Tag Archives: Clostridium difficile R&D

C. diff. Research and Development Community March 2015, Infection and Immunity. Dynamics and Establishment of Clostridium difficile Infection

Microscope - 5

Shared by Dr. David Cook, MS, PhD

C. diff. Research and Development Community:

March 2015

Review of: Konigsknecht MJ, CM Theriot, IL Bergin, CA Schumacher, PD Schloss and VB Young. 2015. Infection and Immunity. Dynamics and establishment of Clostridium difficile infection in the murine gastrointestinal tract. Vol 83 (3): pages 934-41.

In this paper from Vince Young’s lab at the University of Michigan Medical School,              Konigsknecht et al measure the early events associated with C. difficile infection in a mouse model. Investigators followed the germination, growth, toxin production and histopathology following infection with C. difficile strain VPI 10463 subsequent to a 5 day antibiotic treatment of cefoperazone in the drinking water. Strengths of the work include the sampling every 6 hours of the mouse GI tract–including stomach, small intestine, cecum and colon–over the first 36 hours post-infection. In this brief time span, the investigators observe evidence of bacterial germination and growth, initially in the cecum and large intestine but later spreading to all regions of the small intestine and stomach, with concomitant pathologic effects and mortality. Both spores and toxin are detectable by 24 hours post-infection, consistent with the observation that the transcriptional program associated with sporulation is also likely involved in toxin production. This observation suggests that disease caused by toxin requires a minimal titer in the GI tract, and is consistent with the observation in humans that some individuals are colonized by low levels of C. difficile without evidence of clinical symptoms.
By 30-36 hours post-infection, the levels of vegetative organisms and spores are comparable in stomach and cecum-colon, with lower amounts in the small intestine.Despite the (unexpectedly) high levels of C. difficile observed in stomach and small intestine, tissue damage in the mouse is confined to the cecum and colon, consistent with the site of C. difficile pathology in humans. The authors demonstrate that bile acid profiles are shifted away from detectable secondary bile acids. In addition, microbiota diversity is dramatically decreased in the colon, primarily as a result of the antibiotic regimen, to favor an abundance of Lactobacillus. Both of these observations are consistent with previously published results from the Young lab                   (Theriot et al, 2014. Nature Communications).

These results further refine our understanding of infection in the mouse model and will enable other researchers to make more precise use of the model in developing new therapies. It should be noted, however, that there are important differences between C. difficile infection and disease in mice and in humans. In the mouse, C. difficile infection leads to rapid mortality. In humans, disease is slower, more chronic due to relapse, and is fatal only in a minority of cases. The microbiome changes are also different. Depending on the antibiotic used, mice can become dominated by a single microbe unlike humans. In the present case, mice were completely dominated by Lactobacillus, a normal commensal in the mouse but one that is mostly absent in humans. The observation in the Konigsknecht study that C. difficile grows in the stomach of mice is also at odds with our understanding of C. difficile infection in humans. Despite these caveats, this is an important work that furthers the                                                                  science behind understanding C. difficile infection.



Clostridium difficile Research and Development; April/May 2014

Here’s the latest from the Clostridium difficile research community:

April/May 2014
The role of probiotics in the treatment or the prevention of C. difficile infection (CDI) has not been clearly defined as yet. To study the role of Lactobacillus strains on the quorum-sensing signals and toxin production of C. difficile, Yun et al. looked at in vitro and in vivo effects of L. acidophilus strain or cell extracts. The results show that L. acidophilus GP1B can inhibit the growth of C. difficile and contribute to the survival of mice given C.difficile.

The endospores of Bacillus subtilis (B. subtilis) can serve as a tool for surface presentation of heterologous proteins in addition to acting in the role of a probiotic. The utility of B .subtilis as a probiotic was studied in a mouse model of CDI to show that oral administration of B. subtilis spores, especially when administered post infection, was able to attenuate symptomatic disease.
C. difficile flagellar proteins play a myriad role in pathogenesis from adherence, toxin production, and biofilm formation. Barketi-Klai et al looked at the global gene expression profiles of C.difficile fliC mutants and compared gene expression levels to those of the parent wild-type strain. fliC mutants strains led to the up-regulation of genes involved in mobility, expression of virulence factors and sporulation which was not seen with the wild-type mutant strain. The authors conclude that deregulation of fliC expression could lead to the upregulation or deregulation of other genes that enhance the pathogenecity of such strains.
In addition to a patient’s health, CDI is also a huge financial burden to patients, hospital and society. A recent study looked at the added costs of CDI on cardiac surgical patients using the Nationwide Inpatient Sample (NIS) database, and reported that in cardiac surgery alone, CDI adds an incremental cost of $212 million/year.
The spores of C.difficile are important in the pathogenesis of CDI. Spore proteins present on the outer layer of spore may be essential for CDI. The BclA proteins are glycolipids present on the spore surface and may be glycosylated by sgtA, which is cotranscribed with BclA3. Mutant strains of sgtA were not different from wild-type strains in terms of sensitivity to ethanol or lysozyme, but showed a change in heat-resistance and the ability to be internalized by macrophages.


Chandrabali Ghose-Paul,MS,PhD,  Chairperson of Research and Development

Clostridium difficile; Research and Development April 2014

Here’s the latest from the Clostridium difficile research community:
The importance of biofilm formation in chronic and recurrent infections across a cross-section of pathogens, including C. difficile has been studied in great detail. Whether biofilm formation in CDI is an important marker for recurrence is not clearly understood. Crowther et al compared germination, proliferation and toxin production between planktonic and sessile communities of C.difficile in a triple-stage chemostat gut model and here have reported that planktonic populations of C.difficile may be a reservoir for spore persistence and recurrence.
Ransom et al have identified a gene cluster that is found exclusively in C.difficile and some closely-related species of bacteria that encode three cell division proteins: MldA, MldB and MldC. Mutant strains that lack the Mld proteins are severely attenuated for pathogenesis in a hamster model of CDI and thus are potential targets for therapeutics that can disrupt the spread of CDI.
C.difficile are spores formers and the infectious unit for CDI is the spore. The proteins present on the outerlayer of the spores can be used as potential vaccine targets. Spore proteins present on the outer layer of spores, the exosporium, may be essential for the initiation and persistence of CDI. Three C.difficile collagen-like exosporium proteins (BclA) are expressed on the exosporium of the spore. Mutants of BclA proteins were reported to have aberrant structure and faster germination rates than wild type strains. Infection experiments done in mice suggest that BclA1 plays a role in the early stages of infection.
C.difficile strains express three highly complex cell-surface polysaccharides (PSI, PSII and PSIII). PSII is the more abundantly expressed by most strains and is a potential target for vaccine development. The efficacy of PSII glycoconjugate-based vaccine using recombinant fragments of toxin A and toxin B were studied in mice. This vaccine was immunogenic and able to illicit toxin neutralizing antibody, one of the correlates of protection against CDI.
The last decade has seen a rapid change in the epidemiology of CDI due to the emergence of so called ”hypervirulent”strains of C. difficile BI/NAP1/027 . Although it is still unclear what exactly contributes to this rapid spread of this strain, Robinson et al hypothesize that the rapid spread of these hypervirulent ribotype 027strains is due to increased fitness over the historic strains such as ribotypes (001, 002, 014, and 053). Looking at in vitro as well as an in vivo model of competition in mice, data suggests that these hypervirulent strains may be able to outcompete historic strains in a mixed infection/complex microbiota environment.

Chandrabali Ghose-Paul,MS,PhD, Chairperson of Research and Development


Clostridium difficile Research and Development; February/March 2014

February/March 2014

Here’s the latest from the Clostridium difficile research community:

C. difficile was first identified in the stool of neonates in 1935 by Hall and O’Toole. Approximately 30% of newborns are colonized with C. difficile, although by age 3, only 3-5% are asymptomatically colonized. CDI, historically not a pediatric disease, is now on the rise in this population. Pediatric patients with risk factors for CDI include that antibiotic therapy, immunodeficiency, poor diet, comorbidities should be tested for CDI if presented with persistent diarrhea.  Wendt et al looked at 944 pediatric cases of CDI and found that 71% of the cases were community-acquired and the incidence of CDI was highest among 1-year-olds, with 72% with diarrhea and 8% with severe disease.  With the increased severity of CDI, conscientious use of antimicrobials should be implemented in this patient population.



C.difficile is a spore forming anerobe. Although the vegetative form of the bacterium expresses the disease causing virulence factors, it is the highly-resistant spores that are the infectious units needed for fecal-oral transmission.  Spo0A is a master regulator of sporulation and has now been proven to positively regulate sporulation genes and also several virulence factors such as flagella, as well as factors involved in the metabolic pathway.


C.difficile expresses two major toxins, A and B. The toxins have a tripartite structure with an enzymatically active N-terminal domain, a central translocation section and a C-terminal receptor-binding domain (RBD) consisting of repeating units of 21, 30 or 50 amino acid residues.  The toxins function by internalization into endosomes followed by the release of the activation domain into the cytosol of the cell via the 1,050-aa translocation domain.  By systemic mutagenesis, Zhang et al have identified a region in toxin B between aa 1035 and 1107 that decrease cellular toxicity more than a 1000x due to impaired pore formation.


Given the importance of the role of the human microflora in preventing CDI, svereal groups have been studying the effects of various antibiotics on the microflora of the host. In one study, mice given tigecycline saw decreased Bacteroidetes and increased Proteobacteria and were at a higher risk of developing CDI.

In the second study, 16S rRNA was sequenced from healthy human controls (no antibiotic therapy); individuals receiving antibiotic therapy with subsequent CDI and individuals receiving antibiotic therapy without CDI.  The authors suggest that differential regulation of specific bacterial species may be involved in colonization resistance against CDI and this finding could lead to the identification of potential new therapies via the manipulation of the intestinal microbiome such as probiotic treatment.





Chandrabali Ghose-Paul,MS,PhD,  Chairperson of Research and Development

Clostridium difficile Research and Development; January 2014

Here is the latest from the Clostridium difficile research community:

C. difficile colonization and adherence to the gut are important steps in the pathogenesis of CDI.  Several outer membrane proteins and adherence factors have been identified in C.difficile such as S-layer protein (SLPs), flagella, etc.   Using a bioinformatics approach Kovacs-Simon et al. have been able to identify a novel adhesion factor, C. difficile lipoprotein CD0873. Recombinant lipoprotein CD0873 was able to binding Caco-2 cells in vitro.   Using ClosTron technology, mutants of C. difficile lipoprotein CD0873 showed decreased adherence to Caco-2 cells in culture. This novel target could be used as a method for preventing and treating colonization and infection by C.difficile




The changing epidemiology of C. difficile infection (CDI) is partly due to the emergence of a hypervirulent strain of C. difficile (BI/NAP1/027). One of the novel features of these hypervirulent strains is the production of newly described binary toxin (CDT), in addition to C. difficile’s two major toxins, toxin A (TcdA) and toxin B (TcdB).  The role of CDT in symptomatic disease has been studied (January research update Kuehne et al).  In this publication, Schwan et al. show that CDT-induced protrusions are involved in vesicular transport.  CDT reroutes Rab11-positive vesicles containing fibronectin, which is involved in bacterial adherence, leading to the increased adherence of C.difficile, one of the first steps of colonization.





The use of antibiotics is one of the major risk factors for developing C. difficile infection (CDI).   Antibiotics lead to a loss of colonization resistance and lead to the overgrowth of pathogens such as C.difficile. Theriot et al show that in a murine model of CDI, antibiotic treatment induces substantial changes in the gut microbial community and in the metabolome.  Changes in the level of bile acids, especially taurocholate which is essential for C.difficile spore germination into vegetative cells in the gut favors the growth of C. difficile and makes these mice more susceptible to CDI.





Another source of C. difficile infection (CDI)! The zoo! Alvarez-Perez show that C.difficile was isolated from the following animals: chimpanzee (Pan troglodytes troglodytes), dwarf goat (Capra hircus), an Iberian ibex (Capra pyrenaica hispanica), with and plains zebra (Equus quagga burchellii).  Hypervirulent epidemic PCR ribotype 078, produced toxins A and B, and had the genes encoding binary toxin (i.e. A+B+CDT+ isolates) was the most common isolate followed PCR ribotypes 039 (ABCDT), 042 (A+B+CDT) and 110 (AB+CDT). All isolates were resistant to the fluoroquinolones ciprofloxacin, enrofloxacin and levofloxacin. A ribotype 078 isolate recovered from a male zebra foal initially showed in vitro resistance to metronidazole. 




Host humoral immune responses play an important role in the disease pathogenesis of C. difficile infection (CDI). Immune responses to the toxins have been associated with protection from symptomatic disease and also recurrence.   Whether the protection is due to systemic IgG and IgA as well as to mucosal IgA Ab to the toxins is not well-defined.  Johnston et al. use the murine model of CDI to parse out the individual role of the systemic and mucosal immune responses and the role they play in disease outcome. Wild-type C57BL/6 mice develop protective immunity against CDI and remain uninfected upon rechallenge.  CD4−/− mice also generated both mucosal and serum IgA anti-toxin Abs and were protected from CDI upon rechallenge without the expression of IgG anti-toxin Ab.  pIgR−/− mice, lacking the receptor to transcytose polymeric Ab, were also protected from CDI, suggesting that although mucosal anti-toxin Ab is not essential for protection. Therefore protection against CDI can be mediated via multiple mechanisms, depending on the host.




Chandrabali Ghose-Paul,MS,PhD, Chairperson of Research and Development