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Microbiology
Clostridioides difficile is considered a nosocomial pathogen that flares up in patients exposed to antibiotic treatment. However, four out of ten patients diagnosed with C. difficile infection (CDI) acquired the infection from non-hospitalized individuals, many of whom have not been treated with antibiotics. Treatment of recurrent CDI (rCDI) with antibiotics, especially vancomycin (VAN) and metronidazole (MNZ), increases the risk of experiencing a relapse by as much as 70%. Fidaxomicin, on the other hand, proved more effective than VAN and MNZ by preventing the initial transcription of RNA toxin genes. Alternative forms of treatment include quorum quenching (QQ) that blocks toxin synthesis, binding of small anion molecules such as tolevamer to toxins, monoclonal antibodies, such as bezlotoxumab and actoxumab, bacteriophage therapy, probiotics, and fecal microbial transplants (FMTs).
- colonization
- toxin production
- antibiotics
- Clostridioides difficile
- biofilm formation
1. Introduction
Clostridioides difficile is a Gram-positive, sporulating, rod-shaped cell, and obligatory anaerobic [1]. Although C. difficile is considered a nosocomial pathogen that flairs up in patients exposed to antibiotic treatment [2,3], four out of ten patients diagnosed with Clostridioides difficile infection (CDI) acquired the infection from non-hospitalized individuals [4], many of whom have not been treated with antibiotics [5]. Contracting C. difficile may also be through contact with infected animals, including reptiles and birds [6,7]. One to three percent of adults are asymptomatic carriers of C. difficile [8].
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]. 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]. Biofilm formation protects cells from oxygen and antibiotics, including metronidazole (MNZ) and vancomycin (VAN), which are commonly used to treat CDI [16,17].
Treatment of recurrent CDI (rCDI) with antibiotics, especially vancomycin (VAN) and metronidazole (MNZ), increases the risk of experiencing a relapse by as much as 70% [16,17]. Strains becoming resistant to both antibiotics are on the increase. Fidaxomicin, which prevents the initial transcription of RNA toxin genes, proved more effective than VAN and MNZ. The efficacy of antibiotics is, however, hampered by their poor ability to penetrate biofilms. More research is required on alternatives to antibiotics, such as non-antimicrobial agents sequestering or inactivating toxin production, quenching of genes (quorum quenching, QQ), immunization, and bacteriotherapy, including fecal microbial transplants (FMTs).
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]. 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]. 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].
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]. 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]. Increased c-di-GMP levels reduce the expression of tcdA, tcdB, and tcdR [25,26,27,28,29,30]. The tcdR gene encodes an alternative sigma factor SigD (FliA; σ28) that activates the expression of tcdA and tcdB in response to c-di-GMP [25,30]. Degradation of c-di-GMP is controlled by the protein domain EAL (Figure 1) [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]. 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] 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]. 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 [32,45], 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]. 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].
A mutation in the prkC gene of C. difficile 630Δerm 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 630Δerm 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]. Other genes attributed to C. difficile biofilm formation are spo0A [23,24,54], quorum sensing regulator luxS [44,45], 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].
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]. 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]. 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].
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]. 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].
This entry is adapted from the peer-reviewed paper 10.3390/microorganisms11092161
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