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