Study of food borne pathogens in poultry

Campylobacter and Salmonella are the two main causative agents of bacterial foodborne disease in humans and are a significant public health problem worldwide. In the poultry industry, broilers frequently become colonized by Campylobacter species. As a consequence, Campylobacter can enter the poultry meat supply chain by posing a significant risk to human health. Several “generally recognized as safe” (GRAS) chemicals, such as chlorine and acidified sodium chlorite are used in processing plants to mitigate the load of Campylobacter on point-of-use chicken meat. However, contamination of chicken meat in the food chain remains a significant public health issue and its control is critical for the poultry industry. My research has investigated the physiological, morphological, and cellular responses of Campylobacter jejuni following exposure to chicken meat sanitizers and how this affects bacterial virulence potential. In many countries, chlorine is commonly used as a biocide in processing plants to reduce bacterial loads. In Chapter 2, the effectiveness of chlorine on the inactivation of C. jejuni was investigated. The results revealed the majority of the isolates exhibited minimum inhibitory concentration (MIC) and minimum biocidal concentration (MBC) values of 128 ppm for chlorine, which is higher than the concentration (8ppm) recommended for use in the Australian poultry industry. Experiments performed in Chapter 2 also demonstrated that the efficacy of chlorine is significantly influenced by organic matter as well as bacterial load. The morphological effect of exposure to chlorine was also investigated using scanning and transmission electron microscopy. C. jejuni exposed to chlorine showed changes in shape (coccoid, or elongated), cellular degeneration, and shriveled bacterial cells. The present study suggests that, although chlorine is effective in reducing C. jejuni contamination, the current protocol used in the Australian poultry industry is unable to eliminate completely. Acidified sodium chlorite (ASC) has been previously shown to have greater bacteriocidal capacity compared with chlorine and is most commonly recommended for post-spin chill applications. In Chapter 3, the cellular response of C. jejuni following exposure to ASC was studied. The data showed that 7.03 ppm of ASC was sufficient to inactivate C. jejuni. Interestingly, the effects of ASC were independent of organic matter presence and bacterial load. C. jejuni exposed to ASC could not be resuscitated indicating that this sanitizer induced irreversible cellular damage. Analysis of the C. jejuni transcriptome post-exposure to sanitizer revealed that chlorine induced an adaptive stress response mechanism, which aids in survivability under oxidative stress. ASC, however, induced higher oxidative damage by inhibiting all vital metabolic pathways resulting in cellular death. This study suggests that ASC would be a better alternative to chlorine in reducing C. jejuni contamination in chicken meat. The presence of sub-lethally injured food-borne pathogens such as C. jejuni and Salmonella Typhimurium in the chicken meat juice of thawed or raw poultry packages is a significant risk. In Chapter 4, the survivability, invasion potential, and virulence gene expression of C. jejuni and S. Typhimurium following exposure to chlorine and acidified sodium chlorite (ASC) during storage in chicken meat juice (CMJ) were investigated. The results revealed that CMJ facilitated the survival of both C. jejuni and S. Typhimurium following exposure to chlorine but not ASC. Both chlorine and ASC reduced bacterial invasiveness, motility, and culturability of C. jejuni but not S. Typhimurium. The gene expression data revealed the upregulation of bacterial stress response and virulence genes in C. jejuni (ropB, sobB, flaG, flaA, cadF, racR) and S. Typhimurium (rpoH, rpoS, hilA, fimH, spvR, avrA) over storage time, indicating an increase in virulence potential. Moreover, the data suggested that sub-lethally injured C. jejuni and S. Typhimurium in CMJ remain a significant risk in the food chain due to the likelihood of cross-contamination while handling chicken meat. The understanding of the in-vivo virulence potential sub lethally injured C. jejuni is crucial in evaluating public health risks. In Chapter 5, the virulence of sub-lethally injured C. jejuni during prolonged storage in chicken meat juice in C57BL/6 mice was evaluated. The results revealed that tissue colonization and invasion of C. jejuni were significantly reduced following exposure to ASC. The reduced expression levels of inflammatory cytokine genes, (IL6, IL23, and IL10), Toll-like receptor genes (TLR2, and TLR4), and host stress response genes (CRP, MBL, and NF-B) in mice infected with C. jejuni exposed to chlorine or ASC suggested the reduced virulence potential of sublethally injured C. jejuni . Exposure to chlorine results in sublethal injury of C. jejuni and S. Typhimurium which allows for their persistence in the food supply chain. ASC, however, induces substantial bacterial cell damage which bacteria have limited or no ability to recover from which would contribute to reducing bacterial foodborne pathogens in the food supply chain. A re-evaluation of current Australian protocols is warranted to improve the safety of the chicken meat supply chain.Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 202

PREPARATION AND CHARACTERIZATION OF GLUCOSE NANOSENSORS FOR INTRACELLULAR APPLICATIONS

The dynamics of glucose-stimulated insulin secretion play a key role in normal physiological regulation, with altered temporal profiles observed in diabetic patients. Elucidation of the underlying mechanisms responsible for insulin secretion is key to understanding the development and progression of diabetes, though such studies have been limited due to a dearth of methods with sufficient sensitivity and spatiotemporal resolution to facilitate direct quantification of cellular glucose. In this work, we developed a cell-penetrating, glucose-responsive fluorescence indicator protein (FLIP) capable of detecting dynamic glucose changes in single pancreatic β-cells. Flow cytometry and confocal microscopy revealed that cell penetrating FLIP was directly loaded into single cells via simple incubation, avoiding the requirement for cellular transfection. The cell-penetrating FLIP yielded similar analytical performance in vitro compared to unmodified FLIP, and facilitated direct observation of cellular glucose levels, though with a lower in vivo response compared to FLIP-transfected cells. To minimize intracellular proteolytic degradation and/or compartmentalization that may contribute to the lowered sensor response, FLIP was encapsulated in stabilized, porous phospholipid nanoshells (PPNs). In vitro characterization showed that the FLIPPPN (K(d) = 854 ± 32 μM) yielded similar analytical performance to FLIP with < 1 min response time. FLIP-PPN was readily loaded into live cells upon modification of the PPN with TAT peptide. Intracellular delivery and localization of FLIP-PPN was confirmed using confocal microscopy. FLIP-PPNs exhibited ca. 50% more activity in the intracellular environment compared to non-encapsulated FLIP, suggesting the importance of encapsulated delivery. To obtain optimized glucose transport, the molecular weight cutoff of PPNs was examined using a novel dextran retention method with a mass resolution of 162 Da. Stabilized PPNs exhibited 90 % retention at MW ca. 1800. Kinetic control of permeability with binary lipid mixtures was investigated, and composition dependent permeability of PPNs was observed. Lipid domain formation was evident in PPNs prepared using binary lipid mixtures (30-100% bis-SorbPC), where the MW cutoff was found to be unchanged compared to 100% bis-SorbPC. Proton permeability studies of lipid mixtures below the resolution of the dextran retention assay (< 20% bis-SorbPC) confirmed composition dependent change of permeability

Earthquake protection of masonry shear walls using fibre reinforced polymer strengthening

Research Doctorate - Doctor of Philosophy (PhD)Unreinforced masonry (URM) buildings are highly vulnerable to damage during earthquakes, due to their high mass, limited ductility and low tensile strength. However, being economical, durable, easy to procure and good for thermal and sound insulation ensures that URM is widely used both for low-rise structural walls and for infill to framed structures. In addition to that, many of the existing historically and culturally important buildings have been identified as URM constructions. Therefore, strengthening of URM buildings to resist earthquake damage has a remarkable importance. URM shear wall strengthening with near surface mounted (NSM) fibre reinforced polymer (FRP) strips is a relatively new and effective seismic retrofitting technique to improve their earthquake resistance. This technique involves inserting thin FRP strips into grooves cut into the surface of the wall. The aesthetic impact to the structure is minimal due to this strengthening technique compared with attaching FRP reinforcement to the surface of the wall (External Bonding (EB) technique). The other advantages of NSM FRP are the ability to develop higher strains in the FRP before debonding compared to EB techniques, and protection from vandalism, to some extent from fire and other environmental influences. In this research study an extensive experimental study along with numerical analyses were carried out to investigate the cyclic in-plane shear behaviour of unreinforced masonry (URM) walls retrofitted/strengthened with near surface mounted (NSM) fibre reinforced polymer (FRP) strips. Carbon FRP (CFRP) strips were used in this technique and were designed to enhance the performance of URM walls which fail by diagonal cracking or bed joint sliding within the height of the wall. The bond-slip behaviour between NSM FRP strips and clay brick masonry was investigated using six experimental pull tests under cyclic loading. The results including bond strengths, critical bond length and the local bond-slip behaviour were determined and were compared with a similar monotonically loaded pull test results. The bond-slip curves for monotonic and cyclic loading cases were approximately similar. Two major experimental investigations were carried out in this project to investigate the effectiveness of retrofitting/strengthening of URM walls panels with NSM CFRP strips using previously damaged and newly constructed undamaged wall panels. The effectiveness of NSM CFRP strip retrofitting applied to damaged URM walls was investigated using sixteen previously damaged wall panels with two different damage levels (lightly and highly) subjected to vertical pre-compression combined with increasing reversing cycles of in-plane lateral displacement. The damaged walls were partially repaired, retrofitted with NSM FRP strips and retested. The study assessed the effect on strength, displacement capacity, energy dissipation and ductility achieved by FRP retrofitting compared to the undamaged URM panels under different pre-compression levels. The retrofitted walls displayed higher displacement capacities compared with URM walls. The ultimate loads were not enhanced due to retrofitting under higher pre-compression levels. However the presence of the reinforcement restored the ultimate loads to those observed for the original undamaged URM state. The improvements in the behaviour of the URM walls due to retrofitting were generally similar irrespective of the amount of pre-existing damage in the URM walls. A new test setup representing realistic boundary conditions to simulate the earthquake behaviour of shear walls in actual buildings was designed and built for the series of experiments with newly constructed wall panels. It was designed to impose zero in-plane rotation (fixed-fixed) boundary conditions at the top and bottom of the masonry wall specimens. A representative finite element (FE) model was used to obtain the actual dimensions of the test setup. The design parameters for the experimental series, including test specimen dimensions and pre-compression loads to achieve diagonal cracking failure modes, were obtained using the same FE model. A total of twenty three wall panels constructed with two wall aspect ratios (height : length = 1 and 0.5) were tested. They were strengthened with NSM CFRP strips in six different reinforcement arrangements including vertical, horizontal and a combination of both. Four panels were tested under monotonically increasing in-plane lateral displacement and the others under increasing reversing cycles of in-plane lateral displacement combined with a vertical pre-compression. The expected zero in-plane rotation (fixed-fixed) boundary conditions were achieved from the new setup with classic diagonal failure occurring through the test walls. The displacement capacity, energy dissipation and ductility of the wall panels were enhanced due to the NSM FRP strengthening. The maximum load of the strengthened walls was increased compared to URM when the strengthening contained vertical FRP strips. The reinforcing scheme which used a combination of vertical and horizontal FRP strips performed the best. A finite element model was developed to validate the experimental results. The micro-modelling approach was used in this masonry model. The FRP strips were attached to the masonry model using the bond-slip relationship established from the experimental pull tests. The key behaviours of the experimental test results could be reproduced by the developed FE model

Rhizobiophage Functional Genomics

Rhizobiophages are bacterial viruses that specifically infect nitrogen-fixing, legume-nodulating bacterial group rhizobia. In this study, the whole genome sequences of eight rhizobiophages were generated. These included four Rhizobium leguminosarum myophages (AF3, P9VFCI, RL2RES and RL38JI), two R. leguminosarum siphophages (P11VFA and B1VFA), one R. leguminosarum phage with unknown morphology (V1VFA-S) and one M. japonicum phage (Cp1R7A-A1). Characterization of Cp1R7A-A1 indicated a siphophage morphology with a prolate capsid, a distinct genome, and a close phylogenetic relationship to certain Caulobacter siphophages with similar morphology. The myophages AF3, P9VFCI, RL2RES and RL38JI belong in the ICTV family Ackermanviridae and in a T4-like group of viruses. A recombination dependent replication mechanism and circularly permuted genomes due to a pure headful packaging mechanism were postulated for these phages. B1VFA and V1VFA-S had genomes similar to each other. A theta replication mechanism and phage genome terimini with direct terminal repeats were suggested for these two. P11VFA was similar to the R. leguminosarum phage L338C genome available in databases. Quantitative reverse transcription PCR for selected genes of phages Cp1R7A-A1 and Lo5R7ANS, using RNA extracts from Mesorhizobium japonicum separately infected with these phages, confirmed an early-late type maximum gene expression in Lo5R7ANS. Selected replication genes showed a maximum expression 30 minutes after infection, but structural and packaging gene expression maximized after 90 minutes. Discrete timepoints of maximum expression for any selected gene in Cp1R7A-A1 were not observed during the given incubation periods, probably due to the slow rate of replication and assembly. RNAseq analysis of Cp1R7A-A1 infected M. japonicum indicated the expression of all 237 putative phage genes and gave some insights into host gene expression during phage infection. Attempts were made to engineer R. leguminosarum and M. japonicum to carry a plasmid with a functional CRISPR/Cas9 system. A CRISPR/Cas9 system derived from Streptococcus pyogenes was cloned into the broad-host range vector pRK415. The new vector pRK415Cas9 functioned successfully in E. coli. However, it was not functional in Rhizobium or Mesorhizobium species. Cas9 promoter expression studies and RT-PCR revealed that the Cas9 promoter was expressed in Mesorhizobium. However, whether correct translation occurs has yet to be tested

Determination of Pore Sizes and Relative Porosity in Porous Nanoshell Architectures Using Dextran Retention with Single Monomer Resolution and Proton Permeation

Unilamellar phospholipid vesicles prepared using the polymerizable lipid bis-sorbylphosphatidylcholine (bis-SorbPC) yield three-dimensional nanoarchitectures that are highly permeable to small molecules. The resulting porous phospholipid nanoshells (PPNs) are potentially useful for a range of biomedical applications including nanosensors and nanodelivery vehicles for cellular assays and manipulations. The uniformity and size distribution of the pores, key properties for sensor design and utilization, have not previously been reported. Fluorophore-assisted carbohydrate electrophoresis (FACE) was utilized to assess the nominal molecular weight cutoff limit (NMCL) of the PPN via analysis of retained dextran with single monomer resolution. The NMCL of PPNs prepared from pure bis-SorbPC was equivalent to a 1800 Da linear dextran, corresponding to a maximum pore diameter of 2.6 nm. Further investigation of PPNs prepared using binary mixtures of bis-SorbPC and dioleoylphosphatidylcholine (DOPC) revealed a similar NMCL when the bis-SorbPC content exceeded 30 mol %, whereas different size-dependent permeation was observed below this composition. Below 30 mol % bis-SorbPC, dextran retention provided insufficient mass resolution (162 Da) to observe porosity on the experimental time scale; however, proton permeability showed a marked enhancement for bis-SorbPC ≥ 10 mol %. Combined, these data suggest that the NMCL for native pores in bis-SorbPC PPNs results from an inherent property within the lipid assembly that can be partially disrupted by dilution of bis-SorbPC below a critical value for domain formation. Additionally, the analytical method described herein should prove useful for the challenging task of elucidating porosity in a range of three-dimensional nanomaterials