Enhanced biofilm and extracellular matrix production by chronic carriage versus acute isolates of Salmonella Typhi

Abstract

Salmonella Typhi is the primary causative agent of typhoid fever; an acute systemic infection that leads to chronic carriage in 3-5% of individuals. Chronic carriers are asymptomatic, difficult to treat and serve as reservoirs for typhoid outbreaks. Understanding the factors that contribute to chronic carriage is key to development of novel therapies to effectively resolve typhoid fever. Herein, although we observed no distinct clustering of chronic carriage isolates via phylogenetic analysis, we demonstrated that chronic isolates were phenotypically distinct from acute infection isolates. Chronic carriage isolates formed significantly thicker biofilms with greater biomass that correlated with significantly higher relative levels of extracellular DNA (eDNA) and DNABII proteins than biofilms formed by acute infection isolates. Importantly, extracellular DNABII proteins include integration host factor (IHF) and histone-like protein (HU) that are critical to the structural integrity of bacterial biofilms. In this study, we demonstrated that the biofilm formed by a chronic carriage isolate in vitro, was susceptible to disruption by a specific antibody against DNABII proteins, a successful first step in the development of a therapeutic to resolve chronic carriage.

Fig 1. Phylogenetic relationship of S. Typhi isolates.

(A) Maximum likelihood phylogenetic tree based on core genome sequence variation. This shows the relationship of S. Typhi strains used in this study in the context of a global collection of S. Typhi strains reported in (31). (B) Maximum likelihood phylogenetic tree showing the relationship of S. Typhi clinical isolates and S. Typhi Ty2 based on core genome sequence variation. (C) Single nucleotide polymorphism distance matrix and heatmap of isolates in this study. There was no apparent clustering of the sequenced isolates by the acute versus chronic infection status of the patient.

Fig 2. Chronic carriage isolates formed thicker biofilms than acute isolates.

Biofilms of each of the indicated S. Typhi strains were established in a 8-well chambered coverglass slide for 40 hours. Biofilms were stained with LIVE/DEAD stain and visualized via CLSM. Images were analyzed by COMSTAT to calculate average thickness (A) and biomass (B). Bars represent the standard error of the mean (SEM). The mean of the average thickness or biomass of the chronic carriage isolates was represented by the red dotted line labeled AC and the mean of the average thickness or biomass of the acute isolates was represented by the blue dotted line labeled AA. Statistical significance of average thickness or biomass of each of the strains versus the the lab strain, S. Typhi Ty2 (JSG698), was assessed by a one-way ANOVA followed by Dunnett’s multiple comparison test. Statistical significance between AC and AA were assessed by a one-way ANOVA followed by Tukey’s multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. On average, chronic carriage isolates formed a thicker biofilm with more biomass than acute isolates. Chronic carriage isolates are indicated in red and acute infection isolates are indicated in blue.

Fig 3. Chronic carriage isolates have higher steady levels of extracellular DNA and DNABII proteins within their biofilm EPS than acute isolates.

Biofilms of each of the indicated S. Typhi strains were established in an 8-well chambered coverglass slide for 40 hours. Biofilms were labeled with α-dsDNA monoclonal antibody, α-IHF_Ec and FM 4–64 and visualized via CLSM. (A) Representative immunofluorescence images of a lab wild type strain S. Typhi Ty2 (JSG698), chronic carriage isolate (JSG3980) and acute isolate (JSG3986). eDNA is visualized in teal and DNABII proteins in purple. Images were analyzed by ImageJ to quantify the fluorescence intensity of eDNA (B), DNABII proteins (D), and bacteria. Fluorescence intensity of eDNA and DNABII proteins were normalized to cells and plotted in (C) and (E), respectively. Bars represent the SEM. AA and AC represent the averages of acute infection isolates and chronic carriage isolates, respectively. Statistical significance between AC and AA were assessed by a one-way ANOVA followed by Tukey’s multiple comparison test. **P<0.01. On average, chronic carriage isolates had higher steady state levels of both eDNA and DNABII proteins within the biofilm EPS as compared to acute isolates. Chronic carriage isolates are indicated in red and acute infection isolates are indicated in blue.

Fig 4. DNABII-specific antibody disrupted biofilms formed by chronic carriage isolate of S. Typhi.

(A) Biofilms formed by lab wild type strain S. Typhi Ty2 (JSG698) or chronic carriage isolate (JSG3074) or (B, C) JSG698 were established in an 8-well chambered coverglass slide in TSB (A), chambered coverglass slide coated with 5 mg/ml cholesterol (B), or TSB+0.5% ox bile extract (C) for 24 hours. Biofilms were incubated with medium alone, naive IgG, or α-IHF_Ec IgG at the indicated concentration for 16 hours. Biofilms were stained with LIVE/DEAD stain and visualized via CLSM. Images were analyzed by COMSTAT to calculate biomass. Bars represent the SEM. **P<0.01, ***P<0.001 via unpaired t test. Biofilms formed by S. Typhi Ty2 (JSG698) in the presence of cholesterol and bile, as well as a chronic carriage isolate of S. Typhi, were significantly disrupted by DNABII-specific antibody (α-IHF_Ec).