Tag Archives: C. diff research and development

Belgian Nursing Home Survey of Clostridium difficile Presence and Gut Microbiota Composition

  • Cristina Rodriguez+
  • Bernard Taminiau,
  • Nicolas Korsak,
  • Véronique Avesani,
  • Johan Van Broeck,
  • Philippe Brach,
  • Michel Delmée and
  • Georges Daube
Contributed equally
BMC MicrobiologyBMC series – open, inclusive and trusted201616:229

DOI: 10.1186/s12866-016-0848-7

The Author(s). 2016m,Received: 13 April 2016,Accepted: 23 September 2016

Published: 1 October 2016



Increasing age, several co-morbidities, environmental contamination, antibiotic exposure and other intestinal perturbations appear to be the greatest risk factors for C. difficile infection (CDI). Therefore, elderly care home residents are considered particularly vulnerable to the infection. The main objective of this study was to evaluate and follow the prevalence of C. difficile in 23 elderly care home residents weekly during a 4-month period. A C. difficile microbiological detection scheme was performed along with an overall microbial biodiversity study of the faeces content by 16S rRNA gene analysis.


Seven out of 23 (30.4 %) residents were (at least one week) positive for C. difficile. C. difficile was detected in 14 out of 30 diarrhoeal samples (43.7 %). The most common PCR-ribotype identified was 027. MLVA showed that there was a clonal dissemination of C. difficile strains within the nursing home residents. 16S-profiling analyses revealed that each resident has his own bacterial imprint, which was stable during the entire study. Significant changes were observed in C. difficile positive individuals in the relative abundance of a few bacterial populations, including Lachnospiraceae and Verrucomicrobiaceae. A decrease of Akkermansia in positive subjects to the bacterium was repeatedly found.


A high C. difficile colonisation in nursing home residents was found, with a predominance of the hypervirulent PCR-ribotype 027. Positive C. difficile status is not associated with microbiota richness or biodiversity reduction in this study. The link between Akkermansia, gut inflammation and C. difficile colonisation merits further investigations.


C. difficile Elderly care home residents 16S rRNA gene analysis


Clostridium difficile is a Gram-positive, anaerobic, spore-forming, rod-shaped bacterium that has been widely described in the intestinal tract of humans and animals. In 1978, C. difficile was recognized as a major cause of antibiotic associated diarrhoea and, in the most serious cases pseudomembranous colitis [1, 2, 3]. Since then, many outbreaks have been reported; most of them were associated with the emergence of a specific subtype, hyper-virulent PCR-ribotype 027 [4]. Nowadays, C. difficile is a worldwide public health concern as it is considered the major cause of antibiotic-associated infections in healthcare settings [5]. A recent report of C. difficile infection (CDI) cost-of-illness attributes a mean cost ranging from 8,911 to 30,049 USD for hospitalised patients (per patient/admission/episode/infection) in the USA [6] and annual economic burden estimated around 3,000 million euro in Europe [7].

CDI is more commonly diagnosed among older people in nursing homes. High isolation frequencies have been described in USA, with up to 46 % of elderly residents testing positive for C. difficile, while in Europe or Canada the reported rates are much lower, varying between 0.8 and 10 % [8]. This is partly because elderly people are more commonly in hospitals, have an antibiotic treatment and age-related changes in intestinal flora and host defences, as well as the presence or other underlying health problem [8, 9, 10]. These factors can have an impact on the intestinal microbiota, which may promote C. difficile colonisation and the development of the infection [11]. Therefore, a new concern of several studies has been the identification of the microbial communities implicated in the CDI through the use of new sequencing techniques, like metagenomics [12].

The aim of this study was to evaluate and follow the prevalence of C. difficile among the residents of a Belgian nursing home. Multilocus variable number of tandem repeats analysis (MLVA) was performed to determine the genetic diversity of the C. difficile isolates and possible cross-infection between patients. Additionally, 16S rRNA gene sequencing was used to characterise the faecal microbiota of the elderly residents, to evaluate the global evolutions of the total microbiota and to identify possible relationships between certain bacteria populations and C. difficile colonisation, diarrhoea and antibiotic treatment.


Prevalence of C. difficile

A total of 242 faecal samples were collected from 23 residents in seventeen consecutive weeks (resident number 11 was excluded from the study as he finally did not agree to participate in the survey). Two subjects passed away within the four-month study period. Seven out of 23 monitored residents were positive for C. difficile at least once (Table 1).
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U.Va.’s Division of Infectious Diseases and International Health Could Lead To a New Treatment For C. diff. Infection (CDI)



Every year, about half a million patients are infected by Clostridium difficile, an otherwise harmless bacterium that can multiply out of control when the use of antibiotics upsets the balance of microorganisms in the gut. In 2011, about 15,000 deaths were directly attributable to the infection, according to a recent study by the federal Centers for Disease Control and Prevention (CDC).

Current probiotic treatments, which promote the growth of helpful bacteria, have been ineffective against the infection, also known as C. diff.

But work being done at U.Va.’s Division of Infectious Diseases and International Health could lead to a new treatment by the end of the calendar year, according to Dr. Bill Petri, chief of the division. That’s an unusually optimistic estimate in medical research, where scientific breakthroughs predate new treatments by several years.

“Some of these advanced probiotics are actually being tested today in the clinic for their role,” Petri said. “We’re actually participating in advanced clinical trials at U.Va.”

Immunologist Erica L. Buonomo was the driving force behind the new discovery, Petri said, which has to do with the role of white blood cells in protecting against C. diff.

Buonomo found that a particular type of white blood cells, called eosinophils, act as a barrier against the infection, which breaks down the lining of the gut. These eosinophils are recruited by a protein called IL-25. A serious C. diff infection kills eosinophils, allowing the bacteria to enter the gut.

The researchers found that gut bacteria stimulate the production of IL-25, so the right probiotic could help with the production of protective eosinophils.

“We identified a pathway in the immune response that reduces the severity of an infection,” Buonomo said. “When we activate this pathway, we find mice are a lot less sick.”

The discovery would be especially helpful for elderly patients, who are most at risk. It also could have larger implications in the world of microbiology.

Eosinophils are best known for their role in allergic reactions and asthma attacks, when a high number of eosinophils cause inflammation.

The function of these cells was not entirely clear before Buonomo’s discovery. She believes this knowledge could help doctors fight other types of gastrointestinal disorders, such as irritable bowel syndrome.

U.Va. is now working on a probiotic with a Boston-based firm called Seres Therapeutics 

The finished product will be tested in Charlottesville, Petri said.

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C. diff. Researcher Leads Researchers To Suggest Secondary Bile Salts May Have Potential As a Novel Biomarker For CDI Recurrence

C.diff. Research and Development News:

Patients with recurrent Clostridium difficile infection had distinct bile acid and microbiome profiles compared with healthy controls and patients with first Clostridium difficile infection, according to research published in Alimentary Pharmacology and Therapeutics. These findings led researchers to suggest that secondary bile salts may have potential as a novel biomarker for recurrence.

“The mechanism of recurrent CDI remains unknown, though bile salts have been implicated. The intestinal microbiota metabolizes bile acids, a process that if disrupted by antibiotics may be critical to initiation of CDI,” Jessica R. Allegretti, MD, MPH, from the Crohn’s and Colitis Center and Harvard Medical School, Brigham and Women’s Hospital told Healio Gastroenterology. “This study aimed to assess bile salt profiles in three distinct groups of patients — recurrent CDI, first episode CDI and healthy controls — to better understand the role bile salts play in pathogenesis of recurrent CDI and to gain further understanding as to which bacteria may be responsible for this important function. Additionally, we performed random forest regression to identify predictors of group membership.”

Allegretti and colleagues collected blood and stool samples from 20 patients with first CDI, 19 patients with recurrent CDI being screened for fecal transplantation, and 21 controls. Samples from the first CDI arm were collected before patients received antibiotic treatment, whereas samples from the recurrent CDI arm were collected while they were receiving stable doses of chronic oral vancomycin. Participants in the control arm had not been exposed to antibiotics for at least 3 months.

Researchers then analyzed blood plasma and stool samples for bile salt metabolomics profiles, and performed 16S rRNA amplicon sequencing to determine the microbiota composition of the stool samples.

“We found that secondary bile acids, which are protective, were significantly elevated in controls compared with both CDI groups in stool and blood,” Allegretti said.

The median secondary bile acids lithocholate and deoxycholate were significantly higher in the stool samples of controls compared with both first CDI (P < .0001 and P = .0003, respectively) and recurrent CDI arms (both P < .0001). Deoxycholate was also significantly higher in first vs. recurrent CDI patients (P = .017).

Median deoxycholic acid was significantly higher in the blood samples of controls compared with both first CDI (P < .0001) and recurrent CDI patients (P = .05), and was also significantly higher in the blood samples of first vs. recurrent CDI patients (P = .003).

“Conversely, primary bile acids, which can induce germination, were elevated in the recurrent group,” Allegretti said.

The primary bile acids cholate and chenodeoxycholate were significantly higher in the stool samples of the recurrent CDI arm compared with controls (P = .0002 and P = .02, respectively).

16S rRNA gene analyses showed significant differences in microbial alpha diversity across groups, which were most pronounced in recurrent CDI patients vs. controls (adjusted P < .001), but also significant between first and recurrent CDI patients and between first CDI patients and controls (both adjusted P < .05). There were also significant differences in beta-diversity between all groups (P = .001) and significant differences in relative abundances at the taxa level.

Using PICRUSt analyses, the researchers also found significant differences in predicted abundances of bacterial bile salt hydrolase genes between groups. Finally, using random forest regression, the researchers differentiated recurrent and first CDI patients 84.2% of the time using bile acid ratios, with stool deoxycholate to glycoursodeoxycholate ratio as the best predictor.

“Plasma deoxycholate (a secondary bile acid) was a strong predictor of disease state and may be utilized as a possible biomarker of recurrence,” Allegretti said. “This study further elucidates the role of bile salts in the pathogenesis of recurrent CDI and identifies possible novel biomarkers for recurrent disease.”

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by Adam Leitenberger

C. difficile Infection Study By Yale-led Team Estimate Transmission Rates In Three Healthcare Settings


In The News

A Yale-led study estimates transmission rates inside and outside of hospitals, providing insight into different sources of the infection and how it might be better controlled.

Previous studies found that less than half of C. diff infections in hospitalized patients could be attributed to spread from other infected patients. “It’s traditionally been thought of as a hospital-focused disease, but there is increasing recognition of transmission outside the hospital,” said first author David P. Durham, associate research scientist in epidemiology.

To determine how the remaining infections spread, the Yale-led team developed a dynamic model to estimate transmission rates in three settings: hospitals, long-term care facilities, and the general community.

They found that hospitalized patients with symptoms of C. diff infection transmitted it at a rate 15 times higher than asymptomatic patients, even after accounting for infection control measures.

The rates of transmission among residents in long-term care facilities and in the community were 27% and 0.1% that of hospitalized patients, respectively.

“The latter rates are lower but still important sources of transmission, due to the much larger population outside of the hospital setting,” said co-author Jeffrey Townsend, associate professor of public health.

The findings point to the need to account for asymptomatic carriers and community sources in efforts to prevent and control C. diff infection, the researchers noted.

The study was published on March 16 in Emerging Infectious Diseases. Other authors include Yale professor Alison Galvani and Washington University researchers Erik Dubberke and Margaret Olsen.




FloraSeq, LLC , Looks To Improve Treatment for C. diff. Colitis


In the News:




FloraSeq LLC

Waukesha startup FloraSeq LLC is trying to improve treatments of a colon infection that’s increasingly hitting younger people.

Clostridium difficile colitis is caused by bacteria that damage the lining of the colon, potentially rupturing it. The infection, which affects more than 700,000 people each year, can also spread to the rest of the body.

Once seen primarily in the elderly, C.diff. is appearing more often in younger populations – which means the market for a treatment has expanded.”

Treating this infection can currently be a lengthy and difficult process. Antibiotics can’t cure 30 percent of infected patients, which means the final option for many patients is a fecal transplant. This process is not FDA-approved yet, and as it’s currently in the experimental stage, most insurance companies won’t cover the cost.

These fecal transplants, although they appear to be 94 percent effective, are time-consuming, requiring doctors to find a healthy donor who’s a perfect match for the patient.

That’s where FloraSeq comes in. The Waukesha company wants to create a better alternative to fecal transplants, and has developed an oral microbiota capsule that appears to be significantly more effective than current antibiotics.

The two founders, Steve Visuri and Karen Harrington, met and worked at Prodesse, a medical diagnosis company that was acquired by GenProbe and later rolled up into Hologic.

With 40 years of combined experience, the two looked for a less invasive and more effective treatment option.

Antibiotics currently have a 40 percent success rate for treating  C. diff. but FloraSeq’s capsule will achieve 90 percent effectiveness, its founders say.

This treatment will also be significantly cheaper and more widely available than fecal transplants, they add.

The capsule is on track to get FDA approval through the agency’s orphan drug program for treatment of rare diseases and conditions.

FloraSeq has few direct competitors, although others have tried to create a more durable alternative to fecal transplants. The founders say they can provide therapeutic products that promote gut health and relieve gastrointestinal ailments while offering complementary clinical laboratory services for diagnosing infectious diseases and gut dysbiosis, a microbial imbalance.

FloraSeq is looking for more investors to move beyond the seed stage. The company projects a need for $8 million to $10 million over time, with a current capital raise of $700,000. This money would go towards additional research and development, pre-clinical testing and enhancing the company’s plan.

FloraSeq was among 28 companies selected to present at the Nov. 4-5 Wisconsin Early Stage Symposium.

It has also been selected to pitch at other events featuring tech-based companies and was a finalist in the 2015 Wisconsin Governor’s Business Plan Contest.

The company has been certified as a Qualified New Business Venture by the Wisconsin Economic Development Corp., which means investors may be eligible for state tax credits.

C. diff. Research and Development Community; November/December 2014


Here’s the latest from the

C. diff. Research Community:


Animal models of Clostridium difficile infection (CDI) are essential for the better understanding of this disease. The historical animal model for studying CDI was the Golden Syrian hamster, and the murine model for CDI has been described by Dr. Ciaran Kelly’s group in 2008. In this current study, Koenigsknecht et al. have used the antibiotic-treated murine model to describe in great detail the early dynamics of CDI in mice from ingestion and colonization, germination and colitis.


The role of host microbiota in the development of C. diff. infection (CDI) has been studied in great detail. In the current issue of Cell Host Microbe, two studies have looked at two different pathogens and their interactions with host microbiota.

In the first study, by Ferreyra et al, the authors show that following antibiotic treatment                 C. difficile uses host microbiota produced succinate to colonize the gut and cause diarrhea.

In the second study, Curtis et al. show that Bacteroides thetaiotaomicron enhances the expression of virulence genes of enterohemorrhagic Escherichia coli (EHEC) leading to EHEC colonization. B.thetaiotaomicron leads to increased levels of succinate which in turn is sensed by transcription factor Cra, which then leads to the enhanced expression of virulence genes in EHEC.

Although commensal microbiota often prevents pathogens from gaining a foothold in the gut by providing colonization resistance, gut pathogen have evolved in ways so that they can exploit metabolites produced by commnesals to use for their own advantage.



Several journal articles in December have been published looking at the efficacy of LFF571 in treateing CDI. LFF571 is a novel semi-synthetic thiopeptide antibacterial that inhibits bacterial protein synthesis.

The first article includes results from a Phase 2 exploratory study where the authors compared the safety and efficacy (based on a non-inferiority analysis) of LFF571 to vancomycin in adults with primary episodes or first recurrences of moderate C. difficile. Results show that based on protocol-specified definition, rates of clinical cure for LFF571 were non-inferior to those of vancomycin, recurrences were lower for LFF571 and LFF571 was generally safe and well-tolerated.
The second article compares the pharmacokinetics (PK) of LFF571 and vancomycin in patients with  a C. difficile infection (CDI) as part of an early efficacy study. Patients were randomized to receive 200 mg of LFF571 or 125 mg of vancomycin four times daily for 10 days. The highest LFF571 serum concentration was 41.7 ng/mL. This is in comparison to peak vancomycin serum level at 2.73 μg/mL. CDI patients had high fecal concentrations and low serum levels of LFF571 which is similar to healthy volunteers.



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


Clostridium difficile Research and Development Community; October 2014

Here’s the latest from the Clostridium difficile research community:
The role of hosts gut microbiome plays a critical role in the development of Clostridium difficile infection. Different antibiotic usage leads to the loss of specific bacterial taxa that can lead to different levels of susceptibility to C. difficile. In this study, Buffie et al. report that          Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium, is associated with resistance to CDI which is dependent on secondary bile salts. The major take-home message is that microbiome research can help in identifying potential risks following specific antibiotic treatment and also treatment options such as probiotics.

In the following study, the authors evaluate the safety and rate of resolution of diarrhea following oral FMT with frozen capsules given to patient volunteers with recurrent CDI. Twenty patients received 15 capsules on 2 consecutive days and these patients were followed for 6 months. No SAEs were reported. Diarrhea was resolved in 14 patients following 1 FMT. 4 out of 6 nonresponders resolved after a second FMT, with an overall 90% resolution rate. Larger studies are needed to confirm the current findings.


The use of synthetic polymers as mimics of host-defense antibacterial peptides have been studied by the McBride lab. In vitro analysis of Nylon 3 copolymers against C.difficile shows that peptide LL37 is capable of blocking vegetative cell-growth and inhibiting spore outgrowth and is effective against ribotype 027 and 012 strains, in contrast to vancomycin and nisin. These easy to produce synthetic polymers could be used as a treatment for a CDI.


The following study looks at the role of IL22 and CD160 in the mucosal inflammatory immune response to a CDI. The authors report that in C. difficile-infected mice treated with anti-IL22, anti-CD160 or a combination of the two, STAT3 phosphorylation was significantly reduced compared to infected mice not receieving these antibodies. These treated mice also had reduced influx in neutrophils. These data show that IL22 and CD160 are responsible for a proinflammatory host mucosal response against during CDI in mice.

And lastly, Norway rats (Rattus norvegicus) also known as New York City rats have been found to be widely infected with many common human pathogens such as atypical enteropathogenic Escherichia coli, Clostridium difficile, and Salmonella enterica, as well as infectious agents that have been associated with undifferentiated febrile illnesses, including Bartonella spp., Streptobacillus moniliformis, Leptospira interrogans, and Seoul hantavirus and viruses such as sapoviruses, cardioviruses, kobuviruses, parechoviruses, rotaviruses, and hepaciviruses. Pest control is doubly important in urban settings where these rodents are carriers of such zoonotic diseases and live in close proximity to humans.


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