Tag Archives: Cdiff research

Researchers Find C. diff. a Major Cause of Nosocomial Diarrheal Disease Exhibits Phenotypic Heterogeneity Within a Clonal Population As a Result of Phase Variation

Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation.

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

To read the article in its entirety please click on the following link:

https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000379

CspC Plays a Critical Role in Regulating C. diff. Spore Germination in Response to Multiple Environmental Signals.


Abstract

The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile is thought to use the soluble pseudoprotease, CspC, to detect bile acid germinants. To gain insight into CspC’s unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile acid germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile acid-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since mutations in specific residues altered CspC’s responsiveness to these different signals, CspC plays a critical role in regulating C. difficile spore germination in response to multiple environmental signals. Taken together, these studies implicate CspC as being intimately involved in the detection of distinct classes of co-germinants in addition to bile acids and thus raises the possibility that CspC functions as a signaling node rather than a ligand-binding receptor

Author summary

The major nosocomial pathogen Clostridioides difficile depends on spore germination to initiate infection. Interestingly, C. difficile’s germinant sensing mechanism differs markedly from other spore-forming bacteria, since it uses bile acids to induce germination and lacks the transmembrane germinant receptors conserved in almost all spore-forming organisms. Instead, C. difficile is thought to use CspC, a soluble pseudoprotease, to sense these unique bile acid germinants. To gain insight into how a pseudoprotease senses germinant and propagates this signal, we solved the crystal structure of C. difficile CspC. Guided by this structure, we identified mutations that alter the sensitivity of C. difficile spores to not only bile acid germinant but also to amino acid and calcium co-germinants. Taken together, our study implicates CspC in either directly or indirectly sensing these diverse small molecules and thus raises new questions regarding how C. difficile spores physically detect bile acid germinants and co-germinants.

Authors:

  • Amy E. Rohlfing ,
  • Brian E. Eckenroth ,
  • Emily R. Forster,
  • Yuzo Kevorkian,
  • M. Lauren Donnelly,
  • Hector Benito de la Puebla,
  • Sylvie Doublié,
  • Aimee Shen

To view the Abstract in its entirety – please click on the link provided below:

https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1008224

  • Published: July 5, 2019

University of Virginia School of Medicine Researchers Find Why Certain Patients Are Highly Susceptible to C.diff. Infections

The new finding from the University of Virginia School of Medicine (UVA) explains why certain patients are highly susceptible

to C. diff infections, provides doctors with a way to predict disease severity and points to a new way to treat the often-deadly condition.

The UVA researchers found that the immune response to C. diff causes tissue damage and even death through a type of immune cell called Th17. This solves a longstanding mystery about why disease severity does not correlate with the amount of bacteria in the body but, instead, to the magnitude of the immune response. It also explains why patients with inflammatory bowel disease are more likely to suffer severe C. diff infections and more likely to die from them.

Lingering Effects

The bowel condition colitis, the researchers determined, has a lingering effect on the immune system, priming the patient for a worse C. diff infection.

While scientists have known that C. diff and other bacteria produce toxins that are harmful to the body, they assumed this was a simple matter: more toxin, more sickness.

But UVA’s research reveals that the truth is far more complex. Oftentimes, the type of immune response generated by the body can dictate the outcome of disease independent of bacterial toxin.

“When we, as a lab, started working on this, we were actively discouraged from working

on C. difficile because [some] people in the field thought, ‘Oh, this is a toxin-mediated disease. You don’t need to understand anything more than the fact that the bacteria make toxins,’” UVA’s Dr. William A. Petri Jr. said. “So, it’s been a wonderful opportunity for us because we went in and we sort of countered the prevailing wisdom. Yes, the toxins are important, but the toxins are important because they affect the immune system in dramatic ways.”

Inflammatory Bowel Disease

Seeking to understand why patients with inflammatory bowel disease are so susceptible to C. diff, researcher Mahmoud Saleh created a mouse model of colitis, one of the common forms of inflammatory bowel disease. He was able to determine that mice that recovered from colitis actually had changes in their immune system – an adaptive immune response. Immune cells known as Th17 cells had become hyper-charged, primed to cause a severe reaction to subsequent C. difficile infection. Even the same amount of the bacteria would now cause a dangerous, outsized response. “If we infect a month later, we see that these [T helper cells] alone can cause severe infection,” Saleh said. “So, these cells are sufficient for that increased severity of C. difficile infection.”

The researchers then looked at human samples to determine if their finding would hold true. It did, and they were able to use substances in the blood, including a protein known as interleukin 6 (IL-6), to predict disease severity. Patients with high amounts of IL-6 were almost eight times more likely to die from C. difficile than those with low levels.

Petri, of UVA’s Division of Infectious Diseases and International Health, explained: “Now we know from Mahmoud’s work that if I, as a physician, measure IL-6 in one of my patients with inflammatory bowel disease, I’ll be able to know how severe disease will be in that person and I can make the decision about whether the person needs to be admitted to the hospital … or even go to the intensive care unit.”

Preventing C. diff

The research also suggests a potential new way to treat or prevent severe C. difficile relapses. “We know that in mice by targeting T cells we protect from disease, and that leads to the question, could we do something similar and people to provide better therapy?” Petri said. “It is an interesting and terrible situation right now that C. diff is not resistant to antibiotics, but is resistant to treatment. And so even though we have very, very good antibiotics for this, the [body’s] response is so severe that even though we’re killing the bacteria with the antibiotics, patients are suffering from their own immune response.”

While more research will need to be done to create such a treatment, Petri and Saleh are proud to have solved a big mystery about C. difficile. “When you look at how much bacteria are growing or how much toxin is being produced, a lot of time there is no direct correlation,” Saleh said. “Now we know that what’s making that difference is this immune response.”

Findings Published

The researchers have published their findings in the scientific journal Cell Host & Microbe. The research team consisted of Saleh, Alyse L. Frisbee, Jhansi L. Leslie, Erica L. Buonomo, Carrie A Cowardin, Jennie Z. Ma, Morgan E. Simpson, Kenneth W. Scully, Mayuresh M. Abhyankar and Petri.

The research was supported by the National Institutes of Health, grants T32GM008715, T32AI007496, T32AI007496, T32AI07496, 5F31AI114203, 1R21AI114734 and 1R01AI124214; and the UVA Wagner Fellowship.

 

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https://news.virginia.edu/content/revealed-secret-superpower-makes-c-difficile-so-deadly?utm_source=dlvr.it&utm_medium=twitter

 

Researchers at Chinese University of Hong Kong Develop Fluorescent Microrobots to Detect C.diff.

Researchers at Chinese University of Hong Kong (CUHK) have developed fluorescent microrobots that can spot C. diff in a stool sample within a matter of minutes without relying on expensive laboratory equipment.

The technology relies on fungi spore-inspired microrobots that feature fluorescent functionalised carbon nanodots. When the microrobots encounter toxins produced by C. diff, the brightness of the fluorescence changes, something that can be detected with digital photo equipment.

The process is accelerated by the shape and structure of the microrobots, which spread throughout a diluted stool sample and quickly come in contact with as much as the present toxins as possible. This “active” process also helps to detect low concentrations of toxins, according to the researchers.

Additionally, because the microrobots have iron-based nanoparticles in their structure, they can be manipulated by an external magnetic field and gathered together for best visualization.

Study in journal Science Advances: Real-time tracking of fluorescent magnetic spore–based microrobots for remote detection of C. diff toxins

 

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https://www.medgadget.com/2019/02/microrobots-take-minutes-to-detect-c-diff-in-stool-samples.html

Study Finds Factors That May Help Predict Which Patients Are More Likely to Develop a CDI

A cluster of factors may help predict
which patients are likely to develop Clostridium difficile
infection, a new study has found. And that could help
in efforts to prevent infection, according to the researchers.

Reduced immune function, recent antibiotic use, current or recent hospitalization and prior C. difficile infection predicted risk of subsequent infection, opening the door to potential preventive interventions.

“This could help healthcare providers red-flag those patients who are at high risk of C. diff, and may one day lead to therapeutic or dietary tactics to lower the chances of infection,” said the study’s co-lead author, Vanessa Hale of Ohio State University.

The study appears in the journal Science Translational Medicine.

The research included studies in both humans and mice, and involved the transplant of feces from human study participants to mice to assess differences in susceptibility to
C. difficile infection and molecular-level explanations for that increased risk.

“Microbes in the gut play a critical role in defending against disease, and the really exciting part of this study is that it might help us better identify the risk factors that are linked to problems in the gut and susceptibility to these dangerous infections,” said Hale, an assistant professor of veterinary preventive medicine at Ohio State. The study was conducted at the Mayo Clinic, where she previously worked.

The researchers started by looking at the gut microbes of a group of 115 people who had diarrhea but who did not have C. diff when they first sought medical care, some of whom went on to develop a C. diff infection. They also analyzed the gut microbes of 118 healthy volunteers for comparison.

“About half of the diarrhea patients had gut microbial communities that looked healthy, but the guts of the other half were really intriguing – they had different microbes and very different levels of metabolites. We called this half the ‘dysbiotic’ – or unhealthy – group,” Hale said.

“When we transplanted human stool from the dysbiotic group into mice, we discovered that these mice were more likely to become infected with C. diff than mice that received human stool from the healthy-looking group.”

The researchers then examined potential risk factors found on the medical charts of individuals with “dysbiotic” and healthy-looking gut microbial communities and found a cluster of five factors that were associated with unhealthy communities.

“We knew that dysbiotic microbial communities put mice at higher risk of C. diff infection, and we wanted to see if the five factors could be used to predict C. diff infections in humans,” Hale said.

To do this, the research team went back and looked at the medical charts of more than 17,000 previous patients who were free of C. diff when they initially sought care. In that larger group, there also was a clear connection between the risk factors and subsequent C. diff infection.

Furthermore, the researchers found higher levels of amino acids – particularly proline – in the guts of mice that received transplants from people whose gut microbiomes were unhealthy, or dysbiotic.

That was interesting, and potentially important, because C. diff needs amino acids like proline to proliferate and it cannot make proline on its own. That prompted the team to wonder if reducing dietary amino acids could protect against C. diff, Hale said.

Feeding the mice diets low in protein moderately lowered the growth of C. diff, providing further evidence that amino acids – including proline – play a role in risk of infection and leaving researchers curious about the potential for dietary interventions in at-risk humans, Hale said.

“It’s possible that a dietary strategy could reduce C. diff infection in those patients who are deemed to be susceptible based on the cluster of risk factors we identified,” she said, adding that more study is needed to understand that relationship.

The study also showed that prophylactic fecal transplantation from a healthy donor could protect against C. diff in mice that were initially prone to infection.

“The transplants were fully protective against C. diff infection in all of the animals we tested, which was pretty amazing,” Hale said.

………………………It is unlikely that fecal transplantation would quickly be adopted as a prevention strategy in those deemed to be at elevated risk of infection, Hale said.

 

The National Institutes of Health and the Center for Individualized Medicine at Mayo Clinic supported the study.

Eric Battaglioli of Georgetown College was the co-lead author. Purna Kashyap of Mayo Clinic is the senior author.

Clostridium difficile Infection Incidence In Mainland China – A Systematic Review

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In the news:

Author information

 Abstract

It has been widely reported that the incidence and severity of Clostridium difficile infection (CDI) have increased dramatically in North America and Europe. However, little is known about CDI in Mainland China. In this study, we aimed to investigate the incidence of CDI and the main epidemic and drug-resistant strains of C. difficile in Mainland China through meta-analysis of related studies published after the year 2010. A total of 51 eligible studies were included. The pooled incidence of toxigenic C. difficile among patients with diarrhoea was 14% (95% CI = 12-16%). In Mainland China, ST-37 and ST-3 were the most prevalent strains; fortunately, hypervirulent strains, such as ST-1 (BI/NAP1/027) and ST-11 (RT 078), have only occurred sporadically to date.

The rates of C. difficile resistance to ciprofloxacin (98.3%; 95% CI = 96.9-99.7%), clindamycin (81.7%; 95% CI = 76.1-87.3%) and erythromycin (80.2%; 95% CI = 73.5-86.9%) are higher than in other counties; however, none of the C. difficile isolates reported in Mainland China were resistant to metronidazole (n/N = 0/960), vancomycin (n/N = 0/960), tigecycline (n/N = 0/41) or piperacillin/tazobactam(n/N = 0/288).

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https://www.ncbi.nlm.nih.gov/pubmed/27897206?dopt=Abstract&utm_source=dlvr.it&utm_medium=twitter

C. difficile Researcher Kirk Hevener Of ISU And Research Group At Texas A&M University and University of Hawaii Work On A New Way To Treat Clostridium difficile

Kirk Hevener of the ISU Department of Biomedical and Pharmaceutical Sciences is part of a group that is working on a new way to treat Clostridium difficile, commonly called
C. difficile or C. diff. Working with researchers from Texas A&M University and the University of Hawaii
, Hevener is researching a new target that could change the way C. diff is treated through a $415,000 grant from the National Institute of Health.

C. diff  is a bacteria that commonly causes infection of the colon and can lead to severe damage, and in some cases can even be fatal. It is also highly drug-resistant and extremely transmittable.

Hevener identified C. diff as a possible candidate for research while working with a completely different bacteria during his postdoctoral fellowship in Chicago.

Porphyromonas gingivalis is a bacteria that causes disease in the mouth.

While these two bacteria are unrelated, they have two common traits. Both are pathogenic, meaning they cause disease, and contain an enzyme called FabK.

FabK is not found in many other bacteria, so Hevener decided that C. diff would be a good candidate to extend his work in Chicago to, with FabK being the focus of his current work.

This enzyme is part of the fatty acid synthesis pathway. This creates lipids that are used to create the cell membrane, among other functions of the cell. Within C. diff, it is also is part of the mechanism that creates the bacteria’s spores. These spores are inactive forms of the bacteria and are extremely difficult to kill.  They are the reason that recurrence and transmission rates are so high inside of hospitals.

Hevener is studying ways this enzyme can be targeted specifically, with molecules known as inhibitors.

If he and his team can prove that FabK is targetable, it could lead to the development of new medications specific to the treatment of C. diff.

Hevener wanted to make clear that he is currently working on target validation, and not drug development. He and his team are validating that by inhibiting this enzyme, the bacteria would not be able to able to reproduce and create spores, which would then allow others to develop a medication to leverage this mechanism.

By targeting FabK specifically, Hevener’s team would create a narrow spectrum method of treatment, as opposed to the more common broad spectrum approach.

Broad spectrum antibiotics affect all bacteria, regardless if they are pathogenic or beneficial.

There are many bacteria found inside of the human body that aid in different ways from digestion to preventing harmful organisms causing infection.

This narrow spectrum approach has two benefits for a medication developed using it: it does not kill helpful organisms and it helps slow the development of resistance.

Hevener explained that it is impossible to create an antibiotic that is immune to the development of resistance, but because a medication of this type would affect only C. diff, other bacteria would not develop resistance and transfer that genetic mutation to other bacteria. This would slow the progress of a medication becoming less effective or possibly obsolete over time.

To read article in its entirety please click on the following link:

http://isubengal.com/narrowing-c-difficile/