Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.

Advancing design for sustainable food cultures

Our paper, “HCI & Sustainable Food Culture: A Design Framework for Engagement,” presented at the 2010 NordiCHI conference, introduced a design framework for understanding engagement between people and sustainable food cultures (Choi and Blevis, 2010). Our goal for this chapter “Advancing Design for Sustainable Food Cultures��� is to expand our notion of this design framework and the programme of research it implies. This chapter presents the three elements of design framework for sustainability: (i) engagement across disciplines; (ii) engagement with and amongst users/non-users and; (iii) engagement for sustained usability. The uses a corresponding sample of photographic records of experiences that reflect three key issues in the current sustainable food domain: respectively, (i) context of food cultures, (ii) farmers’ markets, and (iii) producing food.

Synthesis of proteoglycan 4 by chondrocyte subpopulations in cartilage explants, monolayer cultures, and resurfaced cartilage cultures

Objective: To quantify the levels of proteoglycan 4 (PRG4) expression by subpopulations of chondrocytes from superficial, middle, and deep layers of normal bovine calf cartilage in various culture systems. Methods: Bovine calf articular cartilage discs or isolated cells were used in I of 3 systems of chondrocyte culture: explant, monolayer, or transplant, for 1-9 days. PRG4 expression was quantified by enzyme-linked immunosorbent assay of spent medium and localized by immunohistochemistry at the articular surface and within chondrocytes in explants and cultured cells. Results: Superficial chondrocytes secreted much more PRG4 than did middle and deep chondrocytes in all cultures. The pattern of PRG4 secretion into superficial culture medium varied with the duration of culture, decreasing with time in explant culture (from similar to25 mug/cm(2)/day on days 0-1 to similar to3 mug/cm(2)/day on days 5-9), while increasing in monolayer culture (from similar to1 pg/cell/day on days 0-1 to similar to7 pg/cell/day on days 7-9) and tending to increase in transplant culture (reaching similar to2 mug/cm(2)/day by days 7-9). In all of the culture systems, inclusion of ascorbic acid stimulated PRG4 secretion, and the source of PRG4 was immunolocalized to superficial cells. Conclusion: The results described here indicate that the phenotype of PRG4 secretion by chondrocytes in culture is generally maintained, in that PRG4 is expressed to a much greater degree by chondrocytes from the superficial zone than by those from the middle and deep zones. The marked up-regulation of PRG4 synthesis by ascorbic acid may have implications for cartilage homeostasis and prevention of osteoarthritic disease. Transplanting specialized cells that secrete PRG4 to a surface may impart functional lubrication and be generally applicable to many tissues in the body.

Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

Background Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia. Conclusion We describe an Australian origin for B. pseudomallei, characterized by a single introduction event into Southeast Asia during a recent glacial period, and variable levels of lateral gene transfer within populations. These patterns provide insights into mechanisms of genetic diversification in B. pseudomallei and its closest relatives, and provide a framework for integrating the traditionally separate fields of population genetics and phylogenetics for other bacterial species with high levels of lateral gene transfer.

Monitoring mesenchymal stem cell cultures using image processing and pattern recognition techniques

Stem cells have attracted tremendous interest in recent times due to their promise in providing innovative new treatments for a great range of currently debilitating diseases. This is due to their potential ability to regenerate and repair damaged tissue, and hence restore lost body function, in a manner beyond the body's usual healing process. Bone marrow-derived mesenchymal stem cells or bone marrow stromal cells are one type of adult stem cells that are of particular interest. Since they are derived from a living human adult donor, they do not have the ethical issues associated with the use of human embryonic stem cells. They are also able to be taken from a patient or other donors with relative ease and then grown readily in the laboratory for clinical application. Despite the attractive properties of bone marrow stromal cells, there is presently no quick and easy way to determine the quality of a sample of such cells. Presently, a sample must be grown for weeks and subject to various time-consuming assays, under the direction of an expert cell biologist, to determine whether it will be useful. Hence there is a great need for innovative new ways to assess the quality of cell cultures for research and potential clinical application. The research presented in this thesis investigates the use of computerised image processing and pattern recognition techniques to provide a quicker and simpler method for the quality assessment of bone marrow stromal cell cultures. In particular, aim of this work is to find out whether it is possible, through the use of image processing and pattern recognition techniques, to predict the growth potential of a culture of human bone marrow stromal cells at early stages, before it is readily apparent to a human observer. With the above aim in mind, a computerised system was developed to classify the quality of bone marrow stromal cell cultures based on phase contrast microscopy images. Our system was trained and tested on mixed images of both healthy and unhealthy bone marrow stromal cell samples taken from three different patients. This system, when presented with 44 previously unseen bone marrow stromal cell culture images, outperformed human experts in the ability to correctly classify healthy and unhealthy cultures. The system correctly classified the health status of an image 88% of the time compared to an average of 72% of the time for human experts. Extensive training and testing of the system on a set of 139 normal sized images and 567 smaller image tiles showed an average performance of 86% and 85% correct classifications, respectively. The contributions of this thesis include demonstrating the applicability and potential of computerised image processing and pattern recognition techniques to the task of quality assessment of bone marrow stromal cell cultures. As part of this system, an image normalisation method has been suggested and a new segmentation algorithm has been developed for locating cell regions of irregularly shaped cells in phase contrast images. Importantly, we have validated the efficacy of both the normalisation and segmentation method, by demonstrating that both methods quantitatively improve the classification performance of subsequent pattern recognition algorithms, in discriminating between cell cultures of differing health status. We have shown that the quality of a cell culture of bone marrow stromal cells may be assessed without the need to either segment individual cells or to use time-lapse imaging. Finally, we have proposed a set of features, that when extracted from the cell regions of segmented input images, can be used to train current state of the art pattern recognition systems to predict the quality of bone marrow stromal cell cultures earlier and more consistently than human experts.

Occurrence of iron and associated bacterial populations in soils of a forested subtropical coastal catchment

Iron (Fe) biogeochemistry is potentially of environmental significance in plantation-forested, subtropical coastal ecosystems where soil disturbance and seasonal water logging may lead to elevation of Fe mobilization and associated water quality deterioration. Using wet-chemical extraction and laboratory cultivation, we examined the occurrence of Fe forms and associated bacterial populations in diverse soils of a representative subtropical Australian coastal catchment (Poona Creek). Total reactive Fe was abundant throughout 0e30 cm soil cores, consisting primarily of crystalline forms in well-drained sand soils and water-logged loam soils, whereas in water-logged, low clay soils, over half of total reactive Fe was present in poorly-crystalline forms due to organic and inorganic complexation, respectively. Forestry practices such as plantation clear-felling and replanting, seasonal water logging and mineral soil properties significantly impacted soil organic carbon (C), potentially-bioavailable Fe pools and densities of S-, but not Fe-, bacterial populations. Bacterial Fe(III) reduction and abiotic Fe(II) oxidation, as well as chemolithotrophic S oxidation and aerobic, heterotrophic respiration were integral to catchment terrestrial FeeC cycling. This work demonstrates bacterial involvement in terrestrial Fe cycling in a subtropical coastal circumneutral-pH ecosystem.

“Prosuming” across cultures : youth creating digital media across borders

The Scratch Online Community is a space that enables young people to share their creative digital projects internationally with a level of ease that was impossible only a few years ago. Like all creative communities, Scratch is not just a space for sharing products, work, techniques and tips and tricks, but also a space for social interaction. Media literacy educators have unprecedented challenges and opportunities in digital environments like Scratch to harness the vast amount of knowledge in the community to enhance students’ learning. They also have challenges and opportunities in terms of implementing a form of digital media literacy education that is responsive to social and cultural representation. One role of digital media literacy is to help young people to challenge unfair and derogatory portrayals of people and to break down processes of social and cultural ‘othering’ so that all community members feel included and safe to express themselves. This article considers how online community spaces might draw on social interaction to enhance cross-cultural understandings and learning through dialogue and creative practice. The article uses statistics to indicate the amount of international interaction in the Scratch community. It then uses qualitative analysis of forum discussions and creative digital work to analyse the types of cross cultural interaction that occurs.

Blood cultures for assessment of postoperative fever in arthroplasty patients

Postoperative fever in arthroplasty patients is common. The value of diagnostic workup of fever in this instance is of questionable utility. Studies have shown that blood cultures in this scenario add little to clinical management, but sample sizes have been small and the use of blood cultures in this setting continues. This study aimed to examine the value of blood cultures in the assessment of postoperative fever in a large arthroplasty population. The medical records of 101 patients who had 141 blood culture sets taken during a 2-year period were retrospectively analyzed. Of the 141 blood culture sets, only 2 returned positive results. These were both thought to be as a result of skin contamination at the time of venipuncture. No infectious sequelae occurred in either patient. We conclude that blood cultures have no role to play in the assessment of the febrile, otherwise asymptomatic arthroplasty patient in the early postoperative period.

Print media portrayals of giving : exploring national ‘cultures of philanthropy’

In their 2010 study drawing on 500 empirical philanthropy studies, Bekkers and Wiepking identified eight consistently significant giving mechanisms. The pilot study reported here extends what is known about one mechanism, values, as a giving driver, in particular considering how national cultural values apply to giving. Personal values are not formed in a vacuum. They are influenced by the wider culture and society: thus values have a socio-cultural dimension. Accordingly, this pilot research draws on media theory and cultural studies work on national ethos to explore how these national cultural values interact with giving. A directed qualitative content analysis has been undertaken to compare US and Australian print media coverage about philanthropy. The two nations share an Anglo–Saxon orientation but differ significantly in national character and philanthropic activity. This study posits that a nation's media coverage about giving will reflect its national cultural ethos. This coverage can also shape personal values, thus implications exist for theory about the antecedents of personal giving values. Wider national values may drive or stifle giving, so this wider view of values as a driver has implications also for philanthropy promotion and fundraising.

The inherent challenges of knowledge transfer across countries and cultures

Purpose — The purpose of this chapter is to outline the findings of research into knowledge transfer across countries and culture and the implications of the findings for human resource practitioners. Methodology/approach — Use of a qualitative case study methodology approach was employed. Findings — The findings highlight the inherent difficulty of knowledge transfer and the broad range of factors that influence the knowledge transfer process and which are connected together in a complex and non-linear manner. Research implications — The research revealed insight into those managers most likely to be effective at knowledge transfer and what characteristics and experiences had supported this openness to knowledge from outside of their cultural context. The lessons from the research can be incorporated into the selection and development processes for expatriates. Originality value — The study affirmed the critical importance of face-to-face interaction in knowledge transfer. The research also resulted in the development of a practical model and tool, which pulls together the broad range of factors that impact on knowledge transfer.