Study on the promotion of bacterial biofilm formation by a Salmonella conjugative plasmid and the underlying mechanism.

To investigate the effect of the pRST98 plasmid, originally isolated from Salmonella enterica serovar Typhi (S. Typhi), on biofilm (BF) formation, we carried out in vitro experiments using S. Typhi, Salmonella enterica serovar Typhimurium (S. Typhimurium) and Escherichia coli (E. coli). We further explored the effects of pRST98 in vivo by establishing two animal models, a tumor-bearing mouse model and a mouse urethral catheter model. Moreover, we examined the relationship between the quorum-sensing (QS) system and pRST98-mediated BF formation. These studies showed that pRST98 enhanced BF formation in different bacteria in vitro. In both animal models, pRST98 promoted BF formation and caused more severe pathological changes. It was previously reported that Salmonella senses exogenous N-acylhomoserine lactones (AHLs) through the regulatory protein SdiA and regulates the expression of genes including the virulence gene rck, which is located on the virulence plasmid of some serotypes of Salmonella. In this study, we confirmed the locus of the rck gene on pRST98 and found that AHLs increased rck expression in pRST98-carrying strains, thereby enhancing bacterial adherence, serum resistance and bacterial BF formation. In conclusion, the Salmonella conjugative plasmid pRST98 promotes bacterial BF formation both in vitro and in vivo, and the mechanism may relate to the AHL-SdiA-Rck signaling pathway

Bacterial accumulation at the indicated time points and the bacterial load in the organs of CT26 tumor mice.

<p>(<b>A</b>) Tumor-bearing mice were infected with 1×10⁷ CFU of <i>S. Typhimurium</i> χ3337<i>lux</i> and χ3337<i>lux</i>/pR_ST98. The bioluminescence signals were captured by IVIS at the indicated time points. (<b>B</b>) Comparison of χ3337<i>lux</i> and χ3337<i>lux</i>/pR_ST98 accumulated in tumors at 3 d p.i. by SEM. (<b>C</b>) CFU counts of tumors, livers and spleens infected by χ3337<i>lux</i> or χ3337<i>lux</i>/pR_ST98. (**<i>p</i> <0.01); (*<i>p</i> <0.05)</p

Strains used in this study.

<p>Strains used in this study.</p

AHLs on <i>S. Typhi</i> BF formation.

<p><i>S. Typhi</i>, cultured in 24-well polystyrene plates for 24 h by adding 1µM C8-AHLs and 1µM saline and detected by IVIS. a,b: ST₈<i>lux</i>; c,d: ST₈-ΔpR_ST98<i>lux</i>; e,f: ST₈-c-pR_ST98<i>lux</i>.</p

Quantification of BF by CLSM.

<p>Different bacteria were cultured in 24-well plates for 3 d, and the developed pellicles were harvested, placed on glass slides, and subjected to 3D image reconstruction by CLSM.</p

Electrophoresis plasmid profile of pR_ST98.

<p>Lane M1, <i>S. flexneri₂₄₅₇₀</i>, plasmid size marker; Lane M2, <i>E. coli</i> V₅₁₇, plasmid size marker; Lane 1–3, multi-drug resistant <i>S. Typhi</i> used as representative strains that naturally harbored pR_ST98 and were resistant to chloramp henicol, streptomycin, trimethoprim and sulphonamide, gentamicin, neomycin, kanamycin, cephalosporin ampicillin, carbenicillin and tetracycline; Lane 4, antibiotic-sensitive <i>S. Typhi</i>, which were plasmid free, and used as the negative control.</p

Observation of BF by SEM.

<p>Different bacteria were cultured in 24-well plates for 3 d, and the developed pellicles were harvested, placed on glass slides, and subjected to SEM.</p

The effect of AHLs on <i>rck</i> expression and its related function.

<p>(A) The adherence rate of <i>S. Typhi</i> to HeLa cells in the presence of AHLs (*<i>p</i> <0.05). (B and C) Quantification by CFU of surviving bacteria after incubation with sera from rabbits (B) and guinea pigs (C) in the presence of AHLs and saline (*<i>p</i> <0.05).</p

Comparison of BF developed by different bacteria.

<p>(<b>A</b>) Different bacteria cultured <i>in vitro</i> for 3 d in microtiter plates at 30°C and stained by crystal violet (400×). (<b>B</b>) Different bacteria cultured <i>in vitro</i> for 3 d in 96-well plates at 30°C and stained by crystal violet. (<b>C</b>) Optical density of cultures measured at a wavelength of 570 nm (<i>OD₅₇₀</i>) after crystal violet staining (*<i>p</i> <0.05).</p

The locus of <i>rck</i> and its expression.

<p>(A) PCR of <i>rck</i> gene in pR_ST98. M: 1000 bp DNA ladder; Lane 1: <i>S. Typhi</i> ST₈; Lane 2: <i>S. Typhi</i> ST₈-ΔpR_ST98; Lane 3: <i>S. Typhi</i> ST₈-c-pR_ST98. (B) The effect of AHLs on the expression of the <i>rck</i> gene. M: 1000 bp DNA ladder; Lane 1: <i>S. Typhi</i> ST₈ treated with AHLs; Lane 2: <i>S. Typhi</i> ST₈ treated with saline; Lane 3: <i>S. typhi</i> ST₈-c-pR_ST98 treated with AHLs; Lane 4: <i>S. Typhi</i> ST₈-c-pR_ST98 treated with saline; Lane 5: <i>S. Typhi</i> ST₈-ΔpR_ST98 treated with AHLs; Lane 6: <i>S. Typhi</i> ST₈-ΔpR_ST98 treated with saline.</p