Exploring bet-hedging in Salmonella enterica serovar Typhimurium with a dual reporter strain

Non-typhoidal Salmonella are a leading cause of foodborne illness. These pathogens cycle between infecting hosts and persisting in the environment as biofilms. Unfortunately, this transition stage is not well understood. In non-typhoidal Salmonella, bistable synthesis of the master biofilm regulator CsgD results in a population split into CsgD-positive biofilm aggregates that synthesize a protective extracellular matrix and CsgD-negative single cells that synthesize the Salmonella pathogenicity island 1 type III secretion system (SPI-1 T3SS), which facilitates host cell invasion. We hypothesize that this is a bet-hedging strategy evolved to improve transmission, providing a way for NTS to cause disease immediately (single cells) or persist over extended periods (biofilms). We built a fluorescent reporter strain of S. Typhimurium to simultaneously track biofilm⁺ cells (GFP) and SPI-1 T3SS⁺ cells (mCherry) to better understand the dynamics of bet-hedging. Four S. Typhimurium cell populations were quantified in an in vitro flask model of biofilm development: CsgD⁺/SPI-1⁻, CsgD⁻/SPI-1⁺, CsgD⁺/SPI-1⁺, and CsgD⁻/SPI-1⁻. We demonstrate that Salmonella population splitting can occur in vivo during infection of Caenorhabditis elegans. At early stages of infection, the worm intestine was mainly dominated by SPI-1 T3SS⁺ Salmonella cells, while at later time points, both SPI-1 T3SS⁺ cells and biofilm⁺ cells are present. The use of the S. Typhimurium dual reporter will allow us to track and better understand the importance of bet-hedging in the Salmonella lifecycle.IMPORTANCESalmonella strains that cause gastroenteritis remain a leading cause of illness and deaths related to foodborne disease. These bacteria follow a cyclical life cycle (i.e., host → environment → host) that is key to their continued success as pathogens. Here, we built a dual reporter strain to study the infectious single cells and biofilm cell types of Salmonella enterica serovar Typhimurium, one of the most common gastroenteritis-causing serovars worldwide. Studying infectious and persistent cell types simultaneously and how they interact with host tissues in vivo will provide new insights into the Salmonella lifecycle and will be important to develop interventions that reduce outbreaks and disease.