Examining the Link between Biofilm Formation and the Ability of Pathogenic Salmonella Strains to Colonize Multiple Host Species

Abstract

Salmonella are important pathogens worldwide and a predominant number of human infections are zoonotic in nature. The ability of strains to form biofilms, which is a multicellular behavior characterized by the aggregation of cells, is predicted to be a conserved strategy for increased persistence and survival. It may also contribute to the increasing number of infections caused by ingestion of contaminated fruits and vegetables. There is a correlation between biofilm formation and the ability of strains to colonize and replicate within the intestines of multiple host species. These strains predominantly cause localized gastroenteritis infections in humans. In contrast, there are salmonellae that cause systemic, disseminated infections in a select few host species; these "invasive" strains have a narrowed host range, and most are unable to form biofilms. This includes host-restricted Salmonella serovar Typhi, which are only able to infect humans, and atypical gastroenteritis strains associated with the opportunistic infection of immunocompromised patients. From the perspective of transmission, biofilm formation is advantageous for ensuring pathogen survival in the environment. However, from an infection point of view, biofilm formation may be an anti-virulence trait. We do not know if the capacity to form biofilms prevents a strain from accessing the systemic compartments within the host or if loss of the biofilm phenotype reflects a change in a strain's interaction with the host. In this review, we examine the connections between biofilm formation, Salmonella disease states, degrees of host adaptation, and how this might relate to different transmission patterns. A better understanding of the dynamic lifecycle of Salmonella will allow us to reduce the burden of livestock and human infections caused by these important pathogens.

Keywords: Salmonella; biofilms; cellulose; curli; gastroenteritis; host adaptation.

Figure 1

Salmonella taxonomy and general classifications. The genus Salmonella is classified into species, subspecies, and serovars based on the White–Kauffman–Le Minor scheme. Serovars are often grouped into non-typhoidal or typhoidal categories; however, this referencing approach is not a part of the official Salmonella classification scheme. For a expanded version of the taxonomical distribution of Salmonella, readers are referred to Ref. (9).

Figure 2

Examples of Salmonella biofilm formation. (A) Colonies grown for 48 h at 28°C on solid 1% tryptone media form the characteristic surface patterns of the red, dry, and rough (rdar) morphotype. The colony appears red when the media is supplemented with the dye Congo red. (B) Pellicle formation at the air–liquid interface of a 1% tryptone liquid culture [adapted from Ref. (46)]. (C) Salmonella form multicellular aggregates and planktonic cells within the bulk liquid phase of a flask culture. (D) The number of colony forming units (CFU) present in aggregate or planktonic cell subpopulations from (C) was calculated using conversion factors determined from serial dilution plating after homogenization (1.92 × 10⁹ CFU per 1.0 OD₆₀₀ for planktonic cells; 1.73 × 10⁸ CFU/mg for aggregates). The green bars and blue bars represent the proportion of planktonic cells and aggregates comprising the total number of cells in the population; points on the right side of the graph represent total CFU values for each cell type from nine replicate flask cultures. The percentage values represent the average proportion of each cell type.

Figure 3

Salmonella phenotypes that result from the activity of CsgD and c-di-GMP. (A) Synthesis of biofilm-associated polymers is regulated at the genetic level by CsgD and the secondary messenger molecule c-di-GMP. Multiple environmental conditions act as inducing signals for csgD expression, CsgD synthesis, and c-di-GMP production. These environmental conditions (represented here as a lightning bolt) are transduced into intracellular signals via outer membrane proteins, two-component signal transduction systems, regulatory proteins, and enzymes associated with c-di-GMP production. A select set of diguanylate cyclases (STM1283, STM2123, STM2672, and STM1987) can contribute to the c-di-GMP pool that induces BcsE and BcsA activity, resulting in cellulose biosynthesis. CsgD is the master transcriptional regulator associated with Salmonella biofilm formation. In its unphosphorylated active state, CsgD promotes the expression of adrA, a potent diguanylate cyclase associated with promoting cellulose biosynthesis. CsgD is additionally responsible for activating the transcription of genes and operons associated with the biosynthesis of curli fimbriae, O-antigen capsule, and BapA protein. Genes and operons are shown as open arrows, proteins as ovals, and c-di-GMP molecules as dark blue circles. Positive regulation is denoted as green arrows, while regulatory inhibition is shown as flat-headed red arrows. Activation via c-di-GMP molecules is shown as blue arrows. (B) Multiple environmental signals can induce the biosynthesis of biofilm polymers. However, some conditions can activate c-di-GMP production and cellulose biosynthesis independently from other biofilm polymers. Under biofilm-inducing conditions, only a subset of Salmonella cells in the total population will synthesize high levels of CsgD. This subpopulation enters a CsgD-ON state, which results in significant c-di-GMP production and biosynthesis of biofilm matrix polymers. Cells within the biofilm are able to survive and persist in harsh environmental conditions. In contrast, some Salmonella cells in the population do not have sufficient synthesis of CsgD, resulting in a CsgD-OFF state and subsequently low intracellular concentrations of c-di-GMP. These cells remain in a planktonic state, are highly motile, and synthesize the type-three secretion system (T3SS)-1, resulting in a virulent cell subpopulation. Due to the CsgD-independent activity of some diguanylate cyclases, Salmonella cells can have high intracellular levels of c-di-GMP while in a CsgD-OFF state. As such, these cells may synthesize cellulose in the absence of other major biofilm matrix polymers. The relatively low expression/activity of virulence-associated factors in cells aggregated together within cellulose or other biofilm polymers is due at least in part to the state of c-di-GMP pools within the cells.

Figure 4

Host interactions, lifecycles, and biofilm-forming ability of Salmonella strains. Host-generalist Salmonella strains have a varied lifecycle, in which several host species and environments are encountered, and zoonotic transfer to humans may occur. Transfer may also occur through ingestion of contaminated vegetables (i.e., tomatoes, sprouts) or processed foods. Infections are localized to the intestine and the selection pressures are on intestinal replication and transmission. In contrast, host-adapted and host-restricted Salmonella strains have an evolutionary narrowed lifecycle, in which transmission is primarily between individual hosts. Selection pressures are on immune avoidance with the objective of long-term persistence within the host. The loss of biofilm formation in host-adapted and host-restricted strains is thought to reflect a shift in selection pressures caused by a change in lifecycle.