Approximately half a million people in the USA are hospitalized with CDI annually, and 5 to 6% die within the first month of diagnosis
[9]. According to Mada and Alam
[9], antibiotic use remains the leading risk factor for
C. difficile infection. Of these, penicillins, cephalosporins, fluoroquinolones, and clindamycin have been implicated as the most possible cause
[9]. Other risk factors associated with CDI include advanced age, chemotherapy, use of proton pump inhibitors, chronic renal disease, chronic liver disease, and malnutrition
[9]. Based on the latest report from the Center for Disease Control (CDC), recurrent
C. difficile infection (RCDI) was reported in 12.0% of the 4301 cases studied, with a sharp increase in 2020 during the onset of the COVID-19 pandemic
[10]. An earlier report by Miranda-Katz et al.
[11] stated that only 17 to 24 in 100,000 children develop CDI, which is eightfold lower than that reported in adults over the age of 65. The resistance of infants and young children to CDI may be ascribed to the immunoglobulin in breast milk that inhibits the binding of TcdA to intestinal receptors, along with the lack of intestinal receptors in newborns that recognize the toxin
[12,13][12][13]. With aging, changes in diet and the secretion of bile acids render intestinal cells more susceptible to
C. difficile. Drastic changes in the gut microbiota, as observed with prolonged antibiotic treatment, prevent the conversion of primary bile acids to secondary bile acids. This favors
C. difficile colonization
[14,15][14][15]. Biofilm formation protects cells from oxygen and antibiotics, including metronidazole (MNZ) and vancomycin (VAN), which are commonly used to treat CDI
[16,17][16][17].
2. Colonization of C. difficile to Human Intestinal Cells
CDI is contracted through the ingestion of endospores
[18]. Germination of spores is controlled by the concentration and type of primary bile salts in the upper part of the GIT
[19,20][19][20]. Chenodeoxycholate (CDCA) represses spore germination, whereas cholate (CA) induces germination
[20]. Most of the primary bile acids (95%), conjugated with taurine and glycine or unconjugated, are absorbed in the terminal ileum and through the hepatic system
[19,21][19][21]. Primary bile acids that reach the large intestine are converted by gut microbiota into secondary bile acids, for example, ω-muricholate (ωMCA), hyodeoxycholate (HDCA), ursodeoxycholate (UDCA), lithocholate (LCA), and deoxycholate (DCA)
[19,22][19][22].
Physiological conditions, such as an excess of fermentable carbohydrates or an increase in deoxycholate (DOC,
Figure 1), stimulate
C. difficile to form biofilms in the human GIT, which may lead to recurrent episodes of CDI
[23,24][23][24]. Biofilm formation is also regulated by quorum sensing (QS) signals such as cyclic diguanosine monophosphate (c-di-GMP;
Figure 1).
Figure 1. Cyclic di-GMP (c-di-GMP)-mediated riboswitches control the colonization of
C. difficile to the host’s epithelial cells. Type I and II riboswitches control the expression of factors that are involved in motility, surface attachment, and virulence, including production of TcdA and TcdB. Type I riboswitches (off switches) inhibit translation following the binding of c-di-GMP, whereas type II riboswitches (on switches) promote the translation of target genes when bound to c-di-GMP. Increased levels of c-di-GMP stimulate the expression of adhesion factors, such as type IV pili and collagen-binding proteins (CBPs), and inhibit the expression of flagellar genes and CBP protease. Biofilm formation is altered by antimicrobials, QS signals, and the transcription factor spo0A that regulates sporulation. This illustration was made using BioRender (
https://biorender.com/), accessed on 3 April 2023.
Increased production of c-di-GMP represses motility and stimulates biofilm formation
[25]. The synthesis of c-di-GMP is controlled by the protein domain GGDEF, which is widely present in free-living bacteria
[26,27,28,29][26][27][28][29]. Increased c-di-GMP levels reduce the expression of
tcdA, tcdB, and
tcdR [25,26,27,28,29,30][25][26][27][28][29][30]. The
tcdR gene encodes an alternative sigma factor SigD (FliA; σ
28) that activates the expression of
tcdA and
tcdB in response to c-di-GMP
[25,30][25][30]. Degradation of c-di-GMP is controlled by the protein domain EAL (
Figure 1)
[26,27,28,29][26][27][28][29].
Changes in c-di-GMP levels influence the response of riboswitches, which in turn control the expression of flagellar genes. Biological functions have been assigned to 11 of the 16 riboswitches described for
C.
difficile [31,32][31][32]. Seven of the riboswitches, classified as class I, behave in an “off” position in the presence of high levels of c-di-GMP, that is, they terminate gene transcription. The remaining four functional riboswitches, defined as class II, react as “on” switches and trigger gene expressions. Elevated levels of c-di-GMP prevent the transcription of flagellar genes
[33,34][33][34] and result in biofilm formation (
Figure 1, left panel). Strains with mutations in flagellar genes
fliC and
fliD produced higher levels of TcdA and TcdB
[35]. A decrease in the transcription of
sigD, located on operon
flgB, represses the expression of genes encoding the synthesis of chemotaxis proteins, cell wall proteins (e.g., collagen-binding protein CbpA), and putative membrane transport proteins
[36]. Mutations in
sigD and flagellar genes
fliF,
fliG, and fliM resulted in loss of motility and a significant decrease in the expression of toxin genes
[30].
Binding of c-di-GMP to Cdi-2-4, one of the four class II c-di-GMP riboswitches located directly upstream of the type IV pili (T4P) primary locus, upregulates the transcription of TFP (type IV pilus) genes and stimulates the aggregation of
C. difficile cells
[37]. In the absence of c-di-GMP, Cdi-2-4 induces transcription termination and prevents the expression of TFP genes
[38]. A 2.54-fold reduction in the expression of
fliC in the hypervirulent
C.
difficile strain R20291 stimulated flagellin production and biofilm formation on glass beads after 7 days
[39,40][39][40]. Downregulation of other flagellar biosynthesis genes such as
flhA,
flbD,
flgE, and
flgD also resulted in biofilm formation
[41].
Our understanding of biofilm formation by
C.
difficile is far from complete, as stimuli for the aggregation of hypervirulent strains (e.g., strains 630 and R20291) differ
[42]. Valiente et al.
[43] reported an increase in cell hydrophobicity for strain R20291 that lacked flagellar post-transcriptional modification. Only two of the five mutants studied had reduced motility; however, all five mutants showed an increase in biofilm formation. This led the authors to conclude that biofilm formation by
C. difficile is not influenced by motility but by hydrophobicity due to the presence of glycan on the flagella. Dapa et al.
[44] suggested that flagella play an important role in the late stages of biofilm formation, as shown with the mutant R20291
fliC ClosTron. Since the pilin
pilA1 gene (CD3513) in
C. difficile is regulated by c-di-GMP acting on the upstream riboswitch Cdi-2-4
[32[32][45],
45], pili may be required for initial adhesion to epithelial cells and initiate biofilm formation
[34]. Pili does, however, not promote late-stage biofilm formation
[39].
The importance of cell structure in biofilm formation cannot be ignored. Poquet et al.
[41] showed that cells in biofilms have upregulated phospholipid metabolism, active acyl carrier proteins, and increased fatty acid synthesis. Increased production of fatty acids was also reported for cells of
Bacillus subtilis in biofilms
[46]. Mutants of
C. difficile with a deficient
lcpB gene and inability to deposit PSII teichoic acids at the cell surface
[41] were elongated, larger in diameter, formed abnormal septa, and grew slower
[47,48,49][47][48][49]. Cell wall-binding protein Cwp11 (CD2795), cell surface protein Cwp10 (CD2796), and calcium-binding adhesion protein (CD2797) are regulated by c-di-GMP
[32]. Cwp11 is released in the “secretome” during biofilm formation
[41,50][41][50].
A mutation in the
prkC gene of
C. difficile 630Δ
erm resulted in increased biofilm formation after 24 h, but only in the presence of bile salt DOC
[51]. The function of
prkC in
C.
difficile remains unknown
[51]. Mutation of the
dnaK gene of strain 630Δ
erm resulted in the disruption of DnaK synthesis and thus protein folding but also led to a significant increase in biofilm formation and cell elongation
[52]. Similar results were recorded when
lexA, which encodes the transcriptional repressor LexA in
C. difficile R20291, was disrupted
[47,53][47][53]. Other genes attributed to
C. difficile biofilm formation are
spo0A [23[23][24][54],
24,54], quorum sensing regulator
luxS [44[44][45],
45], and germination receptor
sleC [54]. Inactivation of the chaperones
dnaK and
hfq changes the cells to become temperature-sensitive and increases biofilm formation
[52,55][52][55].
Iron plays a key role in the growth of pathogenic bacteria, including
C. difficile [56]. Ferrous iron is required for
C. difficile to colonize the large bowel
[57].
C. difficile regulates iron transport with three membrane-bound ferrous iron transporters (FeoBs), of which FeoB1 is produced in the highest quantity under iron-limiting conditions
[58]. Although iron stimulates the growth of
C. difficile and renders the species more resistant to MNZ
[59], it is not known whether an increase in FeoB1 leads to elevated levels of TcdA and TcdB.
Extracellular DNA (eDNA) is a major component of
C.
difficile biofilms
[44,60][44][60]. Hypervirulent
C. difficile 027 strains are rich in prophages and mobile genetic elements
[61]. DNA released from lysed cells may support biofilm formation, as observed for
Staphylococcus aureus and
Pseudomonas aeruginosa. In both these species, cell lysis in biofilms is controlled by signals regulating quorum sensing
[62,63,64][62][63][64]. In
C.
difficile biofilms, S-ribosylhomocysteinase (LuxS) induces prophages, which likely contribute to biofilm formation
[55]. In LuxS mutants, on the other hand, downregulated prophage loci are conserved among
C.
difficile strains, specifically region 2 encoding a phiC2-like phi-027 phage
[40,61,65][40][61][65].
In a complex system, such as the human GIT, metabolites and enzymes produced by bacteria have a profound influence on the microbial population. Bile salt hydrolase (BSH), for instance, produced by
Bacteroides ovatus, inhibited the growth of
C. difficile [66].
Bacteroides fragilis inhibited the growth of wild-type strains of
C. difficile in biofilms when cultured together
[55]. Elevated levels of succinate produced by
B.
fragilis increased the regulation of succinate metabolism by
C.
difficile [41,67][41][67]. With the upregulation of
sucD (succinate-CoA ligase [ADP-forming] subunit alpha), increased expressions of
accB (biotin carboxyl carrier protein of acetyl-CoA carboxylase),
abfH (4-hydroxybutyrate dehydrogenase),
abfT (4-hydroxybutyrate CoA-transferase),
abfD (4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA-delta-isomerase), and
cat1 (catalase-1) were noted
[55]. Other genes of
C. difficile were downregulated, e.g.,
bcd2 and
idhA, encoding butyryl-CoA dehydrogenase and (r)-2-hydroxyisocaproate dehydrogenase, respectively
[55].
When cells of
C. difficile deficient in
luxS were co-cultured with
B.
fragilis, biofilm formation by the mutant was much weaker than when the same experiment was performed with the wild-type strain of
C. difficile [55]. This indicated that AI2/LuxS is involved in facilitating
B.
fragilis-induced inhibition of
C.
difficile. Poquet et al.
[41] also reported the downregulation of genes involved in carbohydrate metabolism. Both studies have shown that changes in carbohydrate metabolism favor the growth of
B.
fragilis at the expense of
C.
difficile. Thus, repression of carbohydrate metabolism plays a major role in biofilm formation. However, the signaling molecules orchestrating the downregulation of genes involved in key metabolic pathways are unknown. Planktonic cells of
B. fragilis and
C. difficile have no effect on each other’s growth
[55], suggesting that the cells must be in close contact with each other. Biofilm formation by
C. difficile is a multifaceted and complex process. For more information on biofilms and hypervirulence, refer to Taggart et al.
[42].