; 4000;or 6000 IU/ml nisin, and buffered solutions of 2.5; 3.0; or 3.5% SCSD (Study I), and 6000 IU/ml nisin followed by 3.5% SCSD, or 3.5% SCSD followed by 6000 IU/ml nisin, or a combined solution containing both 6000 IU/ml nisin and 3.5% SCSD(Study II). Treated hot dogs were vacuum packaged and stored at 4 and 10°C. L. monocytogenes counts were determined on Modified Oxford Agar on 0, 14, 28, and 42 days at 4°C and 0, 4, 8, 12, 16, and 20 days at 10°C. Nisin (6000 IU/ml) initially reduced L. monocytogenes population by 2.1 and 2.5 logs (at 4 and 10°C, respectively on day 0), buffered SCSD (3.5%) by 1.1 and 0.2 logs (at 4 and 10°C, respectively on day 0) and the combined solution by 1.7 and 2.0 logs (at 4 and 10°C, respectively on day 0). The combined solution was the most effective treatment compared to nisin and buffered SCSD, used in sequence or alone, since it inhibited the growth of L. monocytogenes during 28 days at 4°C. The buffered SCSD was effective against L. monocytogenes at 4°C but not at 10°C. Further research is needed to investigate the effectiveness of buffered SCSD with other antimicrobials. The results of this study may be useful for further research on combinations of antimicrobials.