**Assays for Measuring** *C. difficile* **Toxin Activity and Inhibition in Mammalian Cells**

Mary Ann Cox, Lorraine D. Hernandez, Pulkit Gupta, Zuo Zhang, Fred Racine and Alex G. Therien

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/68127

#### **Abstract**

*Clostridium difficile* infections (CDIs) are the leading cause of hospital-acquired infectious diarrhea. The symptoms of CDI are caused by two exotoxins, TcdA and TcdB, which are structurally and functionally highly homologous. Both toxins bind to specific receptors on mammalian cells, are internalized through endocytosis, translocate to the cytoplasm, and inactivate Rho-type GTPases via covalent glucosylation. This leads to downstream events that include morphological changes and disruption of epithelial tight junctions, release of pro-inflammatory mediators, and cell death. Assays used to assess the effects of toxins on cells have historically relied on evaluation of cell rounding or quantitation of ATP levels to estimate cell death—assays which can be qualitative and variable. In this chapter, several assays are described that robustly and quantitatively measure early and late toxin-dependent events in cells, including (i) toxin binding, (ii) Rac1 glucosylation, (iii) changes in cellular morphology (measured as dynamic mass redistribution), (iv) loss of epithelial integrity (measured as transepithelial electrical resistance), and (v) cell death (measured as total cellular protein using a colorimetric assay). The assays were validated using the highly specific monoclonal antitoxin antibodies, actoxumab and bezlotoxumab, which neutralize TcdA and TcdB, respectively.

**Keywords:** *C. difficile*, toxins, cell-based assays, epithelial cells, antitoxins

## **1. Introduction**

*Clostridium difficile* (*C. difficile*) is an anaerobic, gram-positive, spore-forming bacterium that colonizes the lower intestinal tract of patients whose normal gut microflora has been disrupted by treatment with broad-spectrum antibiotics [1]. The symptoms of *C. difficile*

infection (CDI)—which include diarrhea and, in severe cases, pseudomembranous colitis, colonic rupture, and death [1, 2]—are caused by two exotoxins, toxin A (TcdA) and toxin B (TcdB) [3]. Both toxins have similar structural and functional characteristics. After binding to specific receptors on the surface of gut epithelial cells, they are internalized through endocytosis, translocate to the cytoplasm, and inactivate Rho-type GTPases via covalent glucosylation [4–7]. This leads to a variety of downstream events, including morphological changes associated with disruption of epithelial tight junctions, release of pro-inflammatory mediators (including interleukin-1β, tumor necrosis factor alpha, and interleukin-8), and eventually cell death [3, 8]. The damaging effects on the gut epithelium and initiation of a host inflammatory response are thought to underlie the clinical manifestation of CDI.

for 30 min at 37°C; these mixtures were then chilled on ice and added to plates of pre-chilled Vero cells (ATCC, Rockville, MD). Plates were incubated for 30 min on ice to allow binding of toxins. Following incubation, plates were washed three times with cold phosphate buffered saline (PBS) and cells were harvested by scraping. Cell membranes were isolated at 4°C with the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Scientific, Grand Island, NY), according to the manufacturer's instructions, and solubilized in a total volume of 100 μL solubilization buffer with HALT protease/phosphatase inhibitors (Thermo Scientific). Following addition of Laemmli sample buffer, samples were incubated for 5 min at 95°C and resolved by SDS PAGE in 4–12% polyacrylamide gels and transferred to a nitrocellulose membrane. The nitrocellulose membrane containing transferred protein was blocked in Odyssey blocking buffer (Li-Cor) followed by incubation with actoxumab, bezlotoxumab, or an anti-cadherin antibody (Cell Signaling Technology, Beverly, MA) as the primary antibody for 1 h at room temperature (RT). After washing, the nitrocellulose membrane was incubated with a goat antihuman IgG antibody coupled to IRDye® 800CW (Li-Cor) for 30 min at RT. After additional

Assays for Measuring *C. difficile* Toxin Activity and Inhibition in Mammalian Cells

http://dx.doi.org/10.5772/68127

113

washing, bands were visualized using the Odyssey imaging system (Li-Cor).

antibody sample was added to separate vials containing 3 × 10<sup>5</sup>

488 and 530 nm, respectively. 10,000 events were measured for each sample.

TcdA, from ribotype 087 (The Native Antigen Company, Upper Heyford, the UK), was fluorescently labeled using the Lightning Link Atto488 Antibody Labeling kit (Novus Biosciences, Littleton, CO) as directed by the manufacturer. About 50 μg of lyophilized TcdA was reconstituted for a minimum of 30 min in sterile ddH2O at RT before adding the LL-modifier buffer. The toxin/LL-modifier buffer solution was transferred to a vial containing the lyophilized Lightning Link mix. The mixture was pipetted up and down and incubated at RT in the dark. After 5 h, LL-quencher buffer was added and incubated at RT in the dark for 30 min and then stored at 4°C until use the following day. Several concentrations of TcdA-Atto488 were incubated with or without 200 μg/ml actoxumab at RT for 60 min, protected from light. Samples were then chilled on ice. Adherent HT29 cells (ATCC, Rockville, MD) were resuspended in the cell medium (McCoy's 5A Modified medium supplemented with 10% FBS, 2 mM glutamine, 0.75% sodium bicarbonate, 100 U/ml penicillin, and 100 U/ml streptomycin), following treatment with Accutase (Innovative Cell Technologies), washed once with cold Dulbecco's phosphate-buffered saline with calcium and magnesium (DPBS++) containing 1% bovine serum albumin (BSA), and then chilled on ice. 100 μL of each toxin/

were incubated on ice in the dark. After 30 min, 1 ml of ice cold DPBS++/1% BSA was added to each sample. To remove unbound toxin, cell suspensions were washed twice with ice cold DPBS++/1% BSA by centrifuging for 5 min at 4°C at 200 × g and removing the supernatant. Washed cells were resuspended in 500 μl cold DPBS++/1% BSA and analyzed by flow cytometry using an LSRII instrument (BD Biosciences) with excitation and emission wavelengths of

cells. After mixing, samples

cells/well in a 384-well collagen-coated plate

. TcdA and TcdB (The Native Antigen Company,

**2.2. TcdA-binding assay (flow cytometry)**

**2.3. Rac1 glucosylation assay**

Vero cells were seeded at a cell density of 5 × 10<sup>3</sup>

and grown overnight at 37°C in 5% CO<sup>2</sup>

Current treatment for *C. difficile* infections includes discontinuing the offending broad-spectrum antibiotic and initiating therapy with narrower spectrum agents such as vancomycin, metronidazole, or fidaxomicin [9, 10]. Unfortunately, these treatments do not directly address the damaging effects of the toxins on the gut and perpetuate the gut dysbiosis that caused CDI in the first place. As a result, up to 25% or more patients successfully cured of an initial episode of CDI with these antibiotics suffer a recurrent episode within days to weeks. To address this, recent approaches to CDI treatment, including vaccines and monoclonal antibodies, have focused on neutralizing the effects of TcdA and TcdB, specifically, rather than the organisms itself [11–13]. Foremost among these novel therapies is bezlotoxumab, the anti-TcdB antibody recently approved by the Food and Drug Administration for reducing recurrence of CDI in patients 18 years of age or older who are receiving antibacterial drug treatment of CDI and are at a high risk for CDI recurrence.

The renewed interest in toxin-directed therapies underscores the importance of having robust quantitative assays in place to assess the activity of the *C. difficile* toxins. Historically, studying the effects of TcdA and TcdB on mammalian cells has been hampered by time-consuming and subjective assays that rely, for example, on visualization of cells to assess cell rounding or on the variable quantitation of ATP levels to measure cell death [13]. Thus, there is a scarcity of robust quantitative assays that measure the various cellular events associated with the intoxication cascade, making it difficult to evaluate new toxin-directed agents. In this chapter, we describe multiple quantitative cell-based assays that were newly developed, or adapted and optimized from previous reports, and used to interrogate the effect of the *C. difficile* toxins on epithelial cells. The assays are validated using the highly specific and potent antitoxin antibodies, actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively [13–15].

## **2. Materials and methods**

#### **2.1. TcdA- and TcdB-binding assay (Western blot)**

TcdA (1 μg/ml) or TcdB (0.1 μg/ml) (The Native Antigen Company, Upper Heyford, the UK and tgcBIOMICS, Bingen, Germany) was incubated with or without 200 μg/ml actoxumab or bezlotoxumab in Vero cell culture medium (Eagle's minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin) for 30 min at 37°C; these mixtures were then chilled on ice and added to plates of pre-chilled Vero cells (ATCC, Rockville, MD). Plates were incubated for 30 min on ice to allow binding of toxins. Following incubation, plates were washed three times with cold phosphate buffered saline (PBS) and cells were harvested by scraping. Cell membranes were isolated at 4°C with the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Scientific, Grand Island, NY), according to the manufacturer's instructions, and solubilized in a total volume of 100 μL solubilization buffer with HALT protease/phosphatase inhibitors (Thermo Scientific). Following addition of Laemmli sample buffer, samples were incubated for 5 min at 95°C and resolved by SDS PAGE in 4–12% polyacrylamide gels and transferred to a nitrocellulose membrane. The nitrocellulose membrane containing transferred protein was blocked in Odyssey blocking buffer (Li-Cor) followed by incubation with actoxumab, bezlotoxumab, or an anti-cadherin antibody (Cell Signaling Technology, Beverly, MA) as the primary antibody for 1 h at room temperature (RT). After washing, the nitrocellulose membrane was incubated with a goat antihuman IgG antibody coupled to IRDye® 800CW (Li-Cor) for 30 min at RT. After additional washing, bands were visualized using the Odyssey imaging system (Li-Cor).

#### **2.2. TcdA-binding assay (flow cytometry)**

infection (CDI)—which include diarrhea and, in severe cases, pseudomembranous colitis, colonic rupture, and death [1, 2]—are caused by two exotoxins, toxin A (TcdA) and toxin B (TcdB) [3]. Both toxins have similar structural and functional characteristics. After binding to specific receptors on the surface of gut epithelial cells, they are internalized through endocytosis, translocate to the cytoplasm, and inactivate Rho-type GTPases via covalent glucosylation [4–7]. This leads to a variety of downstream events, including morphological changes associated with disruption of epithelial tight junctions, release of pro-inflammatory mediators (including interleukin-1β, tumor necrosis factor alpha, and interleukin-8), and eventually cell death [3, 8]. The damaging effects on the gut epithelium and initiation of a

host inflammatory response are thought to underlie the clinical manifestation of CDI.

are at a high risk for CDI recurrence.

112 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

**2. Materials and methods**

**2.1. TcdA- and TcdB-binding assay (Western blot)**

Current treatment for *C. difficile* infections includes discontinuing the offending broad-spectrum antibiotic and initiating therapy with narrower spectrum agents such as vancomycin, metronidazole, or fidaxomicin [9, 10]. Unfortunately, these treatments do not directly address the damaging effects of the toxins on the gut and perpetuate the gut dysbiosis that caused CDI in the first place. As a result, up to 25% or more patients successfully cured of an initial episode of CDI with these antibiotics suffer a recurrent episode within days to weeks. To address this, recent approaches to CDI treatment, including vaccines and monoclonal antibodies, have focused on neutralizing the effects of TcdA and TcdB, specifically, rather than the organisms itself [11–13]. Foremost among these novel therapies is bezlotoxumab, the anti-TcdB antibody recently approved by the Food and Drug Administration for reducing recurrence of CDI in patients 18 years of age or older who are receiving antibacterial drug treatment of CDI and

The renewed interest in toxin-directed therapies underscores the importance of having robust quantitative assays in place to assess the activity of the *C. difficile* toxins. Historically, studying the effects of TcdA and TcdB on mammalian cells has been hampered by time-consuming and subjective assays that rely, for example, on visualization of cells to assess cell rounding or on the variable quantitation of ATP levels to measure cell death [13]. Thus, there is a scarcity of robust quantitative assays that measure the various cellular events associated with the intoxication cascade, making it difficult to evaluate new toxin-directed agents. In this chapter, we describe multiple quantitative cell-based assays that were newly developed, or adapted and optimized from previous reports, and used to interrogate the effect of the *C. difficile* toxins on epithelial cells. The assays are validated using the highly specific and potent antitoxin antibodies, actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively [13–15].

TcdA (1 μg/ml) or TcdB (0.1 μg/ml) (The Native Antigen Company, Upper Heyford, the UK and tgcBIOMICS, Bingen, Germany) was incubated with or without 200 μg/ml actoxumab or bezlotoxumab in Vero cell culture medium (Eagle's minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin) TcdA, from ribotype 087 (The Native Antigen Company, Upper Heyford, the UK), was fluorescently labeled using the Lightning Link Atto488 Antibody Labeling kit (Novus Biosciences, Littleton, CO) as directed by the manufacturer. About 50 μg of lyophilized TcdA was reconstituted for a minimum of 30 min in sterile ddH2O at RT before adding the LL-modifier buffer. The toxin/LL-modifier buffer solution was transferred to a vial containing the lyophilized Lightning Link mix. The mixture was pipetted up and down and incubated at RT in the dark. After 5 h, LL-quencher buffer was added and incubated at RT in the dark for 30 min and then stored at 4°C until use the following day. Several concentrations of TcdA-Atto488 were incubated with or without 200 μg/ml actoxumab at RT for 60 min, protected from light. Samples were then chilled on ice. Adherent HT29 cells (ATCC, Rockville, MD) were resuspended in the cell medium (McCoy's 5A Modified medium supplemented with 10% FBS, 2 mM glutamine, 0.75% sodium bicarbonate, 100 U/ml penicillin, and 100 U/ml streptomycin), following treatment with Accutase (Innovative Cell Technologies), washed once with cold Dulbecco's phosphate-buffered saline with calcium and magnesium (DPBS++) containing 1% bovine serum albumin (BSA), and then chilled on ice. 100 μL of each toxin/ antibody sample was added to separate vials containing 3 × 10<sup>5</sup> cells. After mixing, samples were incubated on ice in the dark. After 30 min, 1 ml of ice cold DPBS++/1% BSA was added to each sample. To remove unbound toxin, cell suspensions were washed twice with ice cold DPBS++/1% BSA by centrifuging for 5 min at 4°C at 200 × g and removing the supernatant. Washed cells were resuspended in 500 μl cold DPBS++/1% BSA and analyzed by flow cytometry using an LSRII instrument (BD Biosciences) with excitation and emission wavelengths of 488 and 530 nm, respectively. 10,000 events were measured for each sample.

#### **2.3. Rac1 glucosylation assay**

Vero cells were seeded at a cell density of 5 × 10<sup>3</sup> cells/well in a 384-well collagen-coated plate and grown overnight at 37°C in 5% CO<sup>2</sup> . TcdA and TcdB (The Native Antigen Company, Upper Heyford, the UK and tgcBIOMICS, Bingen, Germany) were serially diluted in Vero cell culture medium, and 50 μl was added to each well. For assays determining neutralization effects of actoxumab and bezlotoxumab, TcdA and TcdB were pre-incubated at 90% effective concentrations (EC90) with actoxumab and bezlotoxumab, respectively (various concentrations), for 1 h at RT in Vero cell culture medium, prior to addition of cells as above. Following incubation at 37°C in 5% CO<sup>2</sup> for 3 h, medium containing toxin alone or toxin+antibody was removed by aspiration. Cells were immediately fixed with 50 μl/well fixing solution (4% paraformaldehyde in modified Dulbecco's phosphate-buffered saline (DPBS/modified)) for 1 h at RT. Following fixation, cells were washed four times for 5 min with 50 μl/well permeabilization solution (0.1% Triton-X-100 in DPBS/modified) at RT with gentle shaking. Cells were then blocked with 50 μl/well Odyssey blocking buffer (Li-Cor) overnight at 4°C. After removing blocking buffer, cells were incubated with 25 μl/well mouse anti-Rac1 (BD Biosciences #610651, recognizing non-glucosylated Rac1), or anti-Rac1 clone 23A8 (Millipore #05-389, recognizing total Rac1), diluted at 1:75 and 1:200, respectively, in Odyssey blocking buffer and incubated for 2 h at RT with gentle shaking. Cells were washed four times for 5 min with 50 μl/well wash solution (0.1% tween 20 in DPBS/modified) at RT with gentle shaking. Cells were then incubated with 25 μl/well secondary antibodies (IRDye 800 CW goat anti-mouse and CellTag 700 stain, diluted at 1:800 and 1:1000, respectively, in Odyssey blocking buffer) at RT for 1 h with gentle shaking protected from light. Cells were again washed four times for 5 min with 50 μl/well wash solution at room temperature with gentle shaking. After the final wash, any remaining solution was removed from the wells, and the plates were scanned on the Li-Cor Odyssey classic (Li-Cor) with detection in both 700 and 800 nm channels (A700 and A800). Cell number normalization/well was calculated using the ratio of A800/A700, and remaining percent of non-glucosylated Rac1 was determined using the ratio of normalized A800 of treated cells/normalized A800 of untreated cells multiplied by 100. Analysis was performed with GraphPad Prism (version 6.04) using the 4-parameter nonlinear regression formula.

**2.5. Transepithelial electrical resistance (TER) assay**

≥ 600 Ω cm<sup>2</sup>

**2.6. Sulforhodamine B assay**

with 5% CO<sup>2</sup>

To initiate the 2-dimensional culture system, 0.5–1 × 105

were seeded into each well of the 24-well insert plates (Falcon #351181 HTS Multiwell Insert System—1.0 um pore size/PET membrane), with 250 μl Caco-2 cell culture medium (EMEM supplemented with 10% FBS, 1× non-essential amino acid, 0.075% sodium bicarbonate, 100 U/ml penicillin, and 100 U/ml streptomycin) in the apical chamber and 800 μl in the basolateral

differentiation and confluency, which were confirmed by plateauing of the TER reading at

Millipore, Billerica, MA, USA). To assess the effect of toxins on the cell monolayer, TcdA and TcdB (The Native Antigen Company, Upper Heyford, UK and tgcBIOMICS, Bingen, Germany) were added to the apical chamber. To evaluate the ability of the antibodies to neutralize toxin effects, actoxumab or bezlotoxumab was added to the apical chamber immediately before addition of TcdA or TcdB to the apical chamber. For neutralization studies, 10 ng/ml TcdA was combined with various concentrations (from 0 to 50 μg/ml) of actoxumab, and 100 ng/ml TcdB was combined with various concentrations (from 0 to 100 μg/ml) of bezlotoxumab. TER measurements were obtained immediately before and, at 6, 24, and 48 h, after addition of toxins/antibodies to the apical chamber. TER values were normalized to values obtained in the

. TER was measured using the Epithelial Volt-Ohm Meter Millicell ERS-2 (EMD

Assays for Measuring *C. difficile* Toxin Activity and Inhibition in Mammalian Cells

chamber. Caco-2 cells were cultured for at least 14 days at 37°C with 5% CO<sup>2</sup>

absence of toxin at each time point to account for minor time-dependent variability.

respectively, and incubated overnight at 37°C with 5% CO<sup>2</sup>

To study the effects of *C. difficile* toxins on cytotoxicity and the ability of actoxumab and bezlotoxumab to neutralize those effects, the sulforhodamine B (SRB) assay was employed to measure total cellular protein as a surrogate of cell number [16]. Vero or T-84 (T-84 growth medium—DMEM/F-12K supplemented with 5% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin) cells were seeded into 96-well plates at 2000 and 3000 cells/well,

fied TcdA and TcdB (tgcBIOMICS, Bingen, Germany) were diluted in the appropriate growth media, incubated at 37°C for 2 h, and added to cells. Following a 24-h incubation at 37°C

200 μl per well of complete medium was added, and plates were incubated for an additional 48 (Vero cells) or 72 h (T-84 cells). After incubation, the medium was removed, and cells were fixed with 100 μl/well of 10% cold trichloroacetic acid (TCA) for 1 h at 4°C. The TCA was then removed and plates were washed four times with distilled water. After washing, 100 μl/well of 100 μg/ml SRB in 10% acetic acid was added, and plates were incubated for 15 min at room temperature (RT). The plates were then washed four times with 10% acetic acid and air-dried. Addition of 150 μl/well of 10 mM tris was followed by a 10-min incubation at RT with shaking. Absorbance was then measured at 570 nm with a SpectraMax plate reader (Molecular Biosystems). Treated and untreated cells were compared, and 90% lethal concentrations (LC90 , that is, concentrations of TcdA or TcdB required to cause a 90% reduction in cell number) were calculated. Antibody-mediated toxin neutralization was measured by incubating serially diluted actoxumab or bezlotoxumab (at concentrations ranging from 1 ng/ml to 192 μg/ml) with purified TcdA or TcdB at LC90 for 2 h at 37°C. The toxin/antibody

, the medium was aspirated and plates were washed twice with PBS. About

Caco-2 cells (ATCC, Rockville, MD)

http://dx.doi.org/10.5772/68127

. Varying concentrations of puri-

to ensure full

115

#### **2.4. Dynamic mass redistribution (Epic) assay**

Vero cells were seeded at a cell density of 5 × 10<sup>3</sup> cells/well in a 384-well fibronectin-coated Epic plate (Corning #5042) and grown overnight at 37°C in 5% CO<sup>2</sup> . On the day of assay, medium was aspirated and replaced with 40 μl/well assay buffer (HBSS in 20 mM HEPES) and equilibrated at RT for 1 h. TcdA and TcdB (The Native Antigen Company, Upper Heyford, UK and tgcBIOMICS, Bingen, Germany) were serially diluted in assay buffer and equilibrated at RT for approximately 10 min. For assays determining neutralization effects of actoxumab and bezlotoxumab, TcdA and TcdB were pre-incubated at EC90 concentrations for 1 h at RT with actoxumab and bezlotoxumab, respectively (various concentrations). Following pre-incubations, 10 μl/well of the toxins alone or toxin/antibody solutions were added to Vero cells using a Matrix Platemate (Thermo Scientific) and gently mixed. The plate was read every 12 s for 200 min using the Epic BT-157900 (Corning). As a baseline, wells containing assay buffer alone were used. The dynamic mass redistribution (DMR) values were recorded at 180 min at which point the signal had plateaued (not shown). The recorded DMR values (corrected for assay buffer alone) were collected with EpicAnalyzer software and analyzed with GraphPad Prism (version 6.04) using the four-parameter nonlinear regression formula.

#### **2.5. Transepithelial electrical resistance (TER) assay**

Upper Heyford, the UK and tgcBIOMICS, Bingen, Germany) were serially diluted in Vero cell culture medium, and 50 μl was added to each well. For assays determining neutralization effects of actoxumab and bezlotoxumab, TcdA and TcdB were pre-incubated at 90% effective concentrations (EC90) with actoxumab and bezlotoxumab, respectively (various concentrations), for 1 h at RT in Vero cell culture medium, prior to addition of cells as above. Following

was removed by aspiration. Cells were immediately fixed with 50 μl/well fixing solution (4% paraformaldehyde in modified Dulbecco's phosphate-buffered saline (DPBS/modified)) for 1 h at RT. Following fixation, cells were washed four times for 5 min with 50 μl/well permeabilization solution (0.1% Triton-X-100 in DPBS/modified) at RT with gentle shaking. Cells were then blocked with 50 μl/well Odyssey blocking buffer (Li-Cor) overnight at 4°C. After removing blocking buffer, cells were incubated with 25 μl/well mouse anti-Rac1 (BD Biosciences #610651, recognizing non-glucosylated Rac1), or anti-Rac1 clone 23A8 (Millipore #05-389, recognizing total Rac1), diluted at 1:75 and 1:200, respectively, in Odyssey blocking buffer and incubated for 2 h at RT with gentle shaking. Cells were washed four times for 5 min with 50 μl/well wash solution (0.1% tween 20 in DPBS/modified) at RT with gentle shaking. Cells were then incubated with 25 μl/well secondary antibodies (IRDye 800 CW goat anti-mouse and CellTag 700 stain, diluted at 1:800 and 1:1000, respectively, in Odyssey blocking buffer) at RT for 1 h with gentle shaking protected from light. Cells were again washed four times for 5 min with 50 μl/well wash solution at room temperature with gentle shaking. After the final wash, any remaining solution was removed from the wells, and the plates were scanned on the Li-Cor Odyssey classic (Li-Cor) with detection in both 700 and 800 nm channels (A700 and A800). Cell number normalization/well was calculated using the ratio of A800/A700, and remaining percent of non-glucosylated Rac1 was determined using the ratio of normalized A800 of treated cells/normalized A800 of untreated cells multiplied by 100. Analysis was performed with GraphPad Prism (version 6.04) using the 4-parameter

for 3 h, medium containing toxin alone or toxin+antibody

cells/well in a 384-well fibronectin-coated

. On the day of assay,

incubation at 37°C in 5% CO<sup>2</sup>

114 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

nonlinear regression formula.

**2.4. Dynamic mass redistribution (Epic) assay**

Vero cells were seeded at a cell density of 5 × 10<sup>3</sup>

Epic plate (Corning #5042) and grown overnight at 37°C in 5% CO<sup>2</sup>

medium was aspirated and replaced with 40 μl/well assay buffer (HBSS in 20 mM HEPES) and equilibrated at RT for 1 h. TcdA and TcdB (The Native Antigen Company, Upper Heyford, UK and tgcBIOMICS, Bingen, Germany) were serially diluted in assay buffer and equilibrated at RT for approximately 10 min. For assays determining neutralization effects of actoxumab and bezlotoxumab, TcdA and TcdB were pre-incubated at EC90 concentrations for 1 h at RT with actoxumab and bezlotoxumab, respectively (various concentrations). Following pre-incubations, 10 μl/well of the toxins alone or toxin/antibody solutions were added to Vero cells using a Matrix Platemate (Thermo Scientific) and gently mixed. The plate was read every 12 s for 200 min using the Epic BT-157900 (Corning). As a baseline, wells containing assay buffer alone were used. The dynamic mass redistribution (DMR) values were recorded at 180 min at which point the signal had plateaued (not shown). The recorded DMR values (corrected for assay buffer alone) were collected with EpicAnalyzer software and analyzed with GraphPad Prism (version 6.04) using the four-parameter nonlinear regression formula.

To initiate the 2-dimensional culture system, 0.5–1 × 105 Caco-2 cells (ATCC, Rockville, MD) were seeded into each well of the 24-well insert plates (Falcon #351181 HTS Multiwell Insert System—1.0 um pore size/PET membrane), with 250 μl Caco-2 cell culture medium (EMEM supplemented with 10% FBS, 1× non-essential amino acid, 0.075% sodium bicarbonate, 100 U/ml penicillin, and 100 U/ml streptomycin) in the apical chamber and 800 μl in the basolateral chamber. Caco-2 cells were cultured for at least 14 days at 37°C with 5% CO<sup>2</sup> to ensure full differentiation and confluency, which were confirmed by plateauing of the TER reading at ≥ 600 Ω cm<sup>2</sup> . TER was measured using the Epithelial Volt-Ohm Meter Millicell ERS-2 (EMD Millipore, Billerica, MA, USA). To assess the effect of toxins on the cell monolayer, TcdA and TcdB (The Native Antigen Company, Upper Heyford, UK and tgcBIOMICS, Bingen, Germany) were added to the apical chamber. To evaluate the ability of the antibodies to neutralize toxin effects, actoxumab or bezlotoxumab was added to the apical chamber immediately before addition of TcdA or TcdB to the apical chamber. For neutralization studies, 10 ng/ml TcdA was combined with various concentrations (from 0 to 50 μg/ml) of actoxumab, and 100 ng/ml TcdB was combined with various concentrations (from 0 to 100 μg/ml) of bezlotoxumab. TER measurements were obtained immediately before and, at 6, 24, and 48 h, after addition of toxins/antibodies to the apical chamber. TER values were normalized to values obtained in the absence of toxin at each time point to account for minor time-dependent variability.

#### **2.6. Sulforhodamine B assay**

To study the effects of *C. difficile* toxins on cytotoxicity and the ability of actoxumab and bezlotoxumab to neutralize those effects, the sulforhodamine B (SRB) assay was employed to measure total cellular protein as a surrogate of cell number [16]. Vero or T-84 (T-84 growth medium—DMEM/F-12K supplemented with 5% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin) cells were seeded into 96-well plates at 2000 and 3000 cells/well, respectively, and incubated overnight at 37°C with 5% CO<sup>2</sup> . Varying concentrations of purified TcdA and TcdB (tgcBIOMICS, Bingen, Germany) were diluted in the appropriate growth media, incubated at 37°C for 2 h, and added to cells. Following a 24-h incubation at 37°C with 5% CO<sup>2</sup> , the medium was aspirated and plates were washed twice with PBS. About 200 μl per well of complete medium was added, and plates were incubated for an additional 48 (Vero cells) or 72 h (T-84 cells). After incubation, the medium was removed, and cells were fixed with 100 μl/well of 10% cold trichloroacetic acid (TCA) for 1 h at 4°C. The TCA was then removed and plates were washed four times with distilled water. After washing, 100 μl/well of 100 μg/ml SRB in 10% acetic acid was added, and plates were incubated for 15 min at room temperature (RT). The plates were then washed four times with 10% acetic acid and air-dried. Addition of 150 μl/well of 10 mM tris was followed by a 10-min incubation at RT with shaking. Absorbance was then measured at 570 nm with a SpectraMax plate reader (Molecular Biosystems). Treated and untreated cells were compared, and 90% lethal concentrations (LC90 , that is, concentrations of TcdA or TcdB required to cause a 90% reduction in cell number) were calculated. Antibody-mediated toxin neutralization was measured by incubating serially diluted actoxumab or bezlotoxumab (at concentrations ranging from 1 ng/ml to 192 μg/ml) with purified TcdA or TcdB at LC90 for 2 h at 37°C. The toxin/antibody mixtures were then added to Vero or T-84 cells as described above and incubated for 24 h at 37°C with 5% CO<sup>2</sup> . The cells were then washed twice with PBS and treated and analyzed as described above.

To assess the cytotoxicity of *C. difficile* toxins derived from bacterial culture supernatants, strain VPI 10463 (ribotype 087) (ATCC) was grown in chopped meat medium (Anaerobe Systems) under anaerobic conditions at 37°C for 72–96 h, and culture supernatants were collected, filtered twice through a 0.22 μm filter, and stored at 4°C. For TcdB immunodepletion, cell culture supernatants were combined and mixed with bezlotoxumab and protein A-agarose beads for 4–6 h at 4°C. After incubation, the beads were removed by centrifugation. Supernatants were then collected, filtered (0.22 μm), and stored at 4°C. Cytotoxicity and antibody-mediated neutralization of the untreated (for determinations on TcdB) or immunodepleted (for determinations on TcdA) supernatants were measured as described above.

## **3. Results**

#### **3.1. Overview of mammalian cell intoxication by TcdA and TcdB**

TcdA and TcdB are large, monomeric proteins (300 and 270 kDa, respectively) with similar structures and functions (**Figure 1**) [17, 18]. The functional domains of the toxins are arranged according to the ABCD model [17]: the N-terminal A domain contains the glucosyltransferase enzymatic activity, the B domain is a putative receptor-binding domain composed of a series of long and short repeats known as combined repetitive oligopeptides (CROPs), the cysteine protease (C) domain is responsible for autocatalytic processing, and the D domain is involved in pore formation and toxin translocation. Both toxins bind to receptors on the surface of the epithelial cells that line the wall of the lower intestine (and possibly other cell types). Once bound, they are internalized via receptor-mediated endocytosis [19]. Acidification of the endosome promotes a conformational change that enables translocation of the N-terminal glucosyltransferase domain of the toxin into the cytoplasm. Cellular inositol hexakisphosphate (InsP6) allows cleavage of the toxin by the cysteine protease domain, releasing the glucosyltransferase domain into the cytoplasm where it inactivates Rho-type GTPases through covalent glucosylation (from UDP-glucose) [20]. This in turn causes changes in epithelial cell morphology due to actin depolymerization, loss of tight junction integrity, and eventually, cell death (**Figure 1**) [21]. The assays described in this chapter measure many of the various steps, described above, involved in the intoxication cascade (steps 1–5, as denoted in **Figure 1**).

#### **3.2. Cell surface binding of TcdA and TcdB (step 1 in Figure 1)**

Binding of toxins to the cell surface of target cells is the first step in TcdA and TcdB cell entry, leading to the downstream effects of the toxins. We assessed cell surface binding of TcdA and TcdB by Western blotting of cell membranes isolated from Vero cells incubated with TcdA or TcdB at 4°C. As shown in **Figure 2**, membrane fractions isolated from cells incubated with TcdA (see **Figure 2A**, top panel) or TcdB (**Figure 2B**, top panel) contain toxins, indicating cell surface binding of the toxins. Actoxumab and bezlotoxumab bind to and neutralize purified TcdA and TcdB, respectively, from a variety of *C. difficile* strains [15]. Pre-incubation of TcdA with actoxumab but not bezlotoxumab efficiently blocked binding of TcdA to cells (**Figure 2A**), while pre-incubation of TcdB with bezlotoxumab but not actoxumab efficiently blocked binding of TcdB to cells (**Figure 2B**), confirming the specificity of

described in this chapter. Figure adapted from Jank and Aktories [17].

**Figure 1.** *Clostridium difficile* toxin structure and mechanism of action. (A) Domain organization of TcdA. (B) Domain organization of TcdB. (C) Mechanism of intoxication of mammalian cells by TcdA and TcdB. Toxins A and B bind to receptors on the surface of target cells (1) and are endocytosed. Endosomal toxins are acidified causing exposure of hydrophobic regions of the protein that allow their insertion into the membrane, forming pore(s). The N-terminal catalytic domain is then translocated from the endosomal compartment into the cytoplasm, where the glucosyltransferase domain is released by inositol hexakisphosphate (InsP6)-dependent auto-cleavage. The toxins then glucosylate Rho-type GTPases (2) from UDP-glucose, causing actin depolymerization, changes in cell morphology (3), disruption of tight junctions (4), and cell death (5). Cellular events numbered 1–5 correspond to the steps assessed by the various assays

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toxins binding to cells.

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mixtures were then added to Vero or T-84 cells as described above and incubated for 24 h at

To assess the cytotoxicity of *C. difficile* toxins derived from bacterial culture supernatants, strain VPI 10463 (ribotype 087) (ATCC) was grown in chopped meat medium (Anaerobe Systems) under anaerobic conditions at 37°C for 72–96 h, and culture supernatants were collected, filtered twice through a 0.22 μm filter, and stored at 4°C. For TcdB immunodepletion, cell culture supernatants were combined and mixed with bezlotoxumab and protein A-agarose beads for 4–6 h at 4°C. After incubation, the beads were removed by centrifugation. Supernatants were then collected, filtered (0.22 μm), and stored at 4°C. Cytotoxicity and antibody-mediated neutralization of the untreated (for determinations on TcdB) or immunodepleted (for determinations on TcdA) supernatants were measured as

TcdA and TcdB are large, monomeric proteins (300 and 270 kDa, respectively) with similar structures and functions (**Figure 1**) [17, 18]. The functional domains of the toxins are arranged according to the ABCD model [17]: the N-terminal A domain contains the glucosyltransferase enzymatic activity, the B domain is a putative receptor-binding domain composed of a series of long and short repeats known as combined repetitive oligopeptides (CROPs), the cysteine protease (C) domain is responsible for autocatalytic processing, and the D domain is involved in pore formation and toxin translocation. Both toxins bind to receptors on the surface of the epithelial cells that line the wall of the lower intestine (and possibly other cell types). Once bound, they are internalized via receptor-mediated endocytosis [19]. Acidification of the endosome promotes a conformational change that enables translocation of the N-terminal glucosyltransferase domain of the toxin into the cytoplasm. Cellular inositol hexakisphosphate (InsP6) allows cleavage of the toxin by the cysteine protease domain, releasing the glucosyltransferase domain into the cytoplasm where it inactivates Rho-type GTPases through covalent glucosylation (from UDP-glucose) [20]. This in turn causes changes in epithelial cell morphology due to actin depolymerization, loss of tight junction integrity, and eventually, cell death (**Figure 1**) [21]. The assays described in this chapter measure many of the various steps, described above, involved in the intoxication

Binding of toxins to the cell surface of target cells is the first step in TcdA and TcdB cell entry, leading to the downstream effects of the toxins. We assessed cell surface binding of TcdA and TcdB by Western blotting of cell membranes isolated from Vero cells incubated

**3.1. Overview of mammalian cell intoxication by TcdA and TcdB**

cascade (steps 1–5, as denoted in **Figure 1**).

**3.2. Cell surface binding of TcdA and TcdB (step 1 in Figure 1)**

. The cells were then washed twice with PBS and treated and analyzed as

37°C with 5% CO<sup>2</sup>

116 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

described above.

described above.

**3. Results**

**Figure 1.** *Clostridium difficile* toxin structure and mechanism of action. (A) Domain organization of TcdA. (B) Domain organization of TcdB. (C) Mechanism of intoxication of mammalian cells by TcdA and TcdB. Toxins A and B bind to receptors on the surface of target cells (1) and are endocytosed. Endosomal toxins are acidified causing exposure of hydrophobic regions of the protein that allow their insertion into the membrane, forming pore(s). The N-terminal catalytic domain is then translocated from the endosomal compartment into the cytoplasm, where the glucosyltransferase domain is released by inositol hexakisphosphate (InsP6)-dependent auto-cleavage. The toxins then glucosylate Rho-type GTPases (2) from UDP-glucose, causing actin depolymerization, changes in cell morphology (3), disruption of tight junctions (4), and cell death (5). Cellular events numbered 1–5 correspond to the steps assessed by the various assays described in this chapter. Figure adapted from Jank and Aktories [17].

with TcdA or TcdB at 4°C. As shown in **Figure 2**, membrane fractions isolated from cells incubated with TcdA (see **Figure 2A**, top panel) or TcdB (**Figure 2B**, top panel) contain toxins, indicating cell surface binding of the toxins. Actoxumab and bezlotoxumab bind to and neutralize purified TcdA and TcdB, respectively, from a variety of *C. difficile* strains [15]. Pre-incubation of TcdA with actoxumab but not bezlotoxumab efficiently blocked binding of TcdA to cells (**Figure 2A**), while pre-incubation of TcdB with bezlotoxumab but not actoxumab efficiently blocked binding of TcdB to cells (**Figure 2B**), confirming the specificity of toxins binding to cells.

**Figure 2.** Cell surface binding of TcdA and TcdB as measured by Western blot. Western blots of cell membranes isolated from Vero cells following incubation with (A) TcdA or (B) TcdB, in the presence of vehicle, actoxumab, or bezlotoxumab (200 μg/ml), as indicated. The top blots in each panel show TcdA and TcdB, while the bottom blots show cadherin, used as a loading control.

TcdA- and TcdB-mediated Rac1 glucosylation in a high throughput and quantitative 384-well in-cell Western assay, using antibodies that detect non-glucosylated and total Rac1. A dosedependent decrease in non-glucosylated Rac1 was observed in the presence of TcdA and TcdB from various ribotypes (027, 078, and the control 087 (strain VPI 10463)) (**Figure 4A** and **B**), while total Rac1 was minimally affected (not shown). Vero cells were found to be more sensitive to TcdB than TcdA, consistent with previous observation by Torres et al. [23]. In addition, differences in sensitivity of Vero cells to toxins of the different *C. difficile* ribotypes were noted. For instance, Vero cells were found to be more sensitive to TcdA of ribotype 087 (VPI 10463) than of ribotypes 027 and 078, while TcdB showed the opposite effect, with cells being more

**Figure 3.** Cell surface binding of TcdA as measured by flow cytometry. A representative experiment showing flow cytometry analysis of HT29 cells pre-incubated with a titration of TcdA-Atto488 in the presence or absence of actoxumab. Following incubation, mean fluorescence intensity (MFI) was measured with excitation and emission wavelengths of 488

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Actoxumab and bezlotoxumab neutralized the effects of TcdA and TcdB (at EC90 concentrations), respectively (**Figure 4C** and **D**). Notably, the potency of actoxumab and bezlotoxumab on their respective toxins was lower for toxins of ribotype 027 and 078 compared to ribotype 087. This is consistent with the lower affinities of the antibodies against toxins of these

The cytopathic effects of TcdA and TcdB on gut epithelium are visualized as profound morphological changes, typically cell rounding, due to the glucosylation and inactivation of Rho-type GTPases and subsequent disruption of actin polymerization. Historically, these cytopathic

**3.4. Changes in cell morphology induced by TcdA and TcdB (step 3 in Figure 1)**

sensitive to TcdB of ribotypes 027 and 078 compared to ribotype 087.

ribotypes, as previously described by Hernandez et al. [15].

and 530 nm, respectively.

Binding of TcdA to cells and the prevention, thereof, by actoxumab were also assessed by flow cytometry (**Figure 3**). Incubation of HT29 cells with increasing levels of fluorescently labeled TcdA (TcdA-Atto488) led to an elevated mean fluorescence intensity (MFI), indicating binding of TcdA to the cell surface in a concentration-dependent manner. In the presence of actoxumab, however, the MFI for each toxin concentration was reduced to background levels showing that actoxumab blocked binding of TcdA to the cell surface. No significant changes in MFI were measured in the presence of bezlotoxumab, indicating that the effect of actoxumab is specific (data not shown).

#### **3.3. Glucosylation of Rac1 by TcdA and TcdB (step 2 in Figure 1)**

Inactivation of Rho-type GTPases is a key step in the intoxication of host cells, leading to the downstream cytopathic and cytotoxic effects of the *C. difficile* toxins. Historically, the glucosylation of Rho GTPases was assessed by polyacrylamide gel-based assays that use either radioactively labeled glucose or antibodies to detect the glucosylated and non-glucosylated protein on a gel [22]. These assays are laborious, low throughput, qualitative, and do not detect glucosylation directly in the cell. A novel assay was therefore developed to measure

**Figure 3.** Cell surface binding of TcdA as measured by flow cytometry. A representative experiment showing flow cytometry analysis of HT29 cells pre-incubated with a titration of TcdA-Atto488 in the presence or absence of actoxumab. Following incubation, mean fluorescence intensity (MFI) was measured with excitation and emission wavelengths of 488 and 530 nm, respectively.

TcdA- and TcdB-mediated Rac1 glucosylation in a high throughput and quantitative 384-well in-cell Western assay, using antibodies that detect non-glucosylated and total Rac1. A dosedependent decrease in non-glucosylated Rac1 was observed in the presence of TcdA and TcdB from various ribotypes (027, 078, and the control 087 (strain VPI 10463)) (**Figure 4A** and **B**), while total Rac1 was minimally affected (not shown). Vero cells were found to be more sensitive to TcdB than TcdA, consistent with previous observation by Torres et al. [23]. In addition, differences in sensitivity of Vero cells to toxins of the different *C. difficile* ribotypes were noted. For instance, Vero cells were found to be more sensitive to TcdA of ribotype 087 (VPI 10463) than of ribotypes 027 and 078, while TcdB showed the opposite effect, with cells being more sensitive to TcdB of ribotypes 027 and 078 compared to ribotype 087.

Binding of TcdA to cells and the prevention, thereof, by actoxumab were also assessed by flow cytometry (**Figure 3**). Incubation of HT29 cells with increasing levels of fluorescently labeled TcdA (TcdA-Atto488) led to an elevated mean fluorescence intensity (MFI), indicating binding of TcdA to the cell surface in a concentration-dependent manner. In the presence of actoxumab, however, the MFI for each toxin concentration was reduced to background levels showing that actoxumab blocked binding of TcdA to the cell surface. No significant changes in MFI were measured in the presence of bezlotoxumab, indicating that the effect of actox-

**Figure 2.** Cell surface binding of TcdA and TcdB as measured by Western blot. Western blots of cell membranes isolated from Vero cells following incubation with (A) TcdA or (B) TcdB, in the presence of vehicle, actoxumab, or bezlotoxumab (200 μg/ml), as indicated. The top blots in each panel show TcdA and TcdB, while the bottom blots show cadherin, used

Inactivation of Rho-type GTPases is a key step in the intoxication of host cells, leading to the downstream cytopathic and cytotoxic effects of the *C. difficile* toxins. Historically, the glucosylation of Rho GTPases was assessed by polyacrylamide gel-based assays that use either radioactively labeled glucose or antibodies to detect the glucosylated and non-glucosylated protein on a gel [22]. These assays are laborious, low throughput, qualitative, and do not detect glucosylation directly in the cell. A novel assay was therefore developed to measure

umab is specific (data not shown).

118 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

as a loading control.

**3.3. Glucosylation of Rac1 by TcdA and TcdB (step 2 in Figure 1)**

Actoxumab and bezlotoxumab neutralized the effects of TcdA and TcdB (at EC90 concentrations), respectively (**Figure 4C** and **D**). Notably, the potency of actoxumab and bezlotoxumab on their respective toxins was lower for toxins of ribotype 027 and 078 compared to ribotype 087. This is consistent with the lower affinities of the antibodies against toxins of these ribotypes, as previously described by Hernandez et al. [15].

#### **3.4. Changes in cell morphology induced by TcdA and TcdB (step 3 in Figure 1)**

The cytopathic effects of TcdA and TcdB on gut epithelium are visualized as profound morphological changes, typically cell rounding, due to the glucosylation and inactivation of Rho-type GTPases and subsequent disruption of actin polymerization. Historically, these cytopathic

**3.5. Toxin-induced disruption of epithelial tight junctions (step 4 in Figure 1)**

dose-dependently neutralized the effects of their respective toxins (**Figure 6C** and **D**).

The traditional way of assessing the cytoxic effects of *C. difficile* on host cells involves measuring cellular ATP levels of intoxicated cells. This method is plagued with low signal to noise ratios and variability due to substantial ATP levels remaining in cells that are not yet dead and still undergoing morphological changes due to intoxication [13]. Additionally, normal metabolismrelated fluctuations in ATP levels that are unrelated to cell viability can further affect the assay readout. We developed a more robust colorimetric assay that measures cellular protein content as a surrogate of cell growth and survival [14]. The sulforhodamine B (SRB) assay was used to determine the cytotoxic effects of purified *C. difficile* toxins of the reference strain VPI 10463 (ribotype 087) and from strains of ribotypes from the so-called hyper-virulent ribotypes 027 and 078. All toxins tested caused a robust concentration-dependent decrease in cell viability

**3.6. Toxin-induced cytotoxicity (step 5 in Figure 1)**

on toxin-induced effects on DMR.

To gain an understanding of the effect of *C. difficile* toxins on the integrity of the gut wall epithelium, a two-dimensional cell culture system was utilized wherein a single monolayer of colonic epithelial cells (Caco-2) is grown on a permeable membrane, separating distinct apical and basolateral compartments [25–28]. The system simulates the polarized nature of the intact intestinal mucosal epithelium, which separates the gut lumen (apical side) from the subepithelial/systemic space (basolateral side). The integrity of the epithelial layer is monitored by measuring the transepithelial electrical resistance (TER), with a decrease in TER suggesting that the integrity of the epithelial monolayer has been compromised [26]. In this system, TcdA and TcdB added to the apical side of the cell monolayer (mimicking the presence of toxin on the lumenal side of the gut) caused significant time- and concentration-dependent decreases in TER (**Figure 6A** and **B**). Neutralization of the toxin-induced effects by actoxumab and bezlotoxumab was assessed at EC90 concentrations of TcdA and TcdB, respectively. Both antibodies

**Figure 5.** Effects of TcdA and TcdB on dynamic mass redistribution and neutralization by actoxumab and bezlotoxumab. (A) Concentration-dependent effects of TcdA and TcdB on DMR. (B) Neutralizing effects of actoxumab and bezlotoxumab

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**Figure 4.** TcdA- and TcdB-mediated Rac1 glucosylation and neutralization thereof by actoxumab and bezlotoxumab. Effect of TcdA (A) and TcdB (B) of ribotypes 027, 078, and 087 on glucosylation of Rac1. Neutralization of TcdA-mediated Rac1 glucosylation by actoxumab (C) and of TcdB-mediated Rac1 glucosylation by bezlotoxumab (D).

effects have been assessed qualitatively through visual determination of cell rounding [23]. Improved phenotypic assays used to investigate changes in cell morphology involve the quantification of length-to-width ratios of fluorescently labeled cells [23, 24]. This latter technique is quantitative and has an improved throughput, although it requires consistent staining and substantial data analysis. To better understand and quantify toxin-induced morphological changes in unlabeled cells, an assay was developed to examine dynamic mass distribution (DMR) in Vero cells using the Epic instrument. In this assay, plates containing optical sensors are used to capture translocation of cellular mass of unlabeled cells in response to ligand binding, allowing changes in cell shape to be quantified. The concentration-dependent effects of TcdA and TcdB on mass redistribution were determined at 180 min (at which time the effects have plateaued, not shown) (**Figure 5A**). As with the Rac1 glucosylation assay, Vero cells are much more sensitive to TcdB than TcdA in the DMR assay. The neutralizing effects of actoxumab and bezlotoxumab on toxin-induced morphological changes were assessed at EC90 concentrations of TcdA and TcdB, respectively. Actoxumab and bezlotoxumab fully neutralized the effects of TcdA and TcdB, respectively, on DMR (**Figure 5B**).

**Figure 5.** Effects of TcdA and TcdB on dynamic mass redistribution and neutralization by actoxumab and bezlotoxumab. (A) Concentration-dependent effects of TcdA and TcdB on DMR. (B) Neutralizing effects of actoxumab and bezlotoxumab on toxin-induced effects on DMR.

#### **3.5. Toxin-induced disruption of epithelial tight junctions (step 4 in Figure 1)**

To gain an understanding of the effect of *C. difficile* toxins on the integrity of the gut wall epithelium, a two-dimensional cell culture system was utilized wherein a single monolayer of colonic epithelial cells (Caco-2) is grown on a permeable membrane, separating distinct apical and basolateral compartments [25–28]. The system simulates the polarized nature of the intact intestinal mucosal epithelium, which separates the gut lumen (apical side) from the subepithelial/systemic space (basolateral side). The integrity of the epithelial layer is monitored by measuring the transepithelial electrical resistance (TER), with a decrease in TER suggesting that the integrity of the epithelial monolayer has been compromised [26]. In this system, TcdA and TcdB added to the apical side of the cell monolayer (mimicking the presence of toxin on the lumenal side of the gut) caused significant time- and concentration-dependent decreases in TER (**Figure 6A** and **B**). Neutralization of the toxin-induced effects by actoxumab and bezlotoxumab was assessed at EC90 concentrations of TcdA and TcdB, respectively. Both antibodies dose-dependently neutralized the effects of their respective toxins (**Figure 6C** and **D**).

#### **3.6. Toxin-induced cytotoxicity (step 5 in Figure 1)**

effects have been assessed qualitatively through visual determination of cell rounding [23]. Improved phenotypic assays used to investigate changes in cell morphology involve the quantification of length-to-width ratios of fluorescently labeled cells [23, 24]. This latter technique is quantitative and has an improved throughput, although it requires consistent staining and substantial data analysis. To better understand and quantify toxin-induced morphological changes in unlabeled cells, an assay was developed to examine dynamic mass distribution (DMR) in Vero cells using the Epic instrument. In this assay, plates containing optical sensors are used to capture translocation of cellular mass of unlabeled cells in response to ligand binding, allowing changes in cell shape to be quantified. The concentration-dependent effects of TcdA and TcdB on mass redistribution were determined at 180 min (at which time the effects have plateaued, not shown) (**Figure 5A**). As with the Rac1 glucosylation assay, Vero cells are much more sensitive to TcdB than TcdA in the DMR assay. The neutralizing effects of actoxumab and bezlotoxumab on toxin-induced morphological changes were assessed at EC90 concentrations of TcdA and TcdB, respectively. Actoxumab and bezlotoxumab fully neutral-

**Figure 4.** TcdA- and TcdB-mediated Rac1 glucosylation and neutralization thereof by actoxumab and bezlotoxumab. Effect of TcdA (A) and TcdB (B) of ribotypes 027, 078, and 087 on glucosylation of Rac1. Neutralization of TcdA-mediated

Rac1 glucosylation by actoxumab (C) and of TcdB-mediated Rac1 glucosylation by bezlotoxumab (D).

120 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

ized the effects of TcdA and TcdB, respectively, on DMR (**Figure 5B**).

The traditional way of assessing the cytoxic effects of *C. difficile* on host cells involves measuring cellular ATP levels of intoxicated cells. This method is plagued with low signal to noise ratios and variability due to substantial ATP levels remaining in cells that are not yet dead and still undergoing morphological changes due to intoxication [13]. Additionally, normal metabolismrelated fluctuations in ATP levels that are unrelated to cell viability can further affect the assay readout. We developed a more robust colorimetric assay that measures cellular protein content as a surrogate of cell growth and survival [14]. The sulforhodamine B (SRB) assay was used to determine the cytotoxic effects of purified *C. difficile* toxins of the reference strain VPI 10463 (ribotype 087) and from strains of ribotypes from the so-called hyper-virulent ribotypes 027 and 078. All toxins tested caused a robust concentration-dependent decrease in cell viability

**Figure 6.** Effects of TcdA and TcdB on integrity of Caco-2 cell monolayers and neutralization by actoxumab and bezlotoxumab. Time- and concentration-dependent effects on TER of TcdA (A) or TcdB (B) added to the apical side of Caco-2 monolayers. Time- and dose-dependent neutralization of TcdA by actoxumab (C) and of TcdB by bezlotoxumab (D), added to the apical side.

(**Figure 7A** and **B**). As with other assays described herein and as previously observed by Torres et al. [23], Vero cells are significantly more sensitive to TcdB than to TcdA. The ability of actoxumab and bezlotoxumab to neutralize TcdA and TcdB, respectively, was assessed at toxin concentrations that are associated with a 90% decrease in cell viability (LC90). Both antibodies fully neutralized the effects of their respective toxins from all ribotypes tested (**Figure 7C** and **D**). However, the neutralization potencies of both antibodies for toxins of ribotypes 027 and 078 were significantly lower than toxins of ribotype 087, similar to data obtained in the Rac1 glucosylation assay above (Section 2.3) and consistent with previous data in the SRB assay [15].

by itself or in combination with 10 μg/ml actoxumab significantly shifted the concentrationresponse curve to the right, indicating that most of the cytotoxic activity in the supernatant is due to TcdB (**Figure 8A**). This is not surprising as Vero cells are more sensitive to TcdB than to TcdA. To assess the cytotoxic activity associated with TcdA, TcdB was first removed from the supernatant using an immunodepletion approach (see Section 2). In this case, 10 μg/ml actoxumab, alone or in combination with 10 μg/ml bezlotoxumab, shifted the response curve to the right, whereas bezlotoxumab showed minimal effect, confirming that the cytotoxic activity in immunodepleted supernatants is associated mainly with TcdA (**Figure 8B**). To confirm this finding, full concentration-response curves of actoxumab and bezlotoxumab were generated against dilutions of intact or immunodepleted supernatants associated with ~90% reduction in cell viability (EC90); actoxumab neutralized the cytotoxic activity of immunodepleted supernatants, whereas bezlotoxumab neutralized the cytotoxic activity of intact supernatants, and no cross-neutralization was observed (**Figure 8C** and **D**). This approach has been used successfully to assess the activities of actoxumab and bezlotoxumab on TcdA and TcdB of dozens of clinical isolates of *C. difficile*, covering 18 distinct ribotypes (seven toxinotypes) [15].

AAC.04433-14]).

**Figure 7.** Purified TcdA- and TcdB-mediated effects on cell viability and neutralization by actoxumab and bezlotoxumab. Reduction in Vero cell viability induced by TcdA (A) and TcdB (B) using purified toxins from ribotypes 087, 027, and 078. Neutralization of TcdA by actoxumab (C) and of TcdB by bezlotoxumab (D). Figure reproduced from Hernandez et al. [15] (Copyright © American Society for Microbiology [Antimicrob Agents Chemother. 59, 2015, 1052–1060. DOI:10.1128/

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The robust nature of the SRB assay also allows for the study of the cytotoxic effects of unpurified *C. difficile* toxins directly from culture supernatants for clinical strains for which purified toxins are not available. For these studies, Vero cells were treated with serially diluted culture supernatants of the reference strain VPI 10463, containing both toxins (not shown), in the absence or presence of actoxumab, bezlotoxumab, or the combination of both antibodies. In the absence of antibodies, there was a concentration-dependent decrease in cell viability, presumably due to the presence of toxin in the supernatant. Addition of actoxumab had no effect on the cytotoxicity of supernatant, while addition of 10 μg/ml bezlotoxumab either

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**Figure 7.** Purified TcdA- and TcdB-mediated effects on cell viability and neutralization by actoxumab and bezlotoxumab. Reduction in Vero cell viability induced by TcdA (A) and TcdB (B) using purified toxins from ribotypes 087, 027, and 078. Neutralization of TcdA by actoxumab (C) and of TcdB by bezlotoxumab (D). Figure reproduced from Hernandez et al. [15] (Copyright © American Society for Microbiology [Antimicrob Agents Chemother. 59, 2015, 1052–1060. DOI:10.1128/ AAC.04433-14]).

(**Figure 7A** and **B**). As with other assays described herein and as previously observed by Torres et al. [23], Vero cells are significantly more sensitive to TcdB than to TcdA. The ability of actoxumab and bezlotoxumab to neutralize TcdA and TcdB, respectively, was assessed at toxin concentrations that are associated with a 90% decrease in cell viability (LC90). Both antibodies fully neutralized the effects of their respective toxins from all ribotypes tested (**Figure 7C** and **D**). However, the neutralization potencies of both antibodies for toxins of ribotypes 027 and 078 were significantly lower than toxins of ribotype 087, similar to data obtained in the Rac1 glucosylation assay above (Section 2.3) and consistent with previous data in the SRB assay [15].

**Figure 6.** Effects of TcdA and TcdB on integrity of Caco-2 cell monolayers and neutralization by actoxumab and bezlotoxumab. Time- and concentration-dependent effects on TER of TcdA (A) or TcdB (B) added to the apical side of Caco-2 monolayers. Time- and dose-dependent neutralization of TcdA by actoxumab (C) and of TcdB by bezlotoxumab

(D), added to the apical side.

122 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

The robust nature of the SRB assay also allows for the study of the cytotoxic effects of unpurified *C. difficile* toxins directly from culture supernatants for clinical strains for which purified toxins are not available. For these studies, Vero cells were treated with serially diluted culture supernatants of the reference strain VPI 10463, containing both toxins (not shown), in the absence or presence of actoxumab, bezlotoxumab, or the combination of both antibodies. In the absence of antibodies, there was a concentration-dependent decrease in cell viability, presumably due to the presence of toxin in the supernatant. Addition of actoxumab had no effect on the cytotoxicity of supernatant, while addition of 10 μg/ml bezlotoxumab either by itself or in combination with 10 μg/ml actoxumab significantly shifted the concentrationresponse curve to the right, indicating that most of the cytotoxic activity in the supernatant is due to TcdB (**Figure 8A**). This is not surprising as Vero cells are more sensitive to TcdB than to TcdA. To assess the cytotoxic activity associated with TcdA, TcdB was first removed from the supernatant using an immunodepletion approach (see Section 2). In this case, 10 μg/ml actoxumab, alone or in combination with 10 μg/ml bezlotoxumab, shifted the response curve to the right, whereas bezlotoxumab showed minimal effect, confirming that the cytotoxic activity in immunodepleted supernatants is associated mainly with TcdA (**Figure 8B**). To confirm this finding, full concentration-response curves of actoxumab and bezlotoxumab were generated against dilutions of intact or immunodepleted supernatants associated with ~90% reduction in cell viability (EC90); actoxumab neutralized the cytotoxic activity of immunodepleted supernatants, whereas bezlotoxumab neutralized the cytotoxic activity of intact supernatants, and no cross-neutralization was observed (**Figure 8C** and **D**). This approach has been used successfully to assess the activities of actoxumab and bezlotoxumab on TcdA and TcdB of dozens of clinical isolates of *C. difficile*, covering 18 distinct ribotypes (seven toxinotypes) [15].

*C. difficile* strains for which purified toxins are not available. The assays described were validated with the antitoxin antibodies actoxumab (anti-TcdA) and bezlotoxumab (anti-TcdB) to demonstrate their utility in evaluating pharmacological blockade of toxins. These assays may be useful in future studies aimed at better understanding of *C. difficile* toxin function, as well

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Mary Ann Cox, Lorraine D. Hernandez, Pulkit Gupta, Zuo Zhang, Fred Racine and Alex

[1] Bassetti M, Villa G, Pecori D, Arzese A, Wilcox M. Epidemiology, diagnosis and treatment of *Clostridium difficile* infection. Expert Rev Anti Infect Ther. 2012;10:1405-23. DOI:

[2] Rupnik M, Wilcox MH, Gerding DN. *Clostridium difficile* infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol. 2009;7:526-36. DOI:10.1038/nrmicro

[3] Carter GP, Rood JI, Lyras D. The role of toxin A and toxin B in the virulence of *Clostridium* 

[4] Dingle T, Wee S, Mulvey GL, Greco A, Kitova EN, Sun J, et al. Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by *Clostridium difficile*. Glycobiology. 2008;18:698-706. DOI:10.1093/glycob/

[5] Pruitt, RN, Lacy DB. Toward a structural understanding of *Clostridium difficile* toxins A

[6] Papatheodorou P, Zamboglou C, Genisyuerek S, Guttenberg G, Aktories K. Clostridial glucosylating toxins enter cells via clathrin-mediated endocytosis. PLoS One. 2010;5:e10673.

[7] Pfeifer G, Schirmer J, Leemhuis J, Busch C, Meyer DK, Aktories K, et al. Cellular uptake of *Clostridium difficile* toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells. J Biol Chem. 2003;278:44535-41. DOI:10.1074/jbc.

and B. Front Cell Infect Microbiol. 2012;2:28. DOI:10.3389/fcimb.2012.00028

*difficile*. Trends Microbiol. 2012;20:21-9. DOI: 10.1016/j.tim.2011.11.003

as in characterizing toxin inhibitors as tools or as potential therapeutics.

\*Address all correspondence to: atherien@inceptionsci.com

Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, USA

† Current affiliation: Inception Sciences Canada, Montreal, Quebec, Canada

**Author details**

G. Therien\* †

**References**

2164

cwn048

M307540200

DOI:10.1371/journal.pone.0010673

10.1586/eri.12.135

**Figure 8.** Unpurified TcdA- and TcdB-mediated effects on Vero cell viability and neutralization by actoxumab and bezlotoxumab. Cytotoxic effects of serially diluted intact (A) or immunodepleted (B) supernatants in the presence or absence of actoxumab, bezlotoxumab, or a combination of the two antibodies. Neutralization of cytotoxic activity by bezlotoxumab, but not actoxumab, in intact supernatant at EC90 dilution (C) and by actoxumab but not bezlotoxumab in immunodepleted supernatant at EC90 dilution (D).

#### **4. Conclusions**

In this chapter, we have described novel cell-based assays for analyzing multiple distinct steps in the intoxication cascade associated with TcdA and TcdB. Unlike historical assays that measure toxin effects qualitatively, such as the visual assessment of cell rounding, or are variable and often unreliable, such as quantitation of ATP levels to estimate cell death, the assays presented here can quantitatively and robustly assess the effects of toxins in mammalian cells. We show how the initial event of toxin binding to host cells can be assessed using cell surface binding assays with labeled or unlabeled toxins in flow cytometry and Western blot formats, respectively. The more proximal events that follow internalization of the toxins, namely Rac1 glucosylation and cell rounding, can be studied with novel quantitative assays by in-cell Western and dynamic mass redistribution assays, respectively. Finally, we show how the TER and SRB assays can be utilized to assess the final stages of intoxication, tight junction disruption, and cell death, respectively. We also show how the SRB assay can be used to accurately measure the activities of TcdA and TcdB from unpurified toxins in culture supernatants of *C. difficile* strains for which purified toxins are not available. The assays described were validated with the antitoxin antibodies actoxumab (anti-TcdA) and bezlotoxumab (anti-TcdB) to demonstrate their utility in evaluating pharmacological blockade of toxins. These assays may be useful in future studies aimed at better understanding of *C. difficile* toxin function, as well as in characterizing toxin inhibitors as tools or as potential therapeutics.

## **Author details**

Mary Ann Cox, Lorraine D. Hernandez, Pulkit Gupta, Zuo Zhang, Fred Racine and Alex G. Therien\* †

\*Address all correspondence to: atherien@inceptionsci.com

Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, USA

† Current affiliation: Inception Sciences Canada, Montreal, Quebec, Canada

## **References**

**4. Conclusions**

immunodepleted supernatant at EC90 dilution (D).

124 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

In this chapter, we have described novel cell-based assays for analyzing multiple distinct steps in the intoxication cascade associated with TcdA and TcdB. Unlike historical assays that measure toxin effects qualitatively, such as the visual assessment of cell rounding, or are variable and often unreliable, such as quantitation of ATP levels to estimate cell death, the assays presented here can quantitatively and robustly assess the effects of toxins in mammalian cells. We show how the initial event of toxin binding to host cells can be assessed using cell surface binding assays with labeled or unlabeled toxins in flow cytometry and Western blot formats, respectively. The more proximal events that follow internalization of the toxins, namely Rac1 glucosylation and cell rounding, can be studied with novel quantitative assays by in-cell Western and dynamic mass redistribution assays, respectively. Finally, we show how the TER and SRB assays can be utilized to assess the final stages of intoxication, tight junction disruption, and cell death, respectively. We also show how the SRB assay can be used to accurately measure the activities of TcdA and TcdB from unpurified toxins in culture supernatants of

**Figure 8.** Unpurified TcdA- and TcdB-mediated effects on Vero cell viability and neutralization by actoxumab and bezlotoxumab. Cytotoxic effects of serially diluted intact (A) or immunodepleted (B) supernatants in the presence or absence of actoxumab, bezlotoxumab, or a combination of the two antibodies. Neutralization of cytotoxic activity by bezlotoxumab, but not actoxumab, in intact supernatant at EC90 dilution (C) and by actoxumab but not bezlotoxumab in


[8] Shen A. *Clostridium difficile* toxins: mediators of inflammation. J Innate Immun. 2012; 4:149-58. DOI:10.1159/000332946

[19] Jank T, Giesemann T, Aktories K. Rho-glucosylating *Clostridium difficile* toxins A and B: new insights into structure and function. Glycobiology. 2007;17:15R–22R. DOI:10.1093/

Assays for Measuring *C. difficile* Toxin Activity and Inhibition in Mammalian Cells

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[20] Aktories K. Bacterial protein toxins that modify host regulatory GTPases Nat Rev

[21] May M, Wang T, Müller M, Genth H. Difference in F-Actin depolymerization induced by toxin B from the *Clostridium difficile* strain VPI 10463 and toxin B from the variant *Clostridium* 

[22] Quesada-Gómez C, López-Ureña D, Chumbler N, Kroh HK, Castro-Peña C, Rodríguez C, Orozco-Aguilar J, González-Camacho S, Rucavado A, Guzmán-Verri C, Lawley TD, Lacy DB, Chaves-Olarte E. Analysis of TcdB proteins within the hypervirulent clade 2 reveals an impact of RhoA glucosylation on *Clostridium difficile* proinflammatory activi-

[23] Torres J, Camorlinga-Ponce M, Munoz O. Sensitivity in culture of epithelial cells from rhesus monkey kidney and human colon carcinoma to toxins A and B from *Clostridium* 

[24] Tam J, Beilhartz G, Auger A, Gupta P, Therien A, Melnyk R. Small molecule inhibitors of *Clostridium difficile* toxin B-induced cellular damage. Chem Biol. 2015;22:175-85. DOI:

[25] Du T, Alfa MJ. Translocation of *Clostridium difficile* toxin B across polarized Caco-2 cell

[26] Nusrat A, von Eichel-Streiber C, Turner JR, Verkade P, Madara JL, Parkos CA. *Clostridium difficile* toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins. Infect Immun. 2001;69:1329-36. DOI:10.1128/

[27] Sambuy Y, De Angelis I, Ranaldi G, Scarino ML, Stammati A, Zucco F. The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol. 2005;21:1-26. DOI:10.1007/

[28] Sutton PA, Li S, Webb J, Solomon K, Brazier J, Mahida YR. Essential role of toxin A in C. difficile 027 and reference strain supernatant-mediated disruption of Caco-2 intestinal epithelial barrier function. Clin Exp Immunol. 2008;153:439-47. DOI: 10.1111/j.

*difficile* Serotype F Strain 1470. Toxins 2013;5:106-19. DOI:10.3390/toxins5010106

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glycob/cwm004

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IAI.69.3.1329-1336.2001

s10565-005-0085-6

1365-2249.2008.03690.x.


[19] Jank T, Giesemann T, Aktories K. Rho-glucosylating *Clostridium difficile* toxins A and B: new insights into structure and function. Glycobiology. 2007;17:15R–22R. DOI:10.1093/ glycob/cwm004

[8] Shen A. *Clostridium difficile* toxins: mediators of inflammation. J Innate Immun. 2012;

[9] Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, Pepin J, Wilcox MH. Society for Healthcare Epidemiology of America, Infectious Diseases Society of America. Clinical practice guidelines for *Clostridium difficile* infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). Infect Control Hosp Epidemiol. 2010;31:431-55. [10] Venugopal AA, Johnson, S. Current state of *Clostridium difficile* treatment options. Clin

[11] Stamper P, Alcabasa R, Aird D, Babiker W, Wehrlin J, Ikpeama I, Carroll K. Comparison of a commercial real-time PCR assay for *tcdB* detection to a cell culture cytotoxicity assay and toxigenic culture for direct detection of toxin-producing *Clostridium difficile* in

[12] Babcock, GJ, Broering TJ, Hernandez HJ, Mandell RB, Donahue K, Boatright N, et al. Human monoclonal antibodies directed against toxins A and B prevent *Clostridium difficile* induced mortality in hamsters. Infect Immun. 2006;74:6339-47. DOI:10.1128/

[13] Xie J, Zorman J, Indrawati L, Horton M, Soring K, Antonello J, Zhang Y, Secore S, Miezeiewski M, Wang S, Kanavage A, Skinner J, Rogers I, Bodmer J, Heinrichs J. Development and optimization of a novel assay to measure neutralizing antibodies against *Clostridium difficile* toxins. Clin Vaccine Immunol. 2013;20:517-25. DOI:10.1128/

[14] Orth P, Xiao L, Hernandez LD, Reichert P, Sheth PR, Beaumont M, Yang X, Murgolo N, Ermakov G, DiNunzio E, Racine F, Karczewski J, Secore S, Ingram RN, Mayhood T, Strickland C,Therien AG. Mechanism of action and epitopes of *Clostridium difficile* toxin B-neutralizing antibody bezlotoxumab revealed by X-ray crystallography. J Biol Chem.

[15] Hernandez LD, Racine F, Xiao L, DiNunzio E, Hairston N, Sheth PR, Murgolo NJ, Therien AG. Broad coverage of genetically diverse strains of *Clostridium difficile* by actoxumab and bezlotoxumab predicted by *in vitro* neutralization and epitope modeling.

[16] Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst. 1990;82:1107-12.

[17] Jank T, Aktories K. Structure and mode of action of clostridial glucosylating toxins: the ABCD model. Trends Microbiol. 2008;16:222-9. DOI:10.1016/j.tim.2008.01.011

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**Chapter 6**

*Clostridium difficile* **in the ICU**

David N. Herndon

**Abstract**

megacolon, NAP1

**1. Introduction**

William C. Sherman, Chris Lewis, Jong O. Lee and

*Clostridium difficile* has become an increasingly common infectious agent in the healthcare setting. It is generally associated with antibiotic use and causes diarrhea as well as other complications such as pseudomembranous colitis (PMC) and toxic megacolon. This organism poses a serious threat to patients in the intensive care unit (ICU) as it increases hospital length of stay, morbidity, and mortality. Recurrence rates are typically higher in the ICU population as those patients usually have immunocompromised systems, more exposure to antibiotics and proton pump inhibitors, loss of normal nutritional balance, and alterations in their colonic flora. Emergence of more virulent and pathogenic strains has made combating the infection even more difficult. Newer therapies, chemotherapeutic agents, and vaccinations are on the horizon. However, the most effective treatments to date are ceasing the inciting agent, reduction in the use of proton pump inhibitors, and prevention of the disease. In this chapter, we will explore the risk factors, diagnosis, treatment, and

**Keywords:** *Clostridium difficile*, intensive care unit, pseudomembranous colitis, toxic

*Clostridium difficile* is a gram-positive, spore forming anaerobic bacillus that can survive on environmental surfaces for years in the spore (dormant) stage. First cultured in 1935 by Hall and O'Toole, *C. difficile* was a relatively unknown organism until 1978 [1]. It was initially thought to be a mostly harmless colonizer of the human intestinal tract. In 1893, a young woman died after gastric surgery from a "diphtheric colitis" as described by John Finney and Sir William Osler [2]. In 1978, Dr. John G. Bartlett determined that *C. difficile* was associated

> © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

prevention of *C. difficile* infections (CDI) in the ICU.

http://dx.doi.org/10.5772/intechopen.69212

## **Chapter 6**

## *Clostridium difficile* **in the ICU**

William C. Sherman, Chris Lewis, Jong O. Lee and

David N. Herndon

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69212

#### **Abstract**

*Clostridium difficile* has become an increasingly common infectious agent in the healthcare setting. It is generally associated with antibiotic use and causes diarrhea as well as other complications such as pseudomembranous colitis (PMC) and toxic megacolon. This organism poses a serious threat to patients in the intensive care unit (ICU) as it increases hospital length of stay, morbidity, and mortality. Recurrence rates are typically higher in the ICU population as those patients usually have immunocompromised systems, more exposure to antibiotics and proton pump inhibitors, loss of normal nutritional balance, and alterations in their colonic flora. Emergence of more virulent and pathogenic strains has made combating the infection even more difficult. Newer therapies, chemotherapeutic agents, and vaccinations are on the horizon. However, the most effective treatments to date are ceasing the inciting agent, reduction in the use of proton pump inhibitors, and prevention of the disease. In this chapter, we will explore the risk factors, diagnosis, treatment, and prevention of *C. difficile* infections (CDI) in the ICU.

**Keywords:** *Clostridium difficile*, intensive care unit, pseudomembranous colitis, toxic megacolon, NAP1

## **1. Introduction**

*Clostridium difficile* is a gram-positive, spore forming anaerobic bacillus that can survive on environmental surfaces for years in the spore (dormant) stage. First cultured in 1935 by Hall and O'Toole, *C. difficile* was a relatively unknown organism until 1978 [1]. It was initially thought to be a mostly harmless colonizer of the human intestinal tract. In 1893, a young woman died after gastric surgery from a "diphtheric colitis" as described by John Finney and Sir William Osler [2]. In 1978, Dr. John G. Bartlett determined that *C. difficile* was associated

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

with the ailment that had killed the young woman 85 years prior and was now termed pseudomembranous colitis (PMC) [3].

associated with it in the United States and Europe due to high reinfection rates of approximately 30% and risk of relapse of 60% producing over 900,000 cases and an estimated \$1.1–\$3.2 billion

*Clostridium difficile* in the ICU

131

http://dx.doi.org/10.5772/intechopen.69212

Antibiotic therapy that disrupts the normal flora are usually to blame but proton pump inhibitors and other gastric acid suppression medications are increasingly associated with increases in *C. difficile* overgrowth [17]. Although the cephalosporin class, clindamycin, and the fluoroquinolones are all thought to place a patient at a higher risk of infection, all antibiotics, including oral vancomycin and metronidazole, can induce pseudomembranous colitis due to their ability to eliminate most normal intestinal flora in combination with the increased resistance patterns of more virulent strains of *C. difficile* [3, 14, 18, 19] The NAP1 strain is particularly important as it is associated with fluoroquinolone use and has risen in incidence in Canada, Europe, and the United States with increased virulence, toxin production, mortality,

The incidence and virulence of this pathogen has been steadily increasing over the last several decades contributing to higher morbidity and mortality. The increasingly older patient population with its higher acuity of medical issues and immunosenescence, the increased use of proton pump inhibitors, and the continued use of antibiotics has all allowed *C. difficile* to leave a greater impact in healthcare settings. In this chapter, we will explore the risk factors, diagnosis, treatment, and prevention of *C. difficile* infections in the intensive care

Pseudomembranous colitis became a common complication of antibiotic use in the 1950s at the beginning of the antibiotic era and was found often in postoperative patients with an incidence of 14–27% [22, 23]. *S. aureus* was the suspected pathogen and standard treatment

Tedesco et al. described "clindamycin colitis" in 1974 utilizing culture and endoscopy to diagnose pseudomembranous colitis associated with antibiotic use after 21% of patients given clindamycin developed diarrhea and 10% developed pseudomembranous colitis [25]. Incidentally, *S. aureus* did not grow from stool cultures from any of the patients. This study, more than prior publications, crystallized the connection between antibiotic use and development of pseudomembranous colitis. Green, while studying penicillin-induced death in guinea pigs in 1974 described stool cytopathic changes that he attributed to the activity of a latent virus. In retrospect, this appears to be the first identification of the effects of *C. difficile* cytotoxin [26]. Between 1977 and 1979, using hamster models, multiple teams of researchers identified *C. difficile* as the causative agent of pseudomembranous colitis, including detecting toxin B produced by *C. difficile* [27–30]. "Clindamycin colitis" became known as "antibioticinduced colitis" and most of the studies done in the 1980s demonstrated that cephalosporins were the most frequently implicated agents followed secondly by broad-spectrum penicillins,

per annum burden [15, 16].

treatment failures, and relapse [20, 21].

became oral vancomycin [24].

including amoxicillin [30–33].

**2. A historical perspective on** *Clostridium difficile*

unit (ICU).

*C. difficile* is currently the most common cause of antibiotic-associated pseudomembranous colitis in the healthcare setting and caused 20–30% of those with uncomplicated antibiotic-associated diarrhea [4]. According to the Centers for Disease Control, the number of cases of *C. difficile* infections (CDIs) in patients discharged from acute-care facilities doubled from 149,000 to 300,000 between 2001 and 2005 and based on recent trends has reached nearly 500,000 cases per year [5, 6]. There are occasionally other causes of antibiotic associated colitis due to organisms such as *Staphylococcus aureus, Klebsiella oxytoca*, enterotoxin-producing strains of *Clostridium perferingens*, or *Salmonella* [7]. Treatment duration for most microbial infections is usually around 14 days but prolonged exposure to broad-spectrum antibiotics has been associated with increased rates of both initial *C. difficile* infection and recurrence of *C. difficile* infection [8, 9].

The damage caused by *C. difficile* is due to the ability of the microbe to attach to the mucosa of the colon and release of exotoxins into the mucosa. The toxins may cause diarrhea, dilation of the colon (toxic megacolon), (**Figure 1**) sepsis, and death. Transmission is person to person via the fecal-oral route with ingestion of spores that germinate into vegetative bacteria within the small intestine. *C. difficile* produces two toxins—toxins A and B. These are large proteins (308 and 270 kDa, respectively) that cause severe inflammation and necrosis of the mucosal tissue by inactivating Rho, Rac, and Cdc42 targets within the epithelial cells through irreversible glycosylation [10, 11]. Toxin B is thought to be a gene duplication event of toxin A but is 10 times more cytotoxic than toxin A [12, 13].

The bacteria are normally found in up to 25% of hospitalized adults and up to 70% of the hospitalized pediatric population [14]. It does not cause disease until the normal flora is disrupted and *C. difficile* is allowed to proliferate. *C. difficile* infection has a very high economic cost

**Figure 1.** Toxic megacolon related to *Clostridium difficile* infection. Credit: University of Pittsburgh Department of Pathology.

associated with it in the United States and Europe due to high reinfection rates of approximately 30% and risk of relapse of 60% producing over 900,000 cases and an estimated \$1.1–\$3.2 billion per annum burden [15, 16].

Antibiotic therapy that disrupts the normal flora are usually to blame but proton pump inhibitors and other gastric acid suppression medications are increasingly associated with increases in *C. difficile* overgrowth [17]. Although the cephalosporin class, clindamycin, and the fluoroquinolones are all thought to place a patient at a higher risk of infection, all antibiotics, including oral vancomycin and metronidazole, can induce pseudomembranous colitis due to their ability to eliminate most normal intestinal flora in combination with the increased resistance patterns of more virulent strains of *C. difficile* [3, 14, 18, 19] The NAP1 strain is particularly important as it is associated with fluoroquinolone use and has risen in incidence in Canada, Europe, and the United States with increased virulence, toxin production, mortality, treatment failures, and relapse [20, 21].

The incidence and virulence of this pathogen has been steadily increasing over the last several decades contributing to higher morbidity and mortality. The increasingly older patient population with its higher acuity of medical issues and immunosenescence, the increased use of proton pump inhibitors, and the continued use of antibiotics has all allowed *C. difficile* to leave a greater impact in healthcare settings. In this chapter, we will explore the risk factors, diagnosis, treatment, and prevention of *C. difficile* infections in the intensive care unit (ICU).

## **2. A historical perspective on** *Clostridium difficile*

with the ailment that had killed the young woman 85 years prior and was now termed pseu-

*C. difficile* is currently the most common cause of antibiotic-associated pseudomembranous colitis in the healthcare setting and caused 20–30% of those with uncomplicated antibiotic-associated diarrhea [4]. According to the Centers for Disease Control, the number of cases of *C. difficile* infections (CDIs) in patients discharged from acute-care facilities doubled from 149,000 to 300,000 between 2001 and 2005 and based on recent trends has reached nearly 500,000 cases per year [5, 6]. There are occasionally other causes of antibiotic associated colitis due to organisms such as *Staphylococcus aureus, Klebsiella oxytoca*, enterotoxin-producing strains of *Clostridium perferingens*, or *Salmonella* [7]. Treatment duration for most microbial infections is usually around 14 days but prolonged exposure to broad-spectrum antibiotics has been associated with increased

The damage caused by *C. difficile* is due to the ability of the microbe to attach to the mucosa of the colon and release of exotoxins into the mucosa. The toxins may cause diarrhea, dilation of the colon (toxic megacolon), (**Figure 1**) sepsis, and death. Transmission is person to person via the fecal-oral route with ingestion of spores that germinate into vegetative bacteria within the small intestine. *C. difficile* produces two toxins—toxins A and B. These are large proteins (308 and 270 kDa, respectively) that cause severe inflammation and necrosis of the mucosal tissue by inactivating Rho, Rac, and Cdc42 targets within the epithelial cells through irreversible glycosylation [10, 11]. Toxin B is thought to be a gene duplication event of toxin A but is

The bacteria are normally found in up to 25% of hospitalized adults and up to 70% of the hospitalized pediatric population [14]. It does not cause disease until the normal flora is disrupted and *C. difficile* is allowed to proliferate. *C. difficile* infection has a very high economic cost

**Figure 1.** Toxic megacolon related to *Clostridium difficile* infection. Credit: University of Pittsburgh Department of Pathology.

rates of both initial *C. difficile* infection and recurrence of *C. difficile* infection [8, 9].

domembranous colitis (PMC) [3].

130 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

10 times more cytotoxic than toxin A [12, 13].

Pseudomembranous colitis became a common complication of antibiotic use in the 1950s at the beginning of the antibiotic era and was found often in postoperative patients with an incidence of 14–27% [22, 23]. *S. aureus* was the suspected pathogen and standard treatment became oral vancomycin [24].

Tedesco et al. described "clindamycin colitis" in 1974 utilizing culture and endoscopy to diagnose pseudomembranous colitis associated with antibiotic use after 21% of patients given clindamycin developed diarrhea and 10% developed pseudomembranous colitis [25]. Incidentally, *S. aureus* did not grow from stool cultures from any of the patients. This study, more than prior publications, crystallized the connection between antibiotic use and development of pseudomembranous colitis. Green, while studying penicillin-induced death in guinea pigs in 1974 described stool cytopathic changes that he attributed to the activity of a latent virus. In retrospect, this appears to be the first identification of the effects of *C. difficile* cytotoxin [26]. Between 1977 and 1979, using hamster models, multiple teams of researchers identified *C. difficile* as the causative agent of pseudomembranous colitis, including detecting toxin B produced by *C. difficile* [27–30]. "Clindamycin colitis" became known as "antibioticinduced colitis" and most of the studies done in the 1980s demonstrated that cephalosporins were the most frequently implicated agents followed secondly by broad-spectrum penicillins, including amoxicillin [30–33].

Although there are many causes of pseudomembranous colitis, the majority of cases since the late 1970s have been caused by *C. difficile* infection. Pseudomembranous colitis is limited to the proximal colon in 20–30% of cases and may therefore be missed by sigmoidoscopy, providing more credence to performing a complete colonoscopy to identify anatomic lesions [25, 34]. With the current availability of *C. difficile* toxin assays, colonoscopy is rarely necessary. The first test used to diagnose *C. difficile* involved neutralization of the cytotoxin by *C. sordellii* antitoxin. This remains the most sensitive and specific diagnostic test, but is expensive and requires 24–48 hours for results [35] that has led to the development of latex particle agglutination [36–38], dot immunoblot [39], PCR [40, 41], stool culture on selective media [42, 43], and enzyme immunoassay (EIA) [44, 45]. Because of differences between the hamster model and humans, it was originally believed that toxin A was important in human disease and many early EIA tests only detected toxin A, leading to false negative tests [46, 47].

by exogenous factors that can occur as a result of medications, procedures, or radiation therapy. Most hospitalized patients with *C. difficile* infection have been exposed to antibiotics within the past 30 days. More recently, it has been noted that medications that sup-

of *C. difficile* infection, though study results are not uniform and the mechanism is not known [50–54]. For patients with primary or recurrent *C. difficile* infection, consideration should be given to discontinuation of gastric acid suppressants unless the patient's risk for GI bleeding outweighs the risk of *C. difficile* infection treatment failure. Chemotherapy, medications for autoimmune conditions, transplant medications, and radiation of the bowel increase the risk of *C. difficile* infection by disrupting the normal intestinal mucosal barrier and inhibiting the body's immunodefenses. Nasogastric tubes and enemas, presumably because of alteration of

The second category of risk factors relates to how patients contract *C. difficile* infection. The most common method is by coming in contact with *C. difficile* spores from the hands of health care workers. Risk of contracting *C. difficile* infection is directly related to length of stay (LOS). Patients with longer LOS have multifactorial risk factors that include more severe illnesses that have a higher likelihood that they will require antibiotics and more prolonged exposure and interactions with health care workers [56, 57]. A patient's risk of contracting *C. difficile* infection is also related to *C. difficile* infection pressure that relates to the number of patients with *C. difficile* infection in a given care area [58]. Certain *C. difficile* strains, including the epidemic BI/NAP1/027 strain, have been isolated from prepared foods, pets, and from

The third category of risk factors relates to innate host susceptibility. Age >65 years is related to both an increased risk of primary *C. difficile* infection as well as an increased risk of more severe *C. difficile* infection. It is not known whether this is related to immune senescence, more frequent antibiotic usage, or increased comorbidities. The four comorbidities that place patients at greatest risk are sepsis, pneumonia, urinary tract infections, and skin infections—all of which generally require antibiotics for treatment. Patients hospitalized with higher numbers of conditions are more likely to contract *C. difficile* infection than patients with fewer conditions [48]. More recently, it has been noted that peripartum women and infants also appear to be at increased risk for *C. difficile* infection, including severe *C. difficile* infection related to the epidemic BI/NAP1/027 strain [62, 63]. Patients with inflammatory bowel disease (IBD) are more susceptible to *C. difficile* infection for reasons that are likely multifactorial, including antibiotic exposure, altered gut mucosal integrity, and immunosuppressive therapy. Patients with *C. difficile* infection superimposed on a flare of IBD are at risk for a particularly fulminant course. Because of altered gut physiology, patients with IBD may not develop pseudomembranes and may have a complicated diagnosis. Additionally, administration of glucocorticoids to treat the IBD exacerbation may predispose to *C. difficile* infection progression [64, 65]. Studies have shown that patients with HIV/AIDS or chronic kidney disease requiring hemodialysis are also at increased risk of *C. difficile* infection, possibly due to increased health care worker exposure or less robust

the normal flora and/or pH, increase patients' risk of *C. difficile* infection [55].


http://dx.doi.org/10.5772/intechopen.69212

*Clostridium difficile* in the ICU

133

press gastric acid, including proton-pump inhibitors and H<sup>2</sup>

livestock [59–61].

immune response [66, 67].

## **3. Clinical signs and symptoms**

Watery diarrhea with a distinct odor is usually the hallmark of *C. difficile* infection. Mild disease consists of crampy, watery diarrhea without systemic symptoms. This cohort constitutes 70% of patients with *C. difficile* infection as only about 30% of patients with *C. difficile* infection are febrile and 50% have a leukocytosis [48]. In severe disease, fecal leukocytes are generally high and diagnosis can be confirmed with endoscopy demonstrating pseudomembranous colitis. Other signs and symptoms of severe disease include abdominal pain, leukocytosis, and fever or other systemic symptoms. Leukocytosis is directly correlated with the severity of the disease. The elevation in white blood cell count can be as marginal as 15,000 cells/mL or as high as 50,000 cells/mL. Complications may include paralytic ileus, toxic megacolon, or other life threatening conditions. Postoperative patients and other patients with altered gastrointestinal motility may have pseudomembranous colitis without diarrhea secondary to ileus. Computed tomography is useful with characteristics of colitis readily seen on imaging that may include colonic wall thickening and associated ascites or toxic megacolon [21, 49].

Patients in the ICU tend to demonstrate the same spectrum of disease signs and symptoms as other infected persons. However, due to their illnesses, comorbidities weakened immune system and reduced ability to heal; the progression of the disease may advance more rapidly. Therefore, continual assessment of diarrhea and other symptoms of *C. difficile* infection is necessary as the severity may progress and further impact the already impaired and critical status of the patient in the ICU.

## **4. Risk factors for** *Clostridium difficile* **infection**

Risk factors for *C. difficile* infection fall under three categories. First category includes disruptions of the endogenous intestinal flora, perturbations of the mucosa, or immunomodulation by exogenous factors that can occur as a result of medications, procedures, or radiation therapy. Most hospitalized patients with *C. difficile* infection have been exposed to antibiotics within the past 30 days. More recently, it has been noted that medications that suppress gastric acid, including proton-pump inhibitors and H<sup>2</sup> -receptor blockers, increase risk of *C. difficile* infection, though study results are not uniform and the mechanism is not known [50–54]. For patients with primary or recurrent *C. difficile* infection, consideration should be given to discontinuation of gastric acid suppressants unless the patient's risk for GI bleeding outweighs the risk of *C. difficile* infection treatment failure. Chemotherapy, medications for autoimmune conditions, transplant medications, and radiation of the bowel increase the risk of *C. difficile* infection by disrupting the normal intestinal mucosal barrier and inhibiting the body's immunodefenses. Nasogastric tubes and enemas, presumably because of alteration of the normal flora and/or pH, increase patients' risk of *C. difficile* infection [55].

Although there are many causes of pseudomembranous colitis, the majority of cases since the late 1970s have been caused by *C. difficile* infection. Pseudomembranous colitis is limited to the proximal colon in 20–30% of cases and may therefore be missed by sigmoidoscopy, providing more credence to performing a complete colonoscopy to identify anatomic lesions [25, 34]. With the current availability of *C. difficile* toxin assays, colonoscopy is rarely necessary. The first test used to diagnose *C. difficile* involved neutralization of the cytotoxin by *C. sordellii* antitoxin. This remains the most sensitive and specific diagnostic test, but is expensive and requires 24–48 hours for results [35] that has led to the development of latex particle agglutination [36–38], dot immunoblot [39], PCR [40, 41], stool culture on selective media [42, 43], and enzyme immunoassay (EIA) [44, 45]. Because of differences between the hamster model and humans, it was originally believed that toxin A was important in human disease and

many early EIA tests only detected toxin A, leading to false negative tests [46, 47].

Watery diarrhea with a distinct odor is usually the hallmark of *C. difficile* infection. Mild disease consists of crampy, watery diarrhea without systemic symptoms. This cohort constitutes 70% of patients with *C. difficile* infection as only about 30% of patients with *C. difficile* infection are febrile and 50% have a leukocytosis [48]. In severe disease, fecal leukocytes are generally high and diagnosis can be confirmed with endoscopy demonstrating pseudomembranous colitis. Other signs and symptoms of severe disease include abdominal pain, leukocytosis, and fever or other systemic symptoms. Leukocytosis is directly correlated with the severity of the disease. The elevation in white blood cell count can be as marginal as 15,000 cells/mL or as high as 50,000 cells/mL. Complications may include paralytic ileus, toxic megacolon, or other life threatening conditions. Postoperative patients and other patients with altered gastrointestinal motility may have pseudomembranous colitis without diarrhea secondary to ileus. Computed tomography is useful with characteristics of colitis readily seen on imaging that may include colonic wall thickening and associated ascites or

Patients in the ICU tend to demonstrate the same spectrum of disease signs and symptoms as other infected persons. However, due to their illnesses, comorbidities weakened immune system and reduced ability to heal; the progression of the disease may advance more rapidly. Therefore, continual assessment of diarrhea and other symptoms of *C. difficile* infection is necessary as the severity may progress and further impact the already impaired and critical

Risk factors for *C. difficile* infection fall under three categories. First category includes disruptions of the endogenous intestinal flora, perturbations of the mucosa, or immunomodulation

**3. Clinical signs and symptoms**

132 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

toxic megacolon [21, 49].

status of the patient in the ICU.

**4. Risk factors for** *Clostridium difficile* **infection**

The second category of risk factors relates to how patients contract *C. difficile* infection. The most common method is by coming in contact with *C. difficile* spores from the hands of health care workers. Risk of contracting *C. difficile* infection is directly related to length of stay (LOS). Patients with longer LOS have multifactorial risk factors that include more severe illnesses that have a higher likelihood that they will require antibiotics and more prolonged exposure and interactions with health care workers [56, 57]. A patient's risk of contracting *C. difficile* infection is also related to *C. difficile* infection pressure that relates to the number of patients with *C. difficile* infection in a given care area [58]. Certain *C. difficile* strains, including the epidemic BI/NAP1/027 strain, have been isolated from prepared foods, pets, and from livestock [59–61].

The third category of risk factors relates to innate host susceptibility. Age >65 years is related to both an increased risk of primary *C. difficile* infection as well as an increased risk of more severe *C. difficile* infection. It is not known whether this is related to immune senescence, more frequent antibiotic usage, or increased comorbidities. The four comorbidities that place patients at greatest risk are sepsis, pneumonia, urinary tract infections, and skin infections—all of which generally require antibiotics for treatment. Patients hospitalized with higher numbers of conditions are more likely to contract *C. difficile* infection than patients with fewer conditions [48]. More recently, it has been noted that peripartum women and infants also appear to be at increased risk for *C. difficile* infection, including severe *C. difficile* infection related to the epidemic BI/NAP1/027 strain [62, 63]. Patients with inflammatory bowel disease (IBD) are more susceptible to *C. difficile* infection for reasons that are likely multifactorial, including antibiotic exposure, altered gut mucosal integrity, and immunosuppressive therapy. Patients with *C. difficile* infection superimposed on a flare of IBD are at risk for a particularly fulminant course. Because of altered gut physiology, patients with IBD may not develop pseudomembranes and may have a complicated diagnosis. Additionally, administration of glucocorticoids to treat the IBD exacerbation may predispose to *C. difficile* infection progression [64, 65]. Studies have shown that patients with HIV/AIDS or chronic kidney disease requiring hemodialysis are also at increased risk of *C. difficile* infection, possibly due to increased health care worker exposure or less robust immune response [66, 67].

## **5. Diagnosis**

In the modern era, multiple tools have been developed to identify and detect *C. difficile* to include cultures, polymerase chain reaction (PCR), and enzyme immunoassays (EIA). Culturing *C. difficile* is difficult due to the strict anaerobic nature of the organism and the oxygen sensitivity that can kill the living organism. Utilizing an anaerobic chamber with a composition of 5% CO<sup>2</sup> , 10% H<sup>2</sup> , and 85% N2, along with an air lock, has allowed the culturing, preservation, and storage of the living organism and spores [35, 43]. Once the organism has been cultured, PCR or EIA techniques can be utilized to detect toxin within the culture. These same techniques can be used independently of a culture to detect toxin within the stool sample. PCR has been successfully used since 1985 to amplify the 8.1 kilo base-pairs of the toxin A gene. Using 35 cycles of alternating 95–55°C temperatures and a Southern blot to isolate the 252 base-pair DNA fragment, PCR has become easy and commonplace for identification of the toxins [40, 41]. EIA has similarly been used since the early 80s for detection of both toxin A and B. The early tests were able to detect levels of toxin to 0.1 ng using a double sandwich microtiter plate with specificities of 98.6% and 100% for toxin A and toxin B, respectively [42–45]. More recently, glutamate dehydrogenase-immunoassay has been used as an initial screening tool with a chemiluminescent toxin-immunoassay for confirmation of both toxins A and B. The combined two-step process has a sensitivity and specificity of 100% [68]. The premise of the EIA tests is that antibodies to the toxins are attached to a plate. When the toxins pass over the antibodies, they become bound. A second preparation of antibodies with a marker attached to them is then added and a device to detect the markers allows for quantitative evaluation of the toxins present.

In addition to laboratory tests, computed tomography is useful to evaluate for toxic megacolon and colitis. When there is high clinical suspicion yet laboratory diagnostic tests have yielded negative results, the definitive test is colonoscopy. The appearance of pseudomembranes in the clinical setting of *C. difficile* infection is confirmatory for the diagnosis (**Figure 2**).

Intensivists should be familiar with the tests offered in their institution and be able to interpret the laboratory results in the context of clinical presentation. When clinical suspicion for *C. difficile* infection is high, the intensivist should initiate empiric therapy for *C. difficile* infec-

toxins

available

increased sensitivity

pseudomembranes

*difficile* infections

2–3 hours Very sensitive for detection of toxigenic strains of *Clostridium difficile* using PCR

3–4 days Must have initial growth from culture prior to testing for

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Culture *Clostridium difficile* 34 days Nonspecific and not useful for detection of toxins

Cytotoxin Toxin B 2–3 days Costly and time-consuming. Results not immediately

EIA toxin A & B Toxin A & B 2–3 hours Very quick but not sensitive. Need 3 specimens for

Colonoscopy Pseudomembranes <1 hour Very specific and sensitive for the detection of

**Table 1.** Diagnostic modalities for the identification of *Clostridium difficile* in the ICU.

EIA GDH *Clostridium difficile* 2–3 hours Screening test. Detects presence of bacteria but not specific

CT scan Colitis <1 hour Very sensitive for colitis but not specific for *Clostridium* 

EIA, Enzyme Immunoassay; GDH, glutamine dehydrogenase; PCR, polymerase chain reaction; CT, computed tomography

Once diagnosed, the first line of treatment is to discontinue implicated antibiotics, gastric acid suppression medications, and antiperistaltic medications, including narcotics and antimotility agents. Reduced peristalsis may prolong toxin exposure to the colonic mucosa [7]. Unfortunately, a large proportion of patients who develop *C. difficile* infection have documented infections that require treatment with antibiotics, and in the ICU setting, this proportion may reach 60% [69]. When it is not possible to stop antibiotic therapy, it is best to tailor coverage to more narrow spectrum agents once cultures and sensitivities are available. It is recommended to transition as soon as possible to β-lactams, macrolides, aminoglycosides, antistaphylococcal drugs, tetracyclines, and other agents that have a lower likelihood of causing

**Table 1** displays the various current diagnostic modalities.

tion regardless of the diagnostic test results [48].

Culture-toxins Toxigenic *Clostridium difficile*

Toxin B gene Toxigenic *Clostridium difficile*

**Test Detection Time Usefulness**

**6. Treatment**

*C. difficile* infection [70].

**Figure 2.** *Clostridium difficile* associated pseudomembranous colitis. Credit: North American Society for Pediatric Gastroenterology, Hepatology and Nutrition.

In addition to laboratory tests, computed tomography is useful to evaluate for toxic megacolon and colitis. When there is high clinical suspicion yet laboratory diagnostic tests have yielded negative results, the definitive test is colonoscopy. The appearance of pseudomembranes in the clinical setting of *C. difficile* infection is confirmatory for the diagnosis (**Figure 2**). **Table 1** displays the various current diagnostic modalities.

Intensivists should be familiar with the tests offered in their institution and be able to interpret the laboratory results in the context of clinical presentation. When clinical suspicion for *C. difficile* infection is high, the intensivist should initiate empiric therapy for *C. difficile* infection regardless of the diagnostic test results [48].


EIA, Enzyme Immunoassay; GDH, glutamine dehydrogenase; PCR, polymerase chain reaction; CT, computed tomography

**Table 1.** Diagnostic modalities for the identification of *Clostridium difficile* in the ICU.

## **6. Treatment**

**5. Diagnosis**

134 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

composition of 5% CO<sup>2</sup>

, 10% H<sup>2</sup>

titative evaluation of the toxins present.

enterology, Hepatology and Nutrition.

In the modern era, multiple tools have been developed to identify and detect *C. difficile* to include cultures, polymerase chain reaction (PCR), and enzyme immunoassays (EIA). Culturing *C. difficile* is difficult due to the strict anaerobic nature of the organism and the oxygen sensitivity that can kill the living organism. Utilizing an anaerobic chamber with a

ing, preservation, and storage of the living organism and spores [35, 43]. Once the organism has been cultured, PCR or EIA techniques can be utilized to detect toxin within the culture. These same techniques can be used independently of a culture to detect toxin within the stool sample. PCR has been successfully used since 1985 to amplify the 8.1 kilo base-pairs of the toxin A gene. Using 35 cycles of alternating 95–55°C temperatures and a Southern blot to isolate the 252 base-pair DNA fragment, PCR has become easy and commonplace for identification of the toxins [40, 41]. EIA has similarly been used since the early 80s for detection of both toxin A and B. The early tests were able to detect levels of toxin to 0.1 ng using a double sandwich microtiter plate with specificities of 98.6% and 100% for toxin A and toxin B, respectively [42–45]. More recently, glutamate dehydrogenase-immunoassay has been used as an initial screening tool with a chemiluminescent toxin-immunoassay for confirmation of both toxins A and B. The combined two-step process has a sensitivity and specificity of 100% [68]. The premise of the EIA tests is that antibodies to the toxins are attached to a plate. When the toxins pass over the antibodies, they become bound. A second preparation of antibodies with a marker attached to them is then added and a device to detect the markers allows for quan-

**Figure 2.** *Clostridium difficile* associated pseudomembranous colitis. Credit: North American Society for Pediatric Gastro-

, and 85% N2, along with an air lock, has allowed the cultur-

Once diagnosed, the first line of treatment is to discontinue implicated antibiotics, gastric acid suppression medications, and antiperistaltic medications, including narcotics and antimotility agents. Reduced peristalsis may prolong toxin exposure to the colonic mucosa [7]. Unfortunately, a large proportion of patients who develop *C. difficile* infection have documented infections that require treatment with antibiotics, and in the ICU setting, this proportion may reach 60% [69]. When it is not possible to stop antibiotic therapy, it is best to tailor coverage to more narrow spectrum agents once cultures and sensitivities are available. It is recommended to transition as soon as possible to β-lactams, macrolides, aminoglycosides, antistaphylococcal drugs, tetracyclines, and other agents that have a lower likelihood of causing *C. difficile* infection [70].

Oral vancomycin is the only agent currently approved for treatment of *C. difficile* infection, although metronidazole in both oral and intravenous forms has been shown to be effective in treating *C. difficile* infection. Intravenous vancomycin has not been shown to be effective. Metronidazole has become the preferred agent for initial treatment of *C. difficile* infection because of lower cost [71, 72] and because of concerns over the possibility of increased development of vancomycin-resistant enterococcus [73, 74]. Metronidazole should be considered first-line therapy for mild to moderate *C. difficile* infection; however, it does have disadvantages compared to oral vancomycin. In a study involving 207 patients with *C. difficile* infection, 22% of patients remained symptomatic after 10 day therapy with metronidazole and 27% developed a relapse [75]. In a separate randomized trial involving 150 patients, the cure rate for metronidazole was only 76% compared with a 97% cure rate after treatment with vancomycin for the treatment of severe *C. difficile* infection [49]. Based on these studies and other data, oral vancomycin should be considered superior in the treatment of severe infections when GI motility is intact (**Table 2**) [49]. The pharmacology of oral vancomycin lends itself to being more effective as it is not absorbed by the GI tract and reaches the colon in high concentrations. The usual dosing regimen of 125 mg achieves levels of vancomycin 500–1000 times the minimal inhibitory concentration (MIC) of 90% of *C. difficile* in stool [48].

patients, of which 287 received fidaxomicin and 309 received vancomycin, 88.2% of patients in the fidaxomicin group and 85.8% of those in the vancomycin group met the criteria for clinical cure. In addition, treatment with fidaxomicin was associated with a significantly lower rate of recurrence than was treatment with vancomycin (15.4 vs. 25.3%). More studies are warranted

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Regardless of the type of medication, early treatment has been supported as the most effective pharmacologic treatment. A study by Zahar et al. conducted in three French ICUs has demonstrated that early treatment of ICU-acquired *C. difficile* infection results in mortality rates consistent with a control population of other ICU patients that have developed diarrhea that is not *C. difficile* infection associated. Treatment was initiated within 24 hours of onset and consisted of either metronidazole or oral vancomycin. The study involved 5,260 patients with an incidence of ICU-acquired diarrhea of 9.7%. All those with diarrhea were tested for *C. difficile* infection and 13.5% of those tested had confirmed toxin A or B by EIA and further confirmation by culture. None of the positive cultures produced any of the hypervirulent NAP1/027 strains seen in North American outbreaks. Overall mortality of ICU-acquired *C. difficile* infection was not independently associated with higher mortality rates compared to other patients with diarrhea in the ICU when matched for severity of illness, comorbidities, or complications occurring in the ICU. However, both the overall hospital stay and ICU stay was prolonged in the ICU-acquired *C. difficile* infection patients when compared to ICU patients as a whole (median 4 vs. 20 days) and ICU patients with diarrhea not associated with *C. difficile* infection (median 17 vs. 20 days). Despite these prolonged median stays, analysis did not demonstrate a statistically significant difference in length of stay with an estimated increase in overall ICU stay of 6.3 days ± 4.3, p = 0.14 compared to other ICU

Microbial therapy with fecal transplantation can be accomplished with instillation of liquid preparations of stool from healthy donors. This method has proven successful for treating recurrent *C. difficile* infection in 70–100% of cases [84]. Probiotics may prevent attachment of *C. difficile* to epithelial cells and can reduce the incidence of *C. difficile* infection. *Saccharomyces boulardii* in particular has proven to be effective [49] whereas the use of *Lactobacillus* with conventional antibiotic therapy has shown mixed results including some studies showing no benefit in the treatment of *C. difficile* infection in several randomized

Use of anion exchange resins, such as cholestyramine and colestipol, with the hope of binding *C. difficile* cytotoxins in the treatment of *C. difficile* infection, has not only been shown to be effective [89, 90], but also carries the theoretical risk of binding intraluminal vancomycin, thus resulting in subtherapeutic vancomycin levels [91]. Intravenous immunoglobulins have been suggested for treatment of *C. difficile* infection but due to an insufficient evidence base and conflicting data, its use cannot be generally recommended until further studies have been conducted [92, 93]. Subtotal colectomy should be considered if there is no response to medical therapy within 3–4 days or if the patient remains seriously ill to avoid complications such as

but results are promising [49, 81, 82].

patients with diarrhea [83].

controlled trials [85–88].

colonic perforation and sepsis [7].

If the patient has ileus or severe pseudomembranous colitis and medication cannot be given orally, the use of rectal instillation of vancomycin solutions is supported by case reports [70, 76, 77]. The addition of intravenous metronidazole to either oral or intracolonic vancomycin in severely ill patients with ileus has been described, although this approach has not been adequately studied [78, 79].

Fidaxomicin is the first member in a new class of narrow spectrum macrocyclic antibiotics that are enterally administered and minimally absorbed in the GI tract. Having excellent *in vitro* and *in vivo* activity against *C. difficile*, including NAP1/BI/027 strains, and, while exhibiting limited activity *in vitro* and *in vivo* against components of the normal gut flora, fidaxomicin is an excellent candidate for replacing other agents in the treatment of *C. difficile* infections [80]. In a prospective, multicenter, double-blind, randomized, parallel-group trial involving 596


**Table 2.** Treatment modalities for *Clostridium difficile* infections.

patients, of which 287 received fidaxomicin and 309 received vancomycin, 88.2% of patients in the fidaxomicin group and 85.8% of those in the vancomycin group met the criteria for clinical cure. In addition, treatment with fidaxomicin was associated with a significantly lower rate of recurrence than was treatment with vancomycin (15.4 vs. 25.3%). More studies are warranted but results are promising [49, 81, 82].

Oral vancomycin is the only agent currently approved for treatment of *C. difficile* infection, although metronidazole in both oral and intravenous forms has been shown to be effective in treating *C. difficile* infection. Intravenous vancomycin has not been shown to be effective. Metronidazole has become the preferred agent for initial treatment of *C. difficile* infection because of lower cost [71, 72] and because of concerns over the possibility of increased development of vancomycin-resistant enterococcus [73, 74]. Metronidazole should be considered first-line therapy for mild to moderate *C. difficile* infection; however, it does have disadvantages compared to oral vancomycin. In a study involving 207 patients with *C. difficile* infection, 22% of patients remained symptomatic after 10 day therapy with metronidazole and 27% developed a relapse [75]. In a separate randomized trial involving 150 patients, the cure rate for metronidazole was only 76% compared with a 97% cure rate after treatment with vancomycin for the treatment of severe *C. difficile* infection [49]. Based on these studies and other data, oral vancomycin should be considered superior in the treatment of severe infections when GI motility is intact (**Table 2**) [49]. The pharmacology of oral vancomycin lends itself to being more effective as it is not absorbed by the GI tract and reaches the colon in high concentrations. The usual dosing regimen of 125 mg achieves levels of vancomycin 500–1000 times the minimal inhibitory concentration (MIC) of 90% of *C. difficile* in stool [48]. If the patient has ileus or severe pseudomembranous colitis and medication cannot be given orally, the use of rectal instillation of vancomycin solutions is supported by case reports [70, 76, 77]. The addition of intravenous metronidazole to either oral or intracolonic vancomycin in severely ill patients with ileus has been described, although this approach has not been

Fidaxomicin is the first member in a new class of narrow spectrum macrocyclic antibiotics that are enterally administered and minimally absorbed in the GI tract. Having excellent *in vitro* and *in vivo* activity against *C. difficile*, including NAP1/BI/027 strains, and, while exhibiting limited activity *in vitro* and *in vivo* against components of the normal gut flora, fidaxomicin is an excellent candidate for replacing other agents in the treatment of *C. difficile* infections [80]. In a prospective, multicenter, double-blind, randomized, parallel-group trial involving 596

Metronidazole: 500 mg PO every 8 hours

Metronidazole: 500 mg IV every 8 hours

Vancomycin: 125 mg PO every 6 hours Metronidazole: 500 mg IV every 8 hours

Metronidazole: 500 mg IV every 8 hours

Life-threatening CDI 1st line Vancomycin: 500 mg every 6 hours via NGT or by enema plus

Vancomycin: 125 mg PO every 6 hours or Fidaxomicin: 200 mg PO every 12

adequately studied [78, 79].

136 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

Mild CDI 1st line

Severe CDI 1st line

**Severity Preference Medications**

Alternate (PO) Alternate (IV)

Alternate (IV)

**Table 2.** Treatment modalities for *Clostridium difficile* infections.

hours

Relapsed CDI 1st line Treatment based on severity as above

Regardless of the type of medication, early treatment has been supported as the most effective pharmacologic treatment. A study by Zahar et al. conducted in three French ICUs has demonstrated that early treatment of ICU-acquired *C. difficile* infection results in mortality rates consistent with a control population of other ICU patients that have developed diarrhea that is not *C. difficile* infection associated. Treatment was initiated within 24 hours of onset and consisted of either metronidazole or oral vancomycin. The study involved 5,260 patients with an incidence of ICU-acquired diarrhea of 9.7%. All those with diarrhea were tested for *C. difficile* infection and 13.5% of those tested had confirmed toxin A or B by EIA and further confirmation by culture. None of the positive cultures produced any of the hypervirulent NAP1/027 strains seen in North American outbreaks. Overall mortality of ICU-acquired *C. difficile* infection was not independently associated with higher mortality rates compared to other patients with diarrhea in the ICU when matched for severity of illness, comorbidities, or complications occurring in the ICU. However, both the overall hospital stay and ICU stay was prolonged in the ICU-acquired *C. difficile* infection patients when compared to ICU patients as a whole (median 4 vs. 20 days) and ICU patients with diarrhea not associated with *C. difficile* infection (median 17 vs. 20 days). Despite these prolonged median stays, analysis did not demonstrate a statistically significant difference in length of stay with an estimated increase in overall ICU stay of 6.3 days ± 4.3, p = 0.14 compared to other ICU patients with diarrhea [83].

Microbial therapy with fecal transplantation can be accomplished with instillation of liquid preparations of stool from healthy donors. This method has proven successful for treating recurrent *C. difficile* infection in 70–100% of cases [84]. Probiotics may prevent attachment of *C. difficile* to epithelial cells and can reduce the incidence of *C. difficile* infection. *Saccharomyces boulardii* in particular has proven to be effective [49] whereas the use of *Lactobacillus* with conventional antibiotic therapy has shown mixed results including some studies showing no benefit in the treatment of *C. difficile* infection in several randomized controlled trials [85–88].

Use of anion exchange resins, such as cholestyramine and colestipol, with the hope of binding *C. difficile* cytotoxins in the treatment of *C. difficile* infection, has not only been shown to be effective [89, 90], but also carries the theoretical risk of binding intraluminal vancomycin, thus resulting in subtherapeutic vancomycin levels [91]. Intravenous immunoglobulins have been suggested for treatment of *C. difficile* infection but due to an insufficient evidence base and conflicting data, its use cannot be generally recommended until further studies have been conducted [92, 93]. Subtotal colectomy should be considered if there is no response to medical therapy within 3–4 days or if the patient remains seriously ill to avoid complications such as colonic perforation and sepsis [7].

## **7. Treatment failure and relapse**

Patient characteristics that predispose to metronidazole failure include low serum albumin, continued exposure to the inciting antibiotic, and residence in the ICU [94, 95]. Particularly worrisome and concerning is the finding that relapsing or recurrent infections occur in up to 30% of patients treated for *C. difficile* infection whether the initial treatment was metronidazole or vancomycin [96]. This could be due to reinfection with the same endogenous strain or from a different strain acquired exogenously. Patients that had an initial infection followed by reinfection have a 50–65% chance of further repeated episodes. A metaanalysis by Garey et al. found that reexposure to antimicrobials, gastric acid suppression, and older age are all associated with an increased risk of recurrent *C. difficile* infection [97]. Patients that have three or more episodes of *C. difficile* infection, considered to be multiple *C. difficile* infection recurrence, are best treated with a tapered regimen of oral vancomycin. The initial dose of vancomycin administered is at the usual 125 mg by mouth four times a day for 10–14 days but then one dose per day is removed one week at a time until the patient is taking one dose every 2–3 days. The rationale for this regimen is that as the doses are spaced out, the colonic flora has time to regenerate [48].

There is some evidence to support that plant based diets may reduce the number of pathobionts such as *C. difficile* and increase the number of protective species such as Lactobacillus [100–103]. Altered flora with resulting altered bile metabolism within the gut by flora favored by plant-based diets have implications in colonocyte protection [102]. Intestinal microbiota are able to produce short chain fatty acids (SCFA), such as acetate, propionate, and butyrate, through metabolism of dietary fiber. These SCFA have been shown to be colonocyte protective. A strong positive correlation has been found between *Faecalibacterium prausnitzii* and butyrate production in the gastrointestinal tract, suggesting that this species may be associated with higher fiber intake and reduced risk, not only for *C. difficile* infection, but also for other common comorbidities in the elderly including cardiovascular disease, colon cancer, diabetes, and obesity [104]. A move toward a diet that decreases risk for contracting *C. difficile* infection should be encouraged, not only in the elderly, but also generally, because of the broad implications.

*Clostridium difficile* in the ICU

139

http://dx.doi.org/10.5772/intechopen.69212

Disinfectant products based on quaternary ammonium compounds, commonly used to clean patient rooms, are not sporicidal. Therefore, using sporicidal hypochlorite-based disinfectants on surfaces is recommended. However, use of antisporicidal agents outside an outbreak

Hand hygiene is the most important preventive measure to reduce transmission of *C. difficile* spores. Soap and water has been demonstrated to be superior to alcohol based hand rubs and other forms of hand sanitation with regard to transmission by healthcare workers [21, 107]. Hospital hygiene hand protocols should be followed assiduously at all times. Other precautions that should be utilized include isolation of the patient, barrier precautions, and use of chlorine based chemical wipes [107]. These precautions should not be lifted based on stool studies as there are no diagnostic methods to determine response to treatment. Rather, the decision should be made on clinical signs and symptoms with resolution of diarrhea, fevers, and leukocytosis. A strong antibiotic stewardship program is essential to limit the use of antibiotics that may cause *C. difficile* infection and is generally a good principle to follow. It has been demonstrated that up to 25% of antibiotic administration is not indicated, even in the ICU [108].

Diarrhea is a common problem in the ICU affecting up to 40% of patients admitted. Severely burned patients may have an incidence of greater than 90% [109, 110]. Enteral tube feeding is the most common cause of diarrhea in the ICU; other causes include hypoalbuminemia, intestinal ischemia, and medications. *C. difficile* infection is the most common infectious cause of diarrhea in the ICU [111, 112]. The severity of *C. difficile* infection is increasing which is possibly related to the emergence of more virulent strains such as the BI/NAP1/027 strain, prompting more admissions to the ICU for management of *C. difficile* infection related complications [113].

is not associated with lower rates of *C. difficile* infection [105, 106].

**10.** *Clostridium difficile* **infection in the intensive care unit**

**9. Prevention of hospital spread**

## **8. Generating optimal colonic flora for risk reduction**

There is an urgent need for alternative means of preventing and treating *C. difficile* infection in high-risk individuals. Metagenomics have improved our understanding of the "colonization resistance barrier" and how this could be optimized. The "colonization resistance barrier" in the normal healthy colon consists of high microbial diversity, substrate/area competition, immune response modulation and short-chain fatty acid (SCFA) production [16, 98]. These factors are often missing in the elderly. Decreased pH, oxidation-reduction potentials, and higher concentrations of short-chain fatty acids have been suggested to inhibit *C. difficile* growth and toxin production throughout *in vitro* and *in vivo* studies. There is, therefore, evidence in support of a colonization resistance barrier against *C. difficile* infection [16, 98].

For instance, *in vitro, Bifidobacterium longum* and *Bifidobacterium breve* have been show to significantly reduce the growth of the toxigenic strain *C. difficile* LMG21717 [99]. In a randomized, placebo-controlled, double-blind trial at a long-term elderly care facility, the effectiveness of a *Lactobacillus casei* strain Shirota (LcS) infused beverage was demonstrated by altering *Clostridium* infection rates among the residents. Daily consumption of the beverage resulted in a significantly lower incidence of fever and improved bowel movements. When compared to a resident control group drinking a placebo beverage, stool studies from the experimental LcS group showed significantly higher number of both *Bifidobacterium* and *Lactobacillus* (p < 0.01), significantly lower number of destructive bacteria such as *C. difficile* (p < 0.05), and a higher fecal acetic acid concentration. This study was also conducted among the facility's staff and a significant difference in the intestinal microbiota, fecal acetic acid, and pH was also observed between the LcS and placebo groups [100].

There is some evidence to support that plant based diets may reduce the number of pathobionts such as *C. difficile* and increase the number of protective species such as Lactobacillus [100–103]. Altered flora with resulting altered bile metabolism within the gut by flora favored by plant-based diets have implications in colonocyte protection [102]. Intestinal microbiota are able to produce short chain fatty acids (SCFA), such as acetate, propionate, and butyrate, through metabolism of dietary fiber. These SCFA have been shown to be colonocyte protective. A strong positive correlation has been found between *Faecalibacterium prausnitzii* and butyrate production in the gastrointestinal tract, suggesting that this species may be associated with higher fiber intake and reduced risk, not only for *C. difficile* infection, but also for other common comorbidities in the elderly including cardiovascular disease, colon cancer, diabetes, and obesity [104]. A move toward a diet that decreases risk for contracting *C. difficile* infection should be encouraged, not only in the elderly, but also generally, because of the broad implications.

## **9. Prevention of hospital spread**

**7. Treatment failure and relapse**

138 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

flora has time to regenerate [48].

**8. Generating optimal colonic flora for risk reduction**

observed between the LcS and placebo groups [100].

Patient characteristics that predispose to metronidazole failure include low serum albumin, continued exposure to the inciting antibiotic, and residence in the ICU [94, 95]. Particularly worrisome and concerning is the finding that relapsing or recurrent infections occur in up to 30% of patients treated for *C. difficile* infection whether the initial treatment was metronidazole or vancomycin [96]. This could be due to reinfection with the same endogenous strain or from a different strain acquired exogenously. Patients that had an initial infection followed by reinfection have a 50–65% chance of further repeated episodes. A metaanalysis by Garey et al. found that reexposure to antimicrobials, gastric acid suppression, and older age are all associated with an increased risk of recurrent *C. difficile* infection [97]. Patients that have three or more episodes of *C. difficile* infection, considered to be multiple *C. difficile* infection recurrence, are best treated with a tapered regimen of oral vancomycin. The initial dose of vancomycin administered is at the usual 125 mg by mouth four times a day for 10–14 days but then one dose per day is removed one week at a time until the patient is taking one dose every 2–3 days. The rationale for this regimen is that as the doses are spaced out, the colonic

There is an urgent need for alternative means of preventing and treating *C. difficile* infection in high-risk individuals. Metagenomics have improved our understanding of the "colonization resistance barrier" and how this could be optimized. The "colonization resistance barrier" in the normal healthy colon consists of high microbial diversity, substrate/area competition, immune response modulation and short-chain fatty acid (SCFA) production [16, 98]. These factors are often missing in the elderly. Decreased pH, oxidation-reduction potentials, and higher concentrations of short-chain fatty acids have been suggested to inhibit *C. difficile* growth and toxin production throughout *in vitro* and *in vivo* studies. There is, therefore, evidence in support of a colonization resistance barrier against *C. difficile* infection [16, 98].

For instance, *in vitro, Bifidobacterium longum* and *Bifidobacterium breve* have been show to significantly reduce the growth of the toxigenic strain *C. difficile* LMG21717 [99]. In a randomized, placebo-controlled, double-blind trial at a long-term elderly care facility, the effectiveness of a *Lactobacillus casei* strain Shirota (LcS) infused beverage was demonstrated by altering *Clostridium* infection rates among the residents. Daily consumption of the beverage resulted in a significantly lower incidence of fever and improved bowel movements. When compared to a resident control group drinking a placebo beverage, stool studies from the experimental LcS group showed significantly higher number of both *Bifidobacterium* and *Lactobacillus* (p < 0.01), significantly lower number of destructive bacteria such as *C. difficile* (p < 0.05), and a higher fecal acetic acid concentration. This study was also conducted among the facility's staff and a significant difference in the intestinal microbiota, fecal acetic acid, and pH was also Disinfectant products based on quaternary ammonium compounds, commonly used to clean patient rooms, are not sporicidal. Therefore, using sporicidal hypochlorite-based disinfectants on surfaces is recommended. However, use of antisporicidal agents outside an outbreak is not associated with lower rates of *C. difficile* infection [105, 106].

Hand hygiene is the most important preventive measure to reduce transmission of *C. difficile* spores. Soap and water has been demonstrated to be superior to alcohol based hand rubs and other forms of hand sanitation with regard to transmission by healthcare workers [21, 107]. Hospital hygiene hand protocols should be followed assiduously at all times. Other precautions that should be utilized include isolation of the patient, barrier precautions, and use of chlorine based chemical wipes [107]. These precautions should not be lifted based on stool studies as there are no diagnostic methods to determine response to treatment. Rather, the decision should be made on clinical signs and symptoms with resolution of diarrhea, fevers, and leukocytosis. A strong antibiotic stewardship program is essential to limit the use of antibiotics that may cause *C. difficile* infection and is generally a good principle to follow. It has been demonstrated that up to 25% of antibiotic administration is not indicated, even in the ICU [108].

## **10.** *Clostridium difficile* **infection in the intensive care unit**

Diarrhea is a common problem in the ICU affecting up to 40% of patients admitted. Severely burned patients may have an incidence of greater than 90% [109, 110]. Enteral tube feeding is the most common cause of diarrhea in the ICU; other causes include hypoalbuminemia, intestinal ischemia, and medications. *C. difficile* infection is the most common infectious cause of diarrhea in the ICU [111, 112]. The severity of *C. difficile* infection is increasing which is possibly related to the emergence of more virulent strains such as the BI/NAP1/027 strain, prompting more admissions to the ICU for management of *C. difficile* infection related complications [113].

In a systematic review and metaanalysis of 22 published studies from 1983 to 2015 that included 80,835 ICU patients, the effects of *C. difficile* infection on morbidity and mortality were investigated. Karanika et al. found that prevalence of *C. difficile* infection among ICU patients was 2% but 5-fold greater in those patients with diarrhea (11%). Those patients that were diagnosed with *C. difficile* infection had a 25% incidence of the severe form of the disease and diagnosed with pseudomembranous colitis. ICU mortality was not significantly different between the group with *C. difficile* infection and the non*C. difficile* infection group based on seven studies that enrolled a combined 12,165 patients. However, the overall hospital mortality between those same groups was significantly increased in the *C. difficile* infection group with 32% mortality compared to 24% (p = 0.03). Similarly, length of ICU and hospital stay among *C. difficile* infection patients was longer when compared to non*C. difficile* infection patients. Based on five studies with over 10,000 patients, *C. difficile* infection patients had an average ICU stay of 24 days and overall hospital stay of 50 days compared to 19 days and 30 days, respectively, for the non*C. difficile* infection group (p = 0.001) [114].

thought to be synergistic with the production of toxin A and B. Strain BI/NAP1/027 was found to be highly resistant to fluoroquinolone classes of antibiotics and was also found to produce 16-fold higher concentrations of toxin A and 23-fold higher concentrations of toxin B than less virulent toxinotype 0 strains. The binary toxin has been associated with more severe diarrhea when combined with toxin A and B. When produced alone, binary toxin does not appear to produce disease [3, 10] but does appear to be a marker of both *C. difficile* infection severity and recurrence [120]. The emergence is generally believed to be related to fluoroquinolone

*Clostridium difficile* in the ICU

141

http://dx.doi.org/10.5772/intechopen.69212

*C. difficile* is a very diverse group of toxin producing organisms. Newer technologies have allowed the identification of numerous toxinotypes and ribotypes with varying virulence factors and toxin production. Multiple lineages contain hypervirulent strains. The large degree of horizontal gene transfer through transposons, bacteriophages, and homologous recombination has dispersed genetic material and pathogenic properties among different

The increased prevalence of ribotypes 027, 017, and 078 may be solely due to population expansion over the last decade or due to a nosocomial enrichment of the proper environment and conditions for the expansion and transference of these virulent strains. The sudden rise may also be related to the delay in purifying selection pressures seen in the more recently diverging lineages. However, a more likely explanation for increasing incidence is the right combination of elderly patients in a contaminated environment with antibiotic and acid suppression medications. Given the high incidence of colonized guts in the hospitalized pediatric population (70%), the hospitalized adult population (25%), the animal kingdom (40%), and

The high virulence, along with a highly mobile genome capable of antibiotic resistance, has prompted further research in the development of vaccinations. Sanofi-Aventis is currently undergoing trials with a vaccine containing formalin-inactivated toxins A and B. To date, 100 healthy subjects have been exposed to the vaccine without any serious side

The hardiness of *C. difficile* spores and the ease with which this bacterium alters its genome has allowed it to flourish and survive among a variety of hosts and reservoirs. More virulent strains are a real possibility given the mobility of code sequencing regions within the genome. As the population continues to age and makes an increasingly stronger presence throughout the healthcare system, especially in the ICU, *C. difficile* will continue to plague patients and healthcare providers until further measures are discovered to control transmission. The increased burden will stress the current resources and facilities financially, geographically, and the pool of available care takers. To date, the best treatment modalities include eliminating the implicated antibiotics, early initiation of oral vancomycin and metronidazole, and

exposure though not to the particular type of fluoroquinolone [121, 122].

the natural environment (50%), reducing exposure is near impossible [14].

strict infection-control engineering to prevent the initial infection.

**12. Conclusions**

strains.

effects [123].

Even though only 3% of patients with *C. difficile* infection require subtotal colectomy for fulminant *C. difficile* colitis, 20% of ICU patients with severe *C. difficile* infection will still require partial colectomy or diversion [115, 116]. Colectomy in this setting is associated with a 50% mortality [90]. Mortality rates are lower when surgical intervention is undertaken within 48 hours of lack of response to medical therapy [117]. During NAP1/027 outbreaks, patients with age >65 years, leukocytosis and elevated lactate appear to benefit the most from early colectomy [118].

In a series of 29 patients with severe or severe/complicated *C. difficile* infection refractory to oral vancomycin ± rectal vancomycin and intravenous metronidazole therapy who underwent fecal microbiota transplantation (FMT) plus continued vancomycin, overall treatment response was 93% (27/29), including 100% (10/10) for severe *C. difficile* infection and 89% (17/19) for severe/complicated *C. difficile* infection. A single FMT was performed in 62%, two FMTs were performed in 31%, and three FMTs in 7% of patients. Continued use of non *C. difficile* infection antibiotics predicted repeat FMT. Thirty-day all-cause mortality after FMT was 7%. Of the two patients who died within 30 days, one underwent colectomy and succumbed to sepsis; the other died from septic shock related to *C. difficile* infection [84]. Further research into the use of FMT combined with continued vancomycin is needed.

## **11. Modern outbreaks**

In 2003, a major outbreak of *C. difficile* occurred in Quebec, Canada and was identified as ribotype 027, strain BI/NAP1. This strain has been identified in >50% of all isolates from hospitals in Europe and North America [4, 10, 20]. Prior to the 2003 outbreak, this strain only accounted for 14 of over 6000 (<0.02%) typed strains collected from U.S. cases during the period of 1984 to 1993. Following the 2003 outbreak in Canada, 96 of 187 (51%) strains tested positive for 027 in eight U.S. outbreaks [119].

The BI/NAP1/027 strain belongs to a hypervirulent group of strains along with types 001, 017, and 078. In particular, the binary toxin produced by 027 was not seen previously. It is thought to be synergistic with the production of toxin A and B. Strain BI/NAP1/027 was found to be highly resistant to fluoroquinolone classes of antibiotics and was also found to produce 16-fold higher concentrations of toxin A and 23-fold higher concentrations of toxin B than less virulent toxinotype 0 strains. The binary toxin has been associated with more severe diarrhea when combined with toxin A and B. When produced alone, binary toxin does not appear to produce disease [3, 10] but does appear to be a marker of both *C. difficile* infection severity and recurrence [120]. The emergence is generally believed to be related to fluoroquinolone exposure though not to the particular type of fluoroquinolone [121, 122].

## **12. Conclusions**

In a systematic review and metaanalysis of 22 published studies from 1983 to 2015 that included 80,835 ICU patients, the effects of *C. difficile* infection on morbidity and mortality were investigated. Karanika et al. found that prevalence of *C. difficile* infection among ICU patients was 2% but 5-fold greater in those patients with diarrhea (11%). Those patients that were diagnosed with *C. difficile* infection had a 25% incidence of the severe form of the disease and diagnosed with pseudomembranous colitis. ICU mortality was not significantly different between the group with *C. difficile* infection and the non*C. difficile* infection group based on seven studies that enrolled a combined 12,165 patients. However, the overall hospital mortality between those same groups was significantly increased in the *C. difficile* infection group with 32% mortality compared to 24% (p = 0.03). Similarly, length of ICU and hospital stay among *C. difficile* infection patients was longer when compared to non*C. difficile* infection patients. Based on five studies with over 10,000 patients, *C. difficile* infection patients had an average ICU stay of 24 days and overall hospital stay of 50 days compared to 19 days and 30

Even though only 3% of patients with *C. difficile* infection require subtotal colectomy for fulminant *C. difficile* colitis, 20% of ICU patients with severe *C. difficile* infection will still require partial colectomy or diversion [115, 116]. Colectomy in this setting is associated with a 50% mortality [90]. Mortality rates are lower when surgical intervention is undertaken within 48 hours of lack of response to medical therapy [117]. During NAP1/027 outbreaks, patients with age >65 years, leukocytosis and elevated lactate appear to benefit the most from early colectomy [118]. In a series of 29 patients with severe or severe/complicated *C. difficile* infection refractory to oral vancomycin ± rectal vancomycin and intravenous metronidazole therapy who underwent fecal microbiota transplantation (FMT) plus continued vancomycin, overall treatment response was 93% (27/29), including 100% (10/10) for severe *C. difficile* infection and 89% (17/19) for severe/complicated *C. difficile* infection. A single FMT was performed in 62%, two FMTs were performed in 31%, and three FMTs in 7% of patients. Continued use of non *C. difficile* infection antibiotics predicted repeat FMT. Thirty-day all-cause mortality after FMT was 7%. Of the two patients who died within 30 days, one underwent colectomy and succumbed to sepsis; the other died from septic shock related to *C. difficile* infection [84]. Further research

In 2003, a major outbreak of *C. difficile* occurred in Quebec, Canada and was identified as ribotype 027, strain BI/NAP1. This strain has been identified in >50% of all isolates from hospitals in Europe and North America [4, 10, 20]. Prior to the 2003 outbreak, this strain only accounted for 14 of over 6000 (<0.02%) typed strains collected from U.S. cases during the period of 1984 to 1993. Following the 2003 outbreak in Canada, 96 of 187 (51%) strains tested positive for 027

The BI/NAP1/027 strain belongs to a hypervirulent group of strains along with types 001, 017, and 078. In particular, the binary toxin produced by 027 was not seen previously. It is

days, respectively, for the non*C. difficile* infection group (p = 0.001) [114].

into the use of FMT combined with continued vancomycin is needed.

**11. Modern outbreaks**

140 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

in eight U.S. outbreaks [119].

*C. difficile* is a very diverse group of toxin producing organisms. Newer technologies have allowed the identification of numerous toxinotypes and ribotypes with varying virulence factors and toxin production. Multiple lineages contain hypervirulent strains. The large degree of horizontal gene transfer through transposons, bacteriophages, and homologous recombination has dispersed genetic material and pathogenic properties among different strains.

The increased prevalence of ribotypes 027, 017, and 078 may be solely due to population expansion over the last decade or due to a nosocomial enrichment of the proper environment and conditions for the expansion and transference of these virulent strains. The sudden rise may also be related to the delay in purifying selection pressures seen in the more recently diverging lineages. However, a more likely explanation for increasing incidence is the right combination of elderly patients in a contaminated environment with antibiotic and acid suppression medications. Given the high incidence of colonized guts in the hospitalized pediatric population (70%), the hospitalized adult population (25%), the animal kingdom (40%), and the natural environment (50%), reducing exposure is near impossible [14].

The high virulence, along with a highly mobile genome capable of antibiotic resistance, has prompted further research in the development of vaccinations. Sanofi-Aventis is currently undergoing trials with a vaccine containing formalin-inactivated toxins A and B. To date, 100 healthy subjects have been exposed to the vaccine without any serious side effects [123].

The hardiness of *C. difficile* spores and the ease with which this bacterium alters its genome has allowed it to flourish and survive among a variety of hosts and reservoirs. More virulent strains are a real possibility given the mobility of code sequencing regions within the genome. As the population continues to age and makes an increasingly stronger presence throughout the healthcare system, especially in the ICU, *C. difficile* will continue to plague patients and healthcare providers until further measures are discovered to control transmission. The increased burden will stress the current resources and facilities financially, geographically, and the pool of available care takers. To date, the best treatment modalities include eliminating the implicated antibiotics, early initiation of oral vancomycin and metronidazole, and strict infection-control engineering to prevent the initial infection.

## **Author details**

William C. Sherman, Chris Lewis, Jong O. Lee and David N. Herndon\*

\*Address all correspondence to: dherndon@utmb.edu

Department of Surgery, University of Texas Medical Branch – Galveston, Galveston, Texas, United States of America

[13] Riegler M, Sedivy R, Pothoulakis C, et al. *Clostridium difficile* toxin B is more potent than toxin A in damaging human colonic epithelium *in vitro*. The Journal of Clinical

*Clostridium difficile* in the ICU

143

http://dx.doi.org/10.5772/intechopen.69212

[14] Carroll KC, Bartlett JG. Biology of *Clostridium difficile*: Implications for epidemiology and

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[17] Aseri M, Schroeder T, Kramer J, Kackula R. Gastric acid suppression by proton pump inhibitors as a risk factor for *Clostridium difficile*-associated diarrhea in hospitalized

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**Author details**

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## *Edited by Shymaa Enany*

Clostridium difficile bacteria could be found everywhere around us: in the air, water, and soil and in the feces of humans and animals. You can easily become infected with C. difficile if you touch contaminated clothing, sheets, or other objects and then touch your mouth. Many people have the bacteria in their intestines and never have any symptoms. Still, it can cause symptoms ranging from diarrhea to life-threatening inflammation of the colon. The chance of developing a C. difficile infection increases with the usage of high doses of antibiotics over a prolonged period; thus, it is most often spread in the healthcare facilities between workers, patients, and residents. Each year in the United States, almost a half million people get sick from C. difficile, and approximately 29,000 patients died within 30 days of its initial diagnosis. Nowadays, C. difficile infections have become more frequent, severe, and difficult to treat. Therefore, the early diagnosis and the suitable treatment have become a real demand. In this book, we present the experience of worldwide specialists on the diagnosis and the treatment of C. difficile infections along with its lights and shadows.

Clostridium Difficile - A Comprehensive Overview

Clostridium Difficile

A Comprehensive Overview

*Edited by Shymaa Enany*

Photo by Jezperklauzen / iStock