Microscopy methods for Clostridioides difficile.

Microscopic technologies including light and fluorescent, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and cryo-electron microscopy have been widely utilized to visualize Clostridioides difficile at the molecular, cellular, community, and structural biology level. This comprehensive review summarizes the microscopy tools (fluorescent and reporter system) in their use to study different aspects of C. difficile life cycle and virulence (sporulation, germination) or applications (detection of C. difficile or use of antimicrobials). With these developing techniques, microscopy tools will be able to find broader applications and address more challenging questions to study C. difficile and C. difficile infection.

Entry of spores into intestinal epithelial cells contributes to recurrence of Clostridioides difficile infection.

Clostridioides difficile spores produced during infection are important for the recurrence of the disease. Here, we show that C. difficile spores gain entry into the intestinal mucosa via pathways dependent on host fibronectin-α5ß1 and vitronectin-αvß1. The exosporium protein BclA3, on the spore surface, is required for both entry pathways. Deletion of the bclA3 gene in C. difficile, or pharmacological inhibition of endocytosis using nystatin, leads to reduced entry into the intestinal mucosa and reduced recurrence of the disease in a mouse model. Our findings indicate that C. difficile spore entry into the intestinal barrier can contribute to spore persistence and infection recurrence, and suggest potential avenues for new therapies.

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

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.

Gold Nanoclusters as an Antibacterial Alternative Against Clostridium difficile.

BACKGROUND: Clostridium difficile infection (CDI) has become one of the most important factors threatening human health, and about 20-30% antibiotic-related diarrhea cases and almost all pseudomembranous enteritis cases are related to CDI. The high recurrence of Clostridium difficile (C. difficile) and the emergence of drug resistance make clinical treatment of CDI difficult. Therefore, there is an urgent need to develop a non-antibiotic-alternative therapy against CDI. Gold nanoclusters (AuNCs) can better interact with bacteria due to its ultrasmall size. The aim of the present study was to explore whether AuNCs could be used as an antibacterial agent to kill C. difficile. METHODS: AuNCs and C. difficile were co-cultivated in an anaerobic atmosphere to evaluate the bactericidal effect of AuNCs. The bacterial growth rate was estimated by using two concentrations of AuNCs (50 and 100 µM). The damage of AuNCs to C. difficile is detected by SYTOX Green staining methods and SEM image analysis. The mechanism of AuNCs on C. difficile was explored by reactive oxygen species (ROS) detection. The toxic effect of AuNCs on human cells was evaluated by MTT method. RESULTS: AuNCs (100 µM) killed C. difficile drastically. AuNCs increased the release of ROS by about 5 fold and destroyed the membrane integrity of C. difficile cells without causing significant toxic effect on human cells. CONCLUSION: AuNCs showed great potential as an alternative to traditional antibiotics in killing C. difficile and may prove to be an alternative to treat CDI.

Surface morphology differences in Clostridium difficile spores, based on different strains and methods of purification.

Infections linked to Clostridium difficile are a significant cause of suffering. In hospitals, the organism is primarily acquired through the faecal-oral route as spores excreted by infected patients contaminate the healthcare environment. We previously reported that members of the C. difficile group varied widely in their ability to adhere to stainless steel and proposed that these differences were a consequence of variations in spore architecture. In this study of clinical isolates and spore coat protein mutants of C. difficile we identified three distinct spore surfaces morphotypes; smooth, bag-like and "pineapple-like" using scanning electron microscopy (SEM). The frequency of each morphotype in a spore population derived from a single isolate varied depending on the host strain and the method used to produce and purify the spores. Our results suggest that the inclusion of a sonication step in the purification process had a marked effect on spore structure. In an attempt to link differences in spore appearance with key structural spore proteins we compared the morphology of spores of CD630 to those produced by CD630 variants lacking either CotE or BclA. While SEM images revealed no obvious structural differences between CD630 and its mutants we did observe significant differences (p < 0.001) in relative hydrophobicity suggesting that modifications had occurred but not at a level to be detectable by SEM. In conclusion, we observed significant variation in the spore morphology of clinical isolates of C. difficile due in part to the methods used to produce them. Sonication in particular can markedly change spore appearance and properties. The results of this study highlight the importance of adopting "standard" methods when attempting to compare results between studies and to understand the significance of their differences.

Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence.

Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies and that bacteria derived from the isolates display discrete motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein colony morphology regulators RST (CmrRST), which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of flagella and type IV pili, suggesting a novel mechanism of cell migration in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration on surfaces. In a hamster model of acute C. difficile disease, the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.

Antibacterial Activity of Bifidobacterium breve Against Clostridioides difficile.

Bifidobacterium breve (YH68) is widely used in the fields of food fermentation and biomedicine. In this study, we explored the antibacterial activity of the cell free culture supernatant (CFCS) of YH68 against Clostridioides difficile ATCC 9689 (CD) by measuring multiple indexes, including the growth, spores production, toxin A/B production, and the expression levels of the tcdA and tcdB genes of CD. In addition, we examined the changes in major cellular functional groups, structures, permeability, integrity, and the proton motive force (PMF) of the cytoplasmic membrane. The results showed that double-dilution ratio of YH68-CFCS (3 × 109 CFU/mL) was the MIC value. The cell density, spores production, and the toxin production of CD treated with YH68-CFCS were lower than that of the control (p < 0.05). In addition, the gene expression levels of tcdA and tcdB in CD treated with YH68-CFCS were significant downregulated (p < 0.05). Marked differences were observed in the cell membrane and cell wall by a FT-IR spectroscopy and SEM. Analysis of the cell membrane permeability and integrity of the CD cells revealed that YH68-CFCS induced the leakage of a large amount of intracellular K+, inorganic phosphate, ATP, nucleic acids and proteinaceous substances. Furthermore, PMF analysis indicated that there was a significant change in Δψ and ΔpH. These findings demonstrated that the antibacterial activity of YH68-CFCS against CD involved the inhibition of growth, spore production, toxin production, and virulence genes expression; a consumption of PMF in the cytoplasmic membrane, the formation of pore in the cell membrane, together with the enhanced cell membrane permeability; and, eventually, cell completely disintegration.

Biofilm-derived spores of Clostridioides (Clostridium) difficile exhibit increased thermotolerance compared to planktonic spores.

Biofilm-derived spores of strains of four ribotypes (001, 020, 027 & 078) of Clostridioides (Clostridium) difficile were found to exhibit increased thermotolerance compared to spores produced in planktonic culture. In addition, 'thick' and 'thin' exosporium morphotypes described previously were visualised by electron microscopy in both biofilm and planktonic spores.

Antibacterial activity of rhodomyrtone on Clostridium difficile vegetative cells and spores in vitro.

The increasing incidence and severity of diarrhoea and colitis caused by Clostridium difficile, together with a high rate of relapse following treatment with currently recommended antimicrobials, calls for novel interventions for C. difficile infection (CDI). Rhodomyrtone, a bioactive compound derived from the leaves of the rose myrtle (Rhodomyrtus tomentosa) has demonstrated antibacterial activity against several Gram-positive bacteria. This study compared the in vitro antimicrobial activity of rhodomyrtone on C. difficile with that of vancomycin, a recommended agent for the treatment of CDI. Determination of the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of rhodomyrtone and vancomycin for ten C. difficile isolates showed that the MICs of rhodomyrtone for C. difficile vegetative cells (0.625-2.5 mg/L) were comparable with that of vancomycin (1.25 mg/L), but the MBCs of rhodomyrtone (1.25-5 mg/L) were significantly lower than those for vancomycin (5 mg/L to ˃40 mg/L; P < 0.001). Time-kill assays showed rapid bactericidal activity for rhodomyrtone, with ≥99% killing within 4 h. Rhodomyrtone was also four-fold more potent than vancomycin in inhibiting C. difficile spore outgrowth. Transmission electron microscopy of rhodomyrtone-treated C. difficile revealed cell lysis and evidence of defective cell division and spore formation. These studies indicate that rhodomyrtone should be further investigated as a potential treatment for CDI.

Clostridium difficile Biofilm.

Clostridium difficile infection (CDI) is an important healthcare-associated disease worldwide, mainly occurring after antimicrobial therapy. Antibiotics administered to treat a number of infections can promote C. difficile colonization of the gastrointestinal tract and, thus, CDI. A rise in multidrug resistant clinical isolates to multiple antibiotics and their reduced susceptibility to the most commonly used antibiotic molecules have made the treatment of CDI more complicated, allowing the persistence of C. difficile in the intestinal environment.Gut colonization and biofilm formation have been suggested to contribute to the pathogenesis and persistence of C. difficile. In fact, biofilm growth is considered as a serious threat because of the related increase in bacterial resistance that makes antibiotic therapy often ineffective. However, although the involvement of the C. difficile biofilm in the pathogenesis and recurrence of CDI is attracting more and more interest, the mechanisms underlying biofilm formation of C. difficile as well as the role of biofilm in CDI have not been extensively described.Findings on C. difficile biofilm, possible implications in CDI pathogenesis and treatment, efficacy of currently available antibiotics in treating biofilm-forming C. difficile strains, and some antimicrobial alternatives under investigation will be discussed here.

Inactivation of the dnaK gene in Clostridium difficile 630 Δerm yields a temperature-sensitive phenotype and increases biofilm-forming ability.

Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood. We previously reported that clinically relevant heat-stress (37-41 °C) resulted in a classical heat-stress response with up-regulation of cellular chaperones. We used ClosTron to construct an insertional mutation in the dnaK gene of C. difficile 630 Δerm. The dnaK mutant exhibited temperature sensitivity, grew more slowly than C. difficile 630 Δerm and was less thermotolerant. Furthermore, the mutant was non-motile, had 4-fold lower expression of the fliC gene and lacked flagella on the cell surface. Mutant cells were some 50% longer than parental strain cells, and at optimal growth temperatures, they exhibited a 4-fold increase in the expression of class I chaperone genes including GroEL and GroES. Increased chaperone expression, in addition to the non-flagellated phenotype of the mutant, may account for the increased biofilm formation observed. Overall, the phenotype resulting from dnaK disruption is more akin to that observed in Escherichia coli dnaK mutants, rather than those in the Gram-positive model organism Bacillus subtilis.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment.

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (> 50% reduction in cell length for each antibiotic; P< 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug's MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence.

Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA- and lcpB- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA- and lcpB- strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence.

A Fluorescent Reporter for Single Cell Analysis of Gene Expression in Clostridium difficile.

Genetically identical cells growing under homogeneous growth conditions often display cell-cell variation in gene expression. This variation stems from noise in gene expression and can be adaptive allowing for division of labor and bet-hedging strategies. In particular, for bacterial pathogens, the expression of phenotypes related to virulence can show cell-cell variation. Therefore, understanding virulence-related gene expression requires knowledge of gene expression patterns at the single cell level. We describe protocols for the use of fluorescence reporters for single cell analysis of gene expression in the human enteric pathogen Clostridium difficile, a strict anaerobe. The reporters are based on modified versions of the human DNA repair enzyme O ( 6)-alkylguanine-DNA alkyltransferase, called SNAP-tag and CLIP-tag. SNAP becomes covalently labeled upon reaction with O ( 6)-benzylguanine conjugated to a fluorophore, whereas CLIP is labeled by O ( 6)-benzylcytosine conjugates. SNAP and CLIP labeling is orthogonal allowing for dual labeling in the same cells. SNAP and CLIP cassettes optimized for C. difficile can be used for quantitative studies of gene expression at the single cell level. Both the SNAP and CLIP reporters can also be used for studies of protein subcellular localization in C. difficile.

Isolating and Purifying Clostridium difficile Spores.

The ability for the obligate anaerobe, Clostridium difficile to form a metabolically dormant spore is critical for the survival of this organism outside of the host. This spore form is resistant to a myriad of environmental stresses, including heat, desiccation, and exposure to disinfectants and antimicrobials. These intrinsic properties of spores allow C. difficile to survive long-term in an oxygenated environment, to be easily transmitted from host-to-host, and to persist within the host following antibiotic treatment. Because of the importance of the spore form to the C. difficile life cycle and treatment and prevention of C. difficile infection (CDI), the isolation and purification of spores are necessary to study the mechanisms of sporulation and germination, investigate spore properties and resistances, and for use in animal models of CDI. Here we provide basic protocols, in vitro growth conditions, and additional considerations for purifying C. difficile spores for a variety of downstream applications.

Inducing and Quantifying Clostridium difficile Spore Formation.

The Gram-positive nosocomial pathogen Clostridium difficile induces sporulation during growth in the gastrointestinal tract. Sporulation is necessary for this obligate anaerobe to form metabolically dormant spores that can resist antibiotic treatment, survive exit from the mammalian host, and transmit C. difficile infections. In this chapter, we describe a method for inducing C. difficile sporulation in vitro. This method can be used to study sporulation and maximize spore purification yields for a number of C. difficile strain backgrounds. We also describe procedures for visualizing spore formation using phase-contrast microscopy and for quantifying the efficiency of sporulation using heat resistance as a measure of functional spore formation.

Ultrastructure Variability of the Exosporium Layer of Clostridium difficile Spores from Sporulating Cultures and Biofilms.

UNLABELLED: The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the morphotype responsible for transmission, infection, and persistence, and the outermost exosporium layer is likely to play a major role in spore-host interactions during recurrent infections, contributing to the persistence of the spore in the host. A recent study (M. Pizarro-Guajardo, P. Calderón-Romero, P. Castro-Córdova, P. Mora-Uribe, and D. Paredes-Sabja, Appl Environ Microbiol 82:2202-2209, 2016, http://dx.doi.org/10.1128/AEM.03410-15) demonstrated by transmission electron microscopy the presence of two ultrastructural morphotypes of the exosporium layer in spores formed from the same sporulating culture. However, whether these distinct morphotypes appeared due to purification techniques and whether they appeared during biofilm development remain unclear. In this communication, we demonstrate through transmission electron microscopy that these two exosporium morphotypes are formed under sporulation conditions and are also present in spores formed during biofilm development. In summary, this work provides definitive evidence that in a population of sporulating cells, spores with a thick outermost exosporium layer and spores with a thin outermost exosporium layer are formed. IMPORTANCE: Clostridium difficile spores are recognized as the morphotype of persistence and transmission of C. difficile infections. Spores of C. difficile are intrinsically resistant to all known antibiotic therapies. Development of spore-based removal strategies requires a detailed knowledge of the spore surface for proper antigen selection. In this context, in this work we provide definitive evidence that two types of spores, those with a thick outermost exosporium layer and those with a thin outermost exosporium layer, are formed in the same C. difficile sporulating culture or during biofilm development.

A novel method for imaging the pharmacological effects of antibiotic treatment on Clostridium difficile.

Clostridium difficile is a significant cause of nosocomial-acquired infection that results in severe diarrhea and can lead to mortality. Treatment options for C. difficile infection (CDI) are limited, however, new antibiotics are being developed. Current methods for determining efficacy of experimental antibiotics on C. difficile involve antibiotic killing rates and do not give insight into the drug's pharmacologic effects. Considering this, we hypothesized that by using scanning electron microscopy (SEM) in tandem to drug killing curves, we would be able to determine efficacy and visualize the phenotypic response to drug treatment. To test this hypothesis, supraMIC kill curves were conducted using vancomycin, metronidazole, fidaxomicin, and ridinilazole. Following collection, cells were either plated or imaged using a scanning electron microscope (SEM). Consistent with previous reports, we found that the tested antibiotics had significant bactericidal activity at supraMIC concentrations. By SEM imaging and using a semi-automatic pipeline for image analysis, we were able to determine that vancomycin and to a lesser extent fidaxomicin and ridinilazole significantly affected the cell wall, whereas metronidazole, fidaxomicin, and ridinilazole had significant effects on cell length suggesting a metabolic effect. While the phenotypic response to drug treatment has not been documented previously in this manner, the results observed are consistent with the drug's mechanism of action. These techniques demonstrate the versatility and reliability of imaging and measurements that could be applied to other experimental compounds. We believe the strategies laid out here are vital for characterizing new antibiotics in development for treating CDI.

Ultrastructural Variability of the Exosporium Layer of Clostridium difficile Spores.

The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the transmission, infectious, and persistent morphotype, and the outermost exosporium layer is likely to play a major role in spore-host interactions during the first contact of C. difficile spores with the host and for spore persistence during recurrent episodes of infection. Although some studies on the biology of the exosporium have been conducted (J. Barra-Carrasco et al., J Bacteriol 195:3863-3875, 2013, http://dx.doi.org/10.1128/JB.00369-13; J. Phetcharaburanin et al., Mol Microbiol 92:1025-1038, 2014, http://dx.doi.org/10.1111/mmi.12611), there is a lack of information on the ultrastructural variability and stability of this layer. In this work, using transmission electron micrographs, we analyzed the variability of the spore's outermost layers in various strains and found distinctive variability in the ultrastructural morphotype of the exosporium within and between strains. Through transmission electron micrographs, we observed that although this layer was stable during spore purification, it was partially lost after 6 months of storage at room temperature. These observations were confirmed by indirect immunofluorescence microscopy, where a significant decrease in the levels of two exosporium markers, the N-terminal domain of BclA1 and CdeC, was observed. It is also noteworthy that the presence of the exosporium marker CdeC on spores obtained from C. difficile biofilms depended on the biofilm culture conditions and the strain used. Collectively, these results provide information on the heterogeneity and stability of the exosporium surface of C. difficile spores. These findings have direct implications and should be considered in the development of novel methods to diagnose and/or remove C. difficile spores by using exosporium proteins as targets.

Subinhibitory concentrations of metronidazole increase biofilm formation in Clostridium difficile strains.

Resistance mechanism to metronidazole is still poorly understood, even if the number of reports on Clostridium difficile strains with reduced susceptibility to this antibiotic is increasing. In this study, we investigated the ability of the C. difficile strains 7032994, 7032985 and 7032989, showing different susceptibility profiles to metronidazole but all belonging to the PCR ribotype 010, to form biofilm in vitro in presence and absence of subinhibitory concentrations of metronidazole. The quantitative biofilm production assay performed in presence of metronidazole revealed a significant increase in biofilm formation in both the susceptible strain 7032994 and the strain 7032985 exhibiting a reduced susceptibility to this antibiotic, while antibiotic pressure did not affect the biofilm-forming ability of the stable-resistant strain 7032989. Moreover, confocal microscopy analysis showed an abundant biofilm matrix production by the strains 7032994 and 7032885, when grown in presence of metronidazole, but not in the stable-resistant one. These results seem to demonstrate that subinhibitory concentrations of metronidazole are able to enhance the in vitro biofilm production of the above-mentioned PCR ribotype 010 C. difficile strains, susceptible or with reduced susceptibility to this antibiotic, suggesting a possible role of biofilm formation in the multifactorial mechanism of metronidazole resistance developed by C. difficile.