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

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.

Antibiotic Resistance and Biofilm Production Capacity in Clostridioides difficile

“…To the best of our knowledge, this is the first time that biofilm production capacity is associated with reduced antibiotic susceptibility in C. difficile planktonic cells. Most studies have determined the antibiotic susceptibility of C. difficile biofilms, showing that biofilm cells are more tolerant of several antibiotics, compared to their planktonic counterparts (Dawson et al, 2012;Dapa and Unnikrishnan, 2013;Crowther et al, 2014;Semenyuk et al, 2014;Dawson et al, 2021). In contrast, we defined the antibiotic susceptibility of the different isolates before biofilm production, and associated the susceptibility profile with the biofilm-forming capacity.…”

“…C. difficile biofilm production has been associated with disease recurrence, which poses a major challenge for CDI treatment ( Dapa and Unnikrishnan, 2013 ). Although C. difficile biofilms have been studied both in vitro and in vivo , most of them explored only a limited number of strains ( Lawley et al, 2009 ; Buckley et al, 2011 ; Dawson et al, 2012 ; Dapa and Unnikrishnan, 2013 ; Ethapa et al, 2013 ; Semenyuk et al, 2014 ; Semenyuk et al, 2015 ; Crowther et al, 2016 ; Vuotto et al, 2016 ; Soavelomandroso et al, 2017 ; James et al, 2018 ; Poquet et al, 2018 ; Dubois et al, 2019 ; Dawson et al, 2021 ; Normington et al, 2021 ). Additionally, previous studies have not compared the biofilm production capacity of antibiotic-susceptible versus antibiotic-tolerant isolates.…”

“…Patients with disease recurrence, are prone to episodic reappearances, with risks of 45% and 64% for second and third episodes, respectively ( Surawicz and Alexander, 2011 ). Mostly, recurrence is associated with the original strain ( Figueroa et al, 2012 ), assigned to either resistance to the administered antibiotic or to a protective, antibiotic-impermeable barrier it forms ( Dawson et al, 2021 ). This barrier is composed of sessile, surface-associated microbial communities, known as a biofilm ( Dawson et al, 2012 ).…”

“…This barrier is composed of sessile, surface-associated microbial communities, known as a biofilm ( Dawson et al, 2012 ). Previous studies have demonstrated in vitro and in vivo formation of biofilms by C. difficile ( Lawley et al, 2009 ; Buckley et al, 2011 ; Dawson et al, 2012 ; Donelli et al, 2012 ; Dapa and Unnikrishnan, 2013 ; Ethapa et al, 2013 ; Semenyuk et al, 2014 ; Semenyuk et al, 2015 ; Crowther et al, 2016 ; Vuotto et al, 2016 ; Jain et al, 2017 ; Soavelomandroso et al, 2017 ; James et al, 2018 ; Poquet et al, 2018 ; Dawson et al, 2021 ; Normington et al, 2021 ), which have been shown to include polysaccharides, proteins and extracellular DNA ( Dawson et al, 2021 ), as well as toxins and spores ( Dapa and Unnikrishnan, 2013 ; Semenyuk et al, 2014 ) [Reviewed at ( Frost et al, 2021 )].…”

c-di-GMP inhibits early sporulation in Clostridioides difficile

“…Many of the C. difficile c-di-GMP metabolic proteins have been demonstrated to be enzymatically active (10,12), suggesting that the regulation of c-di-GMP metabolism in C. difficile is physiologically important and tightly controlled. High intracellular concentrations of c-di-GMP have been demonstrated to inhibit C. difficile motility and toxin production while promoting cell aggregation, biofilm formation, and colonization (13)(14)(15)(16)(17)(18)(19)(20).…”