Typhoid fever, a human-specific disease, is primarily caused by the pathogen Salmonella enterica serovar Typhi (S. Typhi). It is estimated that 3-5% of people infected with typhoid fever become chronic carriers. Studies have demonstrated that a mechanism of chronic carriage involves biofilm formation on gallstone surfaces. In the course of a previous study using a chronic carriage mouse model, a S. Typhimurium isolate was recovered from a mouse gallstone that exhibited a 2-fold increase in biofilm formation over the wild type. In order to identify the gene(s) responsible for the phenotype, the genomic sequence of this isolate and others were determined and compared. These sequences identified SNPs in 14 genes. Mutations in the most promising candidates, envZ and rcsB, were created but neither showed increased biofilm-forming ability separately or in combination. The hyper-biofilm isolate did however, present variations in cellular appendages observable using different techniques and a preferential binding to cholesterol. The isolate was also examined for systemic virulence and the ability to colonize the gallbladder/gallstones in a mouse model of chronic infection, demonstrating a systemic virulence defect and decreased gallbladder/gallstone colonization. Finally, to determine if the appearance of hyper-biofilm isolates could be replicated in vitro and if this was a common event, wild type Salmonella was grown long-term in vitro in gallbladder-mimicking conditions, resulting in a high proportion of isolates that replicated the hyper-biofilm phenotype of the original isolate. Thus, Salmonella acquire random mutations in the gallbladder/gallbladder-simulating conditions that may aid persistence but negatively affect systemic virulence.
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