Parallel evolution leading to impaired biofilm formation in invasive Salmonella strains

Pathogenic Salmonella strains that cause gastroenteritis are able to colonize and replicate within the intestines of multiple host species. In general, these strains have retained an ability to form the rdar morphotype, a resistant biofilm physiology hypothesized to be important for Salmonella transmission. In contrast, Salmonella strains that are host-adapted or even host-restricted like Salmonella enterica serovar Typhi, tend to cause systemic infections and have lost the ability to form the rdar morphotype. Here, we investigated the rdar morphotype and CsgD-regulated biofilm formation in two non-typhoidal Salmonella (NTS) strains that caused invasive disease in Malawian children, S. Typhimurium D23580 and S. Enteritidis D7795, and compared them to a panel of NTS strains associated with gastroenteritis, as well as S. Typhi strains. Sequence comparisons combined with luciferase reporter technology identified key SNPs in the promoter region of csgD that either shut off biofilm formation completely (D7795) or reduced transcription of this key biofilm regulator (D23580). Phylogenetic analysis showed that these SNPs are conserved throughout the African clades of invasive isolates, dating as far back as 80 years ago. S. Typhi isolates were negative for the rdar morphotype due to truncation of eight amino acids from the C-terminus of CsgD. We present new evidence in support of parallel evolution between lineages of nontyphoidal Salmonella associated with invasive disease in Africa and the archetypal host-restricted invasive serovar; S. Typhi. We hypothesize that the African invasive isolates are becoming human-adapted and niche specialized' with less reliance on environmental survival, as compared to gastroenteritis-causing isolates. Author summary African clades of nontyphoidal Salmonella cause invasive disease on a daily basis and thousands of deaths each year. Although it is generally accepted that the transmission route for these organisms is fecal-oral, we know very little about their behaviour in the environment between hosts. In this paper, we have identified both a genotype and a phenotype that suggest environmental niche specialization that is distinct from lineages of Salmonella Typhimurium and Salmonella Enteritidis associated with industrialized food supply chains in resource-rich settings. We also compared with strains of Salmonella Typhi, which cause systemic typhoid fever infections exclusively in humans. In each invasive lineage, regulatory or structural gene mutations leading to loss or impairment of biofilm were identified, all associated with curli and cellulose production, the two main structures that comprise the biofilm matrix. This suggests that similar evolutionary pressures are acting on invasive Salmonella isolates. Public health strategies aimed at reducing the burden of invasive Salmonella disease must prevent transmission to vulnerable adults and children via water sanitation and hygiene practices-a process that starts with identification of environmental reservoirs. The results of our study will raise the profile of this neglected aspect of invasive salmonellosis and will challenge researchers and clinicians to search in new places for potential environmental reservoirs of these pathogens.