Molecular and Phenotypic Analysis of Salmonella Biofilm Formation: Exploring the Links Between Survival, Virulence, and Transmission

Pathogenic Salmonella strains are responsible for millions of human and livestock infections each year. The mechanisms of Salmonella pathogenesis are of great interest, along with the capacity of strains to survive in the environment and complete the transmission cycle. This survival is predicted to be related to a specific physiology called a biofilm. Biofilms are communities of cells within a self-produced extracellular matrix that are often associated with a physical surface. For Salmonella, the biofilm phenotype is activated by the transcriptional regulator CsgD and is associated with the production of an extracellular matrix consisting of protein polymers and exopolysaccharides. Salmonella biofilm formation is induced during growth at low temperatures and in conditions of nutrient limitation and low osmolarity. The biofilm phenotype is highly conserved across nontyphoidal Salmonella strains that briefly colonize the host and cause gastroenteritis. It is hypothesized that biofilm formation is important for increasing the transmission success of nontyphoidal Salmonella by enhancing their persistence in non-host environments. Salmonella biofilms have traditionally been studied as a population-level phenotype associated with colony formation, known as the red, dry, and rough (rdar) morphotype. However, Salmonella grown in liquid broth cultures under biofilm-inducing conditions form clonal subpopulations of multicellular aggregates and planktonic cells. This phenomenon is attributed to bistable expression of CsgD, where aggregated cells exist in a CsgD-ON state and planktonic cells are associated with a CsgD-OFF state. We performed comparative transcriptomic sequencing (RNA-seq), which revealed 1856 genes that were differentially expressed between these two S. Typhimurium cell subpopulations. Multicellular aggregates were associated with increased gene expression typical of Salmonella biofilm formation, including nutrient scavenging, reactive oxygen species defenses, and osmoprotection. In contrast, planktonic cells were associated with higher expression of multiple virulence pathways associated with the SPI-1 and SPI-2 type three secretion systems, cell motility, and chemotaxis. Increased synthesis of the SPI-1 type three secretion system in planktonic cells correlated with enhanced invasion of polarized Caco-2 human intestinal cells. We modified an existing Tn7-based transposition system to generate chromosomally marked strains of Salmonella to facilitate tracking of multicellular aggregates and planktonic cells in competitive fitness assays. Planktonic cells were associated with increased virulence in mice compared to multicellular aggregates. However, when these same cell subpopulations were exposed to desiccation, multicellular aggregates were associated with greater cell survival and the virulence advantage of planktonic cells was lost. We hypothesize that bistable CsgD expression and the generation of specialized cell types may represent a form of bet hedging, where planktonic cells are adapted for direct host-to-host transmission, and multicellular aggregates can survive long-term in the environment to cause infections later. This strategy would prepare nontyphoidal Salmonella for the unpredictable nature of the fecal-oral transmission process and improve their potential to cause future infections. Salmonella serovars that cause systemic disease within a restricted range of hosts have been shown to be biofilm-negative. In sub-Saharan Africa, a phylogenetically distinct group of nontyphoidal Salmonella has recently been identified for its role in an emerging epidemic of invasive extraintestinal infections. These invasive nontyphoidal Salmonella are associated with chronic persistence within the human host and do not have an identified environmental reservoir. We compared the biofilm phenotype of two invasive nontyphoidal Salmonella strains (S. Typhimurium D23580 and S. Enteritidis D7795) to a panel of strains consisting of ‘typical’ gastroenteritis-causing, nontyphoidal Salmonella and Salmonella strains that cause systemic typhoid fever. Both strains of invasive nontyphoidal Salmonella demonstrated an impaired biofilm phenotype, which we attributed to strain-specific genetic polymorphisms. We predict that the impaired biofilm phenotype of invasive nontyphoidal Salmonella correlates with their occupation of the systemic niche within the host and a reduced capacity to survive in the environment. My research has brought insight into how pathogenic Salmonella strains are able to navigate through unpredictable areas of their lifecycle and increased our understanding of their potential transmission mechanisms

Uterine Prolapse in an Adult Richardson's Ground Squirrel, Spermophilus richardsonii

During a study of Richardson's Ground Squirrel (Spermophilus richardsonii) populations in southern Saskatchewan, we captured one adult female with a partially prolapsed uterus. This is the first known case of uterine prolapse in a Richardson's Ground Squirrel

FlexType: Flexible Text Input with a Small Set of Input Gestures

In many situations, it may be impractical or impossible to enter text by selecting precise locations on a physical or touchscreen keyboard. We present an ambiguous keyboard with four character groups that has potential applications for eyes-free text entry, as well as text entry using a single switch or a brain-computer interface. We develop a procedure for optimizing these character groupings based on a disambiguation algorithm that leverages a long-span language model. We produce both alphabetically-constrained and unconstrained character groups in an offline optimization experiment and compare them in a longitudinal user study. Our results did not show a significant difference between the constrained and unconstrained character groups after four hours of practice. As expected, participants had significantly more errors with the unconstrained groups in the first session, suggesting a higher barrier to learning the technique. We therefore recommend the alphabetically-constrained character groups, where participants were able to achieve an average entry rate of 12.0 words per minute with a 2.03% character error rate using a single hand and with no visual feedback

Gas Anxiety and the Charging Choices of Plug-In Hybrid Electric Vehicle Drivers

Plug-in hybrid electric vehicles (PHEV) provide an opportunity to reduce petroleum consumption and greenhouse gas emissions without causing range anxiety. As a result, PHEV drivers are commonly assumed to be less dependent on the availability of charging infrastructure than battery electric vehicle (BEV) drivers. However there is also evidence that PHEVs plug in more often than BEVs because the owners have gas anxiety - a strong desire to avoid using gasoline. This work examines the existence of gas anxiety by analyzing the factors influencing charging decision of PHEV owners. A web-based stated preference survey was conducted and the data was analyzed using a latent class logit model. The result shows that there are two classes of decision making patterns among PHEV owners: those who value gasoline cost and recharging expenditure almost the same (class 1) and those who value gasoline cost more heavily than recharging cost (class 2). Among those in class 2, the amount of money spent on gasoline has much bigger influence on the utility of charging than the amount spent on electricity at the recharging station, which can be interpreted as a form of gas anxiety

Prolonged activation of S6K1 does not suppress IRS or PI-3 kinase signaling during muscle cell differentiation

Background: Myogenesis in C2C12 cells requires the activation of the PI3K/mTOR signaling pathways. Since mTOR signaling can feedback through S6K1 to inhibit the activation of PI3K, the aim of this work was to assess whether feedback from S6K1 played a role in myogenesis and determine whether siRNA mediated knockdown of S6K1 would lead to an increased rate of myotube formation. Results: S6K1 activity increased in a linear fashion following plating and was more than 3-fold higher after Day 3 of differentiation (subconfluent = 11.09 ± 3.05, Day 3 = 29.34 ± 3.58). IRS-1 levels tended to increase upon serum withdrawal but decreased approximately 2-fold (subconfluent = 0.88 ± 0.10, Day 3 = 0.42 ± 0.06) 3 days following differentiation whereas IRS-2 protein remained stable. IRS-1 associated p85 was significantly reduced upon serum withdrawal (subconfluent = 0.86 ± 0.07, Day 0 = 0.31 ± 0.05), remaining low through day 1. IRS-2 associated p85 decreased following serum withdrawal (subconfluent = 0.96 ± 0.05, Day 1 = 0.56 ± 0.08) and remained suppressed up to Day 3 following differentiation (0.56 ± 0.05). Phospho-tyrosine associated p85 increased significantly from subconfluent to Day 0 and remained elevated throughout differentiation. siRNA directed against S6K1 and S6K2 did not result in changes in IRS-1 levels after either 48 or 96 hrs. Furthermore, neither 48 nor 96 hrs of S6K1 knockdown caused a change in myotube formation. Conclusions: Even though S6K1 activity increases throughout muscle cell differentiation and IRS-1 levels decrease over this period, siRNA suggests that S6K1 is not mediating the decrease in IRS-1. The decrease in IRS-1/2 associated p85 together with the increase in phospho-tyrosine associated p85 suggests that PI3K associates primarily with scaffolds other than IRS-1/2 during muscle cell differentiation

What is success? Reflections on assisting BTEC students’ transitions into higher education

Presently, one in four students entering higher education (HE) hold a Business and Technology Education Council (BTEC) qualification. These vocational qualifications have played an important role in developing an additional educational pathway into universities for students often associated with under-represented groups and low participation regions, while widening participation initiatives have contributed to the increased number of BTEC students entering HE institutions (HEIs). Following on, potential approaches to support BTEC students during their first year at university were devised. One intervention has involved the development of an online module aimed to assist BTEC students’ transition from college to higher education. This article reflects upon the development of an online module implemented at a ‘selective’ university and examines what we actually define as 'success' within this context. The case study draws upon a variety of sources and provides not just an understanding of the project outcomes, but also an in-depth understanding about the more complex issues surrounding working with students to produce a successful project. Recommendations are provided for further work within this important field

Building the evidence base on the agricultural nutrition nexus: Vanuatu

A rapid scan on the agriculture and nutrition situation in Vanuatu was undertaken in 2017 to build the evidence base for strengthening the linkage between two important sectors – agriculture and health, for improved food and nutrition outcomes. The food and nutrition situation in Vanuatu has changed over the years as dietary patterns and lifestyles transitioned from a dependence on mostly subsistence living to a more urbanised western lifestyle. Since independence in the early 1980s, food crop production has not significantly increased although the population has almost doubled. In 1983 approximately 0.9 kg of food crops were produced and presumably consumed per capita per day compared to 0.5 kg in 2007. This has resulted in increased dependence on imported foods and the consumption of refined foods that contain higher levels of saturated fats and oils, salt and sugar; less physical activity and increased exposure to other risk factors linked to alcohol intake and smoking. Variation between the food and nutrition situation of rural and urban households and between rural households involved in cash cropping and in subsistence farming, and poor and more affluent urban households have been noted

Declining Sex Ratio in a First Nation Community

Members of the Aamjiwnaang First Nation community near Sarnia, Ontario, Canada, voiced concerns that there appeared to be fewer male children in their community in recent years. In response to these concerns, we assessed the sex ratio (proportion of male births) of the Aamjiwnaang First Nation over the period 1984–2003 as part of a community-based participatory research project. The trend in the proportion of male live births of the Aamjiwnaang First Nation has been declining continuously from the early 1990s to 2003, from an apparently stable sex ratio prior to this time. The proportion of male births (m) showed a statistically significant decline over the most recent 10-year period (1994–2003) (m = 0.412, p = 0.008) with the most pronounced decrease observed during the most recent 5 years (1999–2003) (m = 0.348, p = 0.006). Numerous factors have been associated with a decrease in the proportion of male births in a population, including a number of environmental and occupational chemical exposures. This community is located within the Great Lakes St. Clair River Area of Concern and is situated immediately adjacent to several large petrochemical, polymer, and chemical industrial plants. Although there are several potential factors that could be contributing to the observed decrease in sex ratio of the Aamjiwnaang First Nation, the close proximity of this community to a large aggregation of industries and potential exposures to compounds that may influence sex ratios warrants further assessment into the types of chemical exposures for this population. A community health survey is currently under way to gather more information about the health of the Aamjiwnaang community and to provide additional information about the factors that could be contributing to the observed decrease in the proportion of male births in recent years