The persistance of Escherichia coli in the larval, pupal and adult stages of both house flies, Musca domestica (L.), and stable flies, Stomoxys calcitrans (L). was examined. Abundance of E.coli declined over time in immature house flies, but remained constant in immature stable flies, suggesting house fly larvae digest E. coli but stable fly larvae do not. Survival of house fly and stable fly larvae averaged 62% and 25% respectively when reared on pure E. coli cultures. E. coli load in pupae decreased significantly one day before emergence of adult house flies, but remained constant until stable fly emergence. Nevertheless, E. coli was detected in 78% of emerging house flies and in 28% of emerging stable flies. House flies are more important E. coli vectors as adults, whereas stable flies may be overlooked vectors of E. coli during immature development. / ix, 89 leaves ; 28 cm.

Escherichia coli O157:H7 is an important human pathogen that resides primarily in cattle and feedlot environments. E. coli O157:H7 can be divided into phylogenetic groups termed lineages; lineage I strains are responsible for most human illnesses. An understanding of the etiology of these lineages within cattle and the feedlot environment could allow for more effective surveillance and mitigation strategies. There were no lineage associated differences in growth or survival of E. coli O157:H7 in bovine feces at 4°C, 12°C or 25°C. Lineage I strains more readily colonized cattle jejunum tissue and a bovine colonic cell line than lineage II and intermediate type strains. Enhanced colonization of cattle by lineage I strains may increase the persistence of these strains in feedlots via re-infection and increased shedding. This outcome could increase the transmission of lineage I strains to the food supply and increase the potential for these strains to cause human illness. / xiii, 101 leaves ; 29 cm

For pathogenic microorganisms, heme uptake and degradation is a critical mechanism for iron acquisition that enables multiplication and survival within hosts they invade. While the bacterial proteins involved in heme transport had been identified at the initiation of our investigation, the fate of heme once it reached the cytoplasm was largely uncharacterized. Here we report the first crystal structures of two members of the heme utilization operon from the human pathogen Escherichia coli O157:H7. These are the heme oxygenase ChuS in its apo and heme-complexed forms, and the apo form of heme binding protein ChuX. Surprisingly, despite minimal sequence similarity between the N- and C-terminal halves, the structure of ChuS is a structural repeat. Furthermore, the ChuS monomer forms a topology that is similar to the homodimeric structure of ChuX. Based on spectral analysis and carbon monoxide measurement by gas chromatography, we demonstrated that ChuS is a heme oxygenase, the first to be identified in any E. coli strain. We also show that ChuS coordinates heme in a unique fashion relative to other heme oxygenases, potentially contributing to its enhanced activity. As ChuS and ChuX share structural homology, we extended the structural insight gained in our analysis of ChuS to purport a hypothesis of heme binding for ChuX. Furthermore, we demonstrated that ChuX may serve to modulate cytoplasmic stores of heme by binding heme and transferring it to other hemoproteins such as ChuS. Based on sequence and structural comparisons, we designed a number of site-directed mutations in ChuS and ChuX to probe heme binding sites and mechanisms in each. ChuS and ChuX mutants were analyzed through reconstitution experiments with heme and functional analyses, including enzyme catalysis by ChuS and mutants, and in culture development during heme challenge experiments by ChuX and mutants. Taken together, our results suggested that ChuX acts upstream of ChuS, and regulates heme uptake through ChuX-mediated heme binding and release. ChuS can degrade heme as a potential iron source or antioxidant, thereby contributing directly to E. coli O157:H7 pathogenesis. Functional implications that may be revealed from sequence and structure based information will be addressed as they pertained to our evaluation of ChuS and ChuX. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2007-04-27 11:34:50.272

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Refrigeration has been found to cause cell elongation in mesophilic enteric organisms like commensal Escherichia coli and E. coli O157:H7. As elongated cells may divide into multiple daughter cells, they may result in underestimation of pathogen numbers in foods when plate counts are used. When E. coli cultures were incubated at 6°C for ≤10 days, cells grew by elongation, did not divide, and lost viability (LIVE/DEAD vitality stain) at similar rates. Substantial fractions of cells in cultures elongating at 6°C were inactivated by an abrupt shift to 37°C. Direct microscopic observation of cells transferred to 37ºC after 5 days at 6°C showed that few or no cells of normal size (≤4µm) divided, while elongated cells (>4 µm) formed multiple daughter cells. Thus the threat from mesophilic pathogens with a low infective dose may be underestimated in refrigerated foods. It was also found that E. coli O157:H7 cultures containing elongated cells prepared at 6 or 15 °C have greater potential to attach to food contact surfaces than those grown at higher temperatures. Interestingly, at 6°C cell elongation was inhibited by ≥ 100 mg/l cinnamaldehyde and ≥ 200 mg/l cinnamaldehyde was lethal. In contrast, at 37°C 200 mg/l cinnamaldehyde initially delayed multiplication of E. coli cells by causing cell elongation, but from 2 to 4 h, growth resumed and cells reverted to normal length. To understand this transient behaviour, genome-wide transcriptional analysis of E. coli O157:H7 was performed at 2 and 4 h exposure to cinnamaldehyde in conjunction with reverse phase-high performance liquid chromatography analysis for cinnamaldehyde and other cinnamic compounds. At 2 h exposure, cinnamaldehyde induced expression of many oxidative stress-related genes, reduced expression of genes involved in DNA replication, synthesis of protein, O-antigen and fimbriae. At 4 h many repressive effects of cinnamaldehyde on E. coli O157:H7 gene expression were reversed. Data indicated that by 4 h, E. coli O157:H7 was able to convert cinnamaldehyde into the less toxic cinnamic alcohol using alcohol dehydrogenase or aldehyde reductase enzymes (YqhD and DkgA). The results also showed that the antimicrobial activity of cinnamaldehyde was likely attributable to its carbonyl aldehyde group.

E. coli O157:H7 has been found to survive in dry sausages and cause disease. Isothiocyanates have been studied for their capacity to eliminate pathogens from foods and are attractive from the consumer perspective because of their natural origin. There is a need to better understand how isothiocyanates kill microorganisms and their behaviour in food matrices. It was found that glutathione and cysteine naturally present in meat can react with AIT, forming a conjugate with no or low bactericidal activity against an E. coli O157:H7. In addition, AIT presented higher anti-E. coli activity at lower pH values; therefore, it should be more efficient in acid foods. AIT was also found to inhibit the activity of thioredoxin reductase and acetate kinase; hence, enzymatic inhibition may represent a way in which AIT kills E. coli O157:H7. Mustard powder is used as a spice (active myrosinase) and/or binder (inactive myrosinase) in meat products. Both of these powders killed E. coli O157:H7 in dry fermented sausage. This was not expected since the powder lacking myrosinase is not able to produce isothiocyanates. Starter cultures and E. coli were found to consume significant amounts of glucosinolates. Pediococcus pentosaceus UM 121P and Staphylococcus carnosus UM 123M (higher myrosinase-like activity) were compared against P. pentosaceus UM 116P + S. carnosus UM 109M for their ability in reducing E. coli viability in dry sausage. Sausage batches containing powders of hot mustard, cold mustard, autoclaved mustard and no powder were prepared. Both pairs of starters yielded similar results. Reduction >5 log CFU/g of E. coli O157:H7 occurred after 31 d for hot powder and 38 d for cold powder; there was no reduction in the control. E. coli O157:H7 itself has greater effect on glucosinolate degradation than either pair of starters, which may be more important in determining its survival. Autoclaved powder caused >5 log CFU/g reduction after 18 d. This may be the result of synergistic/additive interaction among E. coli O157:H7 myrosinase-like activity, the presence of newly formed/released antimicrobials in the autoclaved powder and the multiple hurdles present in the dry sausage. Autoclaved mustard powder has potential as a novel food ingredient for the meat industry.

Escherichia (E.) coli O157:H7 survival in dry cured (uncooked) meat products leading to human illness outbreaks is an international problem. Their manufacture does not involve a heat kill step to ensure the destruction of the organism, and the adverse conditions created during processing may not be sufficient to prevent E. coli O157:H7 survival. Deodorized yellow mustard powder has antimicrobial properties from glucosinolate (sinalbin) hydrolysis catalyzed by the endogenous enzyme myrosinase, generating antimicrobial isothiocyanate (PHBIT). Previous work has shown that its addition during dry sausage manufacture was capable of eliminating the pathogen. In this study, its use for the same purpose was investigated during dry cured Westphalian ham production. Hams were inoculated with a 7.5 log cfu•g-1 cocktail of E. coli O157:H7, surface applied with 4% or 6% (w/w) deodorized yellow mustard powder, and monitored for E. coli O157:H7 survival during 80d ham maturation. One trial included the inoculation of Staphylococcus (S.) carnosus, a meat starter culture with myrosinase-like activity, onto the hams (after salt equilibration) to accelerate formation of antimicrobial isothiocyanate from mustard glucosinolate and help control the pathogen. In both trials, E. coli O157:H7 was reduced 3 log cfu•g-1 by 21d on hams treated with mustard powder, whereas only a 1 log cfu•g-1 reduction was found in the inoculated control which was not treated with mustard. By 45d, hams treated with mustard powder showed a reduction of >5 log cfu•g-1 E. coli O157:H7, whereas it took 80d to for numbers in control hams to be similarly reduced. Since a 5 log kill of E. coli O157:H7 was achieved in control hams by the end of 80d, dry cured Westphalian ham manufacture would be considered capable of controlling the risk of E. coli O157:H7 survival by North American regulatory agencies. However, deodorized yellow mustard powder at 4%, and to a greater extent at 6%, eliminated the pathogen at a significantly faster rate than the control during ham processing. Addition of the S. carnosus starter culture in trial 2 may have contributed to the maintenance of this effect through isothiocyanate formation. It also helped restore numbers of staphylococci, which were found to be sensitive to deodorized mustard powder.

The prevalence and antibiotic resistant profiles of shiga-toxin producing Escherichia coli 0157 strains isolated from faeces samples of cattle, pigs and human stool samples were determined. The strains were further characterised by molecular methods for the presence of shiga-toxin virulence genes and antibiotic resistant genes. Seventy-six Escherichia coli 0157 strains were isolated and the prevalence was higher among E. coli isolated from faeces from pigs (44.2% to 50%) than those from cattle faeces (5.4% to 20.0%) or human stool samples (7 .5%). On testing E. coli 0157 isolates for their resistance to 9 antimicrobial agents, multiple antibiotic resistance (MAR) was observed in all of the isolates arising from resistance to three or more antibiotics. Seventy (92.1 %) of the E. coli 0157 isolated from humans, cattle and pigs were resistant to tetracycline. 73 (96.1 %) were resistant to sulphamethoxazole, 63 (82.9%) were resistant to erythromycin. 40 (52.6%) were resistant to streptomycin and 26 (34.2%) were resistant to ampicillin. The highest frequency of resistance was observed among the human isolates (n=3 ), where 3 (I 00%) of the isolates were resistant to tetracycline, sulphamethoxazole, erythromycin and ampicillin. Furthermore, among the pig isolates (n=60), 58 (96. 7%) were resistant to tetracycline, 57 (95%) were resistant to sulphamethoxazole, 47 (78.3%) were resistant to erythromycin. 38 (63.3%) were resistant to streptomycin and 22 (36. 7%) were resistant to ampicillin. The MAR phenotypes S-Smx-T-E, Smx-T-Ap and Smx-T-E were the dorminant phenotypes among the E. coli 0157 isolated from the faeces samples of communal pigs in 30.4%, 21 .7% and 17.4% of these isolates, respectively. However, phenotypes Smx-T -E and S-Smx-T-E-Ne were identified at I6.2% and 10.8%, respectively within the isolates obtained from commercial pig faeces. The phenotype Smx-T-E was the only MAR phenotype identified among the E. coli 0157 isolated from the faecal samples of commercial cattle at Lichtenburg. Furthermore, MAR phenotypes Smx-T-E-C, K-S-Smx-T-E, S-Smx-T-E and Smx-T-E-Ap were obtained at 25%, respectively for the isolates obtained from communal cattle at Mogosane while Smx-T-E-Ap was the dorminant (66.7%) phenotype among the isolates of human origin. The phenotype Smx-T fom1ed the basis of all the MAR phenotypes obtained and this was similar to the percentage antibiotic resistance data. The distribution of the resistant determinants for tetracycline was determined by PCR analysis in resistant isolates. A tetB gene was detected in E. coli 0157 of pig origin. Based on the characterisation of 30 isolates for the presence of STEC virulence genes by PCR, 18 (60%) possessed the hlyA gene, 7 (23.7%) possessed the eae gene and 5 ( 16. 7%,) harboured both genes. The average MAR indices for pig, cattle and human E. coli 0157 isolates were 0.4n2, 0.3419 and 0.4814, respectively. Among the cattle isolates, the group MAR index was highest for the communal (Mogosane) population while the values for the commercial populations at Lichtenburg and Rustenburg were 0.33 and 0.22, respectively. £. coli 0157 isolated from pigs revealed MAR index results that were 0.508 and 0.415 for the commercial and communal populations respectively and 0.1851 for the E. coli control strains. Characterisation by cluster analysis to determine the commonness and resolve differences between the E. coli 0157 isolated from the Various sources revealed a close association between pig (Tlapeng and Mareetsane), cattle (Mogosane) and human isolates. Interestingly, E. coli 0157 isolated from pigs occurred at the highest frequency in all the clusters. which suggested their role in the dissemination of resistant determinants. / MSc. (Agric.) North-West University, Mafikeng Campus, 2006

Refrigeration has been found to cause cell elongation in mesophilic enteric organisms like commensal Escherichia coli and E. coli O157:H7. As elongated cells may divide into multiple daughter cells, they may result in underestimation of pathogen numbers in foods when plate counts are used. When E. coli cultures were incubated at 6°C for ≤10 days, cells grew by elongation, did not divide, and lost viability (LIVE/DEAD vitality stain) at similar rates. Substantial fractions of cells in cultures elongating at 6°C were inactivated by an abrupt shift to 37°C. Direct microscopic observation of cells transferred to 37ºC after 5 days at 6°C showed that few or no cells of normal size (≤4µm) divided, while elongated cells (>4 µm) formed multiple daughter cells. Thus the threat from mesophilic pathogens with a low infective dose may be underestimated in refrigerated foods. It was also found that E. coli O157:H7 cultures containing elongated cells prepared at 6 or 15 °C have greater potential to attach to food contact surfaces than those grown at higher temperatures. Interestingly, at 6°C cell elongation was inhibited by ≥ 100 mg/l cinnamaldehyde and ≥ 200 mg/l cinnamaldehyde was lethal. In contrast, at 37°C 200 mg/l cinnamaldehyde initially delayed multiplication of E. coli cells by causing cell elongation, but from 2 to 4 h, growth resumed and cells reverted to normal length. To understand this transient behaviour, genome-wide transcriptional analysis of E. coli O157:H7 was performed at 2 and 4 h exposure to cinnamaldehyde in conjunction with reverse phase-high performance liquid chromatography analysis for cinnamaldehyde and other cinnamic compounds. At 2 h exposure, cinnamaldehyde induced expression of many oxidative stress-related genes, reduced expression of genes involved in DNA replication, synthesis of protein, O-antigen and fimbriae. At 4 h many repressive effects of cinnamaldehyde on E. coli O157:H7 gene expression were reversed. Data indicated that by 4 h, E. coli O157:H7 was able to convert cinnamaldehyde into the less toxic cinnamic alcohol using alcohol dehydrogenase or aldehyde reductase enzymes (YqhD and DkgA). The results also showed that the antimicrobial activity of cinnamaldehyde was likely attributable to its carbonyl aldehyde group.

Yellow and oriental mustards deodorized by a laboratory autoclave method have been shown to reduce the number of E. coli O157:H7 greater than the mandatory 5 log CFU/g during sausage manufacture. However, E. coli O157:H7 was inconsistently controlled by different deodorized mustards. The antimicrobial action of mustard results from the conversion of naturally present glucosinolates into inhibitory isothiocyanates by plant myrosinase in untreated hot mustard and by bacterial myrosinase-like activity when present in thermally-treated (deodorized) mustard. Variable results with deodorized mustards suggested that plant myrosinase might not have been completely inactivated during laboratory thermal treatment using the autoclave. Results obtained showed that when a 2 cm thick layer of mustard was used during autoclave treatment, plant myrosinase activity periodically remained. However, the completely deodorized mustard failed to reduce bacterial viability as effectively as yellow mustard containing residual or slight amount of myrosinase. As a result, a small amount of myrosinase activity was highly likely contribute to the overall antimicrobial activity of deodorized mustard against E. coli O157:H7 in dry sausage.