Extracellular DNA, cell surface proteins and c-di-GMP promote biofilm formation in Clostridioides difficile

Lisa F Dawson,Maria Derakhshan,Catherine L Hall,Brendan W Wren,Mark A Harrison,Johann Peltier,Neil F Fairweather,Helen A Shaw

doi:10.1038/s41598-020-78437-5

Abstract

Clostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhoea worldwide, yet there is little insight into intestinal tract colonisation and relapse. In many bacterial species, the secondary messenger cyclic-di-GMP mediates switching between planktonic phase, sessile growth and biofilm formation. We demonstrate that c-di-GMP promotes early biofilm formation in C. difficile and that four cell surface proteins contribute to biofilm formation, including two c-di-GMP regulated; CD2831 and CD3246, and two c-di-GMP-independent; CD3392 and CD0183. We demonstrate that C. difficile biofilms are composed of extracellular DNA (eDNA), cell surface and intracellular proteins, which form a protective matrix around C. difficile vegetative cells and spores, as shown by a protective effect against the antibiotic vancomycin. We demonstrate a positive correlation between biofilm biomass, sporulation frequency and eDNA abundance in all five C. difficile lineages. Strains 630 (RT012), CD305 (RT023) and M120 (RT078) contain significantly more eDNA in their biofilm matrix than strains R20291 (RT027) and M68 (RT017). DNase has a profound effect on biofilm integrity, resulting in complete disassembly of the biofilm matrix, inhibition of biofilm formation and reduced spore germination. The addition of exogenous DNase could be exploited in treatment of C. difficile infection and relapse, to improve antibiotic efficacy.

Highlights

Clostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhoea worldwide, yet there is little insight into intestinal tract colonisation and relapse
There is evidence that C. difficile biofilms are composed of DNA and protein surrounding adherent spores and vegetative cells, the extent to which they contribute to biofilm formation, biofilm integrity and dispersal remains to be determined[23,24,25,29]
Using scanning electron microscopy (SEM), we show three distinct stages of biofilm formation: initial attachment of bacteria to an abiotic surface via bacterial appendages, through to early biofilm formation, where we observe formation of a matrix, culminating at late biofilm formation, in which the bacteria appear to be encased within an extracellular matrix (Fig. 1 a and Supplementary Fig. S1 online)

Summary

Introduction

Clostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhoea worldwide, yet there is little insight into intestinal tract colonisation and relapse. We demonstrate that C. difficile biofilms are composed of extracellular DNA (eDNA), cell surface and intracellular proteins, which form a protective matrix around C. difficile vegetative cells and spores, as shown by a protective effect against the antibiotic vancomycin. The structure and composition of a biofilm can vary between species, generally the biofilm matrix is comprised of a self-produced Extracellular Polymeric Substance (EPS), usually formed in three primary categories: (1) exopolysaccharide, (2) extracellular and cell surface proteins and (3) extracellular DNA (eDNA)[13]. Mutation of genes encoding the cell surface protease cwp[84] and the flagella filament fliC have been shown to both positively[36,41] and negatively[26,36] impact biofilm biomass This highlights that biofilm formation in C. difficile like other species is a multifaceted complex process

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Results

Conclusion