Tag Archives: Clostridium difficile Research

University of Australia Researchers Find Ramizol as a Potential to Be Standard of Care For Treating C.difficile Infection

Researchers have now developed a new antibiotic that is heralded as a breakthrough against a lethal drug-resistant hospital superbug.

Antibiotic Ramizol was found safe and effective in addressing the Clostridium difficile (C. difficile) infection which is becoming resistant to traditional antibiotics caused by drug-resistant bacteria.

C.difficile is considered one of the most common infections acquired during hospital visits and the most likely cause of diarrhoea for patients and staff in hospitals.

It causes a deadly infection in the large intestine and is most common in people who need to take antibiotics for a long period of time.

“Cases of C.difficile disease are rising and the strains are becoming more lethal. If there is an imbalance in your intestines it can begin to grow and release toxins that attack the lining of the intestines which leads to symptoms,” said Ramiz Boulos, adjunct research associate at Flinders University in Australia.

For the study, the team gave 48 rats a high dose of a new class of antibiotic for 14 days to assess its safety.

The findings, published in the journal Scientific Reports, showed that when doses of the new antibiotic were given to rats infected with the bacteria, a significant proportion of them survived the infection.

“Our research indicates Ramizol is an extremely well-tolerated antibiotic in rats, with good microbiology and antioxidant properties. It also has high chemical stability and is scalable because of the low cost of manufacturing, which could make it a viable treatment option,” Boulos said.

In addition, a very high dose on rats showed no mortalities or side effects.
There were also no changes in mean body weight, weight gain, food consumption or food efficiency for male and female rats attributable to Ramizol.

“We believe Ramizol has the potential to be the standard of care for treating C.difficile infection and has the potential to be a blockbuster drug,” Boulos noted.

 

Source:  https://www.socialnews.xyz/2019/01/19/novel-antibiotic-to-combat-drug-resistant-hospital-superbug/

Dr. Michael Pride, a Pfizer Scientist, Leads a Team Searching For Ways to Improve Diagnosis, Prevention and Treatment of Clostridium difficile Infections

Dr. Pride of Pfizer leads a team that is searching for ways to improve diagnoses & treatment of C. difficile,

Dr. Michael Pride is the Executive Director, Vaccine Research and Development at Pfizer

Challenges, Chance and Looking Forward. Historically, a difficult diagnosis process has posed challenges to treatment for C. difficile infections, as detection is not straightforward. Dr. Pride and his team are working to tackle this issue by developing better ways to diagnose this infection, which will aid efforts to develop a vaccine. Additionally, he is encouraged by recent work that has demonstrated how an antibody can help prevent recurrent diseases, offering insight that an antibody-mediated response, raised by vaccines, may be a way to help reduce a primary episode of a C. difficile infection.

“If our vaccine is successful, we could help have a great impact on global health, reducing morbidity and even mortality worldwide,” he says. “I’m confident in our team, who is working tirelessly so that hopefully no one must suffer from these horrible symptoms again.”

Today, Dr. Pride leads a team of scientists responsible for the development, qualification and validation of various assays that support Pfizer’s vaccine programs.

 

 

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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.

Veteran Affairs Patients with Recurrent C.difficile Infections Participate In Study

 

 

 

 

Though recurrent Clostridium difficile infections (CDI) are common and pose a major clinical concern, data are lacking regarding mortality among patients who survive their initial CDI and have subsequent recurrences. Risk factors for mortality in patients with recurrent CDI are largely unknown.

Methods

Veterans Affairs patients with a first CDI (positive C. difficile toxin(s) stool sample and ≥ 2 days CDI treatment) were included (2010–2014). Subsequent recurrences were defined as additional CDI episodes ≥ 14 days after the stool test date and within 30 days of end of treatment. A matched (1:4) case-control analysis was conducted using multivariable conditional logistic regression to identify predictors of all-cause mortality within 30 days of the first recurrence.

Results

Crude 30-day all-cause mortality rates were 10.6% for the initial CDI episode, 8.3% for first recurrence, 4.2% for second recurrence, and 5.9% for third recurrence. Among 110 cases and 440 controls six predictors of mortality were identified: use of proton pump inhibitors (PPIs, odds ratio [OR] 3.86, 95% confidence interval [CI] 2.14–6.96), any antibiotic (OR 3.33, 95% CI 1.79–6.17), respiratory failure (OR 8.26, 95% CI 1.71–39.92), congitive dysfunction (OR 2.41, 95% CI 1.02–5.72), nutrition deficiency (OR 2.91, 95% CI 1.37–6.21), and age (OR 1.04, 95% CI 1.01–1.07).

Conclusion

In our national cohort of Veterans, crude mortality decreased by 44% from the initial episode to the third recurrence. Treatment with antibiotics, PPIs, and underlying co-morbidities were important predictors of mortality in recurrent CDI. Our study assists healthcare providers in identifying patients at high risk of death after CDI recurrence.

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Researchers Examine Changes to the Microbiota Composition and Metabolic Profiles of Patients with Recurrent Clostridium difficile Infection (rCDI) Following Treatment with Faecal Microbiota Transplant (FMT)

Objectives

This study aimed to examine changes to the microbiota composition and metabolic profiles of seven patients with recurrent Clostridium difficile infection (rCDI), following treatment with faecal microbiota transplant (FMT).

Summary

Objectives

This study aimed to examine changes to the microbiota composition and metabolic profiles of seven patients with recurrent Clostridium difficile infection (rCDI), following treatment with faecal microbiota transplant (FMT).

Methods

16S rDNA sequencing and 1H NMR were performed on faecal samples from the patients (pre-, post-FMT, and follow-up) and the associated donor samples. Sparse partial-least-square analysis was used to identify correlations between the two datasets.

Results

The patients’ microbiota post-FMT tended to shift towards the donor microbiota, specifically through proportional increases of Bacteroides, Blautia, and Ruminococcus, and proportional decreases of Enterococcus, Escherichia, and Klebsiella. However, although cured of infection, one patient, who suffers from chronic alcohol abuse, retained the compositional characteristics of the pre-FMT microbiota. Following FMT, increased levels of short-chain fatty acids, particularly butyrate and acetate, were observed in all patients. Sparse partial-least-square analysis confirmed a positive correlation between butyrate and Bacteroides, Blautia, and Ruminococcus, with a negative correlation between butyrate and Klebsiella and Enterococcus.

Conclusions

Clear differences were observed in the microbiota composition and metabolic profiles between donors and rCDI patients, which were largely resolved in patients following FMT. Increased levels of butyrate appear to be a factor associated with resolution of rCDI.

Introduction

Although Clostridium difficile is present in the intestines of ∼3–5% of healthy adults,1 the occurrence of C. difficile infection (CDI) in healthy individuals is relatively uncommon due to the protective effect of the gut microbiota. The incidents and severity of CDI has risen significantly over the last decade, and it is now recognised as the main causative agent of healthcare-associated infectious diarrhoea in hospitals worldwide.2 The standard treatment for CDI is the administration of metronidazole for mild to moderate infections, and oral vancomycin or fidaxomicin for severe infections and relapses. The ability of C. difficile to form spores, coupled with the increase in antibiotic-resistant strains, can lead to persistence of infection, relapses, and the administration of more antibiotics, which further depletes the commensal bacteria. This creates an environment that is more favourable to C. difficile, thus setting up a cycle of relapse and re-infection. It is estimated that 20-30% of patients who develop a first episode of CDI go on to have at least one relapse, and of these, a further 60% develop further episodes of relapses.3 This increases the need for further antibiotics, the risk of antibiotic-resistance in the gut commensal flora, and costs to the health service, with each episode of CDI estimated to cost approximately £7000 in 2010.4

Faecal microbiota transplants (FMT) represents an effective alternative to antibiotics to treat recurrent CDI (rCDI), with a primary cure rate as high as 91%.5 The central tenet behind FMT is that the introduction of a healthy bacterial community into the intestines produces an environment that is less favourable to C. difficile by increasing colonisation resistance and reinstating a protective effect. The advantages of this treatment are that it is quick, cost-effective, and could help to eradicate antibiotic resistant strains of C. difficile.

It is known that a dysbiotic gut microbiota increases the risk of developing CDI, however whether there is a common element within this community composition that could help to determine if a patient is at greater risk of rCDI is as yet unknown. The reduction in diversity within the dysbiotic gut microbiota would also suggest a reduction in metabolic potential through the loss of gene diversity. The functional redundancy6 within the gut microbiota suggests, that metabolic function is more relevant than which species are present or absent. Whilst a number of studies have looked at the changes in microbiota composition due to FMT,7, 8, 9, 10 we know little about the changes to the metabolic capacities of the altered microbiota. The aim of this study was to assess FMT-induced changes in both the microbial community structure and metabolite profiles of the gut microbiomes of seven patients with rCDI, as well as those of their associated FMT donors.

Patients and methods

Patients

Patients were selected as candidates for the FMT procedure if they had at least two confirmed recurrences of CDI. C. difficile testing was based on a two stage algorithm in line with Public Health England recommendations.11 This involves screening faecal samples by glutamate dehydrogenase enzyme immunoassay (Techlab, USA), followed by C. difficile toxin testing by enzyme immunoassay (Techlab, USA). Glutamate dehydrogenase positive, toxin negative samples were further tested for the presence of toxigenic genes by PCR. FMT exclusion criteria included immunocompromised patients, those aged less than 16, and those with severe comorbidities which would make the patient unfit for endoscopy. FMT was introduced into clinical care at Norfolk and Norwich University Hospital following approval by the New Therapies committee, and was performed in accordance with the Helsinki Declaration of 1975. Patients were consented for the study by a clinician following a detailed discussion of the procedure with the patient or their next of kin. All patient data is fully anonymised.

Donor screening

The faecal donors used for the cohort of patients who underwent FMT in this study were both healthy Caucasian males with a BMI between 24 and 27 kg/m2, aged 36 (D05) and 30 (D03) years of age, respectively. Potential donors were asked to complete a questionnaire adapted from van Nood et al.12 regarding their medical history and lifestyle habits to identify risk factors for potentially transmittable diseases. Eligible candidates provided blood and stool samples for laboratory screening tests. Blood samples were screened for hepatitis A, B, C, and E antibodies, HIV 1 & 2, human T-lymphotropic virus 1 & 2, Epstein-Barr virus, Cytomegalovirus, syphilis, Entamoeba histolytica, Strongyloides stercoralis, and Treponema pallidum. Stool samples were tested for the presence of C. difficile or its toxins, Helicobacter pylori antigen, Norovirus, methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum β-lactamase-producing organisms, carbapenemase-producing Enterobacteriaceae, Escherichia coli O157, Salmonella spp., Shigella spp., and Campylobacter species. In addition, microscopy was used to investigate for ova, cysts, and parasites. Prior to the donation of stool samples for each FMT procedure, donors were asked to refrain from eating peanuts and shellfish, and to complete a short screening questionnaire to confirm no changes to health or lifestyle since the last donor screening that may put the patient at risk.

Faecal suspension preparation

Donor faeces were collected in a sterile container on the day of the procedure, and transferred to a sterilised class II safety cabinet (Walker Ltd, UK). A maximum of 80 g of donor stool was homogenised with sterile saline (0.9%), to a ratio of 5 ml saline per gram of stool, in a strainer bag (BA6141/STR; Seward Limited, UK) using a Stomacher® 400 Circulator (Seward Limited, UK) set to 230 RPM for a duration of 1 min. The filtered faecal preparation was drawn up into labelled sterile 60 ml syringes using nasojejunal tubing connected to the Luer lock. The syringes were secured with sterile Luer lock caps and transported immediately to the hospital. Aliquots of the donor faecal sample were immediately stored at −20 °C until analysis.

Faecal suspension infusion

Patients were prescribed oral vancomycin 500 mg four times daily for 4 days, with the last dose received the night before the procedure. Also, on the day before the FMT procedure, a bowel lavage is performed using 4 l of macrogol solution (Klean-Prep, Norgine). Patients were taken to the endoscopy unit for insertion of nasojejunal tube the night before the procedure. Our FMT protocol was adapted from that of van Nood et al.12 On the day of FMT infusion, the patient’s headrest was elevated to 45°, patency of the nasojejunal tube was checked by flushing with water, and 420 ml of faecal suspension was delivered slowly by the patient’s bedside in the isolation room via a nasojejunal tube using the prefilled syringes. This was performed at a rate of ∼20 ml per minute with a break of 5-10 minutes applied halfway through the procedure. Post-infusion instructions were to monitor observations, and record bowel motions. Patients could take on fluids one hour after the procedure, and were observed overnight before discharge the next day at the earliest. Although there are no agreed durations of follow-up post-FMT,13 van Nood et al.12 used two endpoints to measure cure, namely no relapse after 5 weeks, and no relapse after 10 weeks. Resolution was defined as type 4 or less on the Bristol stool chart or stool normal for the patient e.g. in case of percutaneous endoscopic gastrostomy feeding. We followed patients up by telephone or in person if they were re-admitted into the hospital for an unrelated illness. Post-FMT samples were collected after a minimum of 10 days post-FMT, and postal kits were provided to patients who were willing to donate a ‘follow-up’ sample up to 2 weeks later.

Sample analysis

Faecal microbiota analysis

Faecal samples were collected from recipients within 9 days prior to FMT, however the pre-FMT sample for patient R13 was not collected within this timeframe, and a previously frozen sample obtained whilst the patient was suffering from the same episode of CDI was used. Further samples were collected for all recipients following the procedure (‘post-FMT’ range: 11–141 days; ‘follow-up’ range: 4–14 days after post-FMT sample), and stored at −20 °C until analysis. The DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, UK) with a bead-beating step.14 DNA yield was quantified using the Qubit fluorometer prior to the samples being sent to the Earlham Institute (UK), where the V4 hypervariable region of the 16S rRNA genes were amplified using the 515F and 806R primers with built-in degeneracy.15 The amplicons were sequenced using paired-end Illumina sequencing (2 × 250 bp) on the MiSeq platform (Illumina, USA). Sequencing data, for the 21 samples that had an appropriate level of sequencing depth, were analysed using the Quantitative Insights Into Microbial Ecology (QIIME) 1.9 software and RDP classifier 16S rRNA gene sequence database.16,17 The trimmed reads were filtered for chimeric sequences using ChimeraSlayer, bacterial taxonomy assignment with a confidence value threshold of 50% was performed with the RDP classifier (version 2.10), and trimmed reads clustered into operational taxonomic units at 97% identity level. Alpha diversity and rarefaction plots were computed using the Chao1 index. Weighted and unweighted UniFrac distances were used to generate beta diversity principal coordinates analysis plots, which were visualised using the Emperor tool.

Faecal metabolite analysis

A known mass (∼ 100 mg) of thawed faecal samples were added to sterile tubes. The faecal waters were generated by adding the phosphate buffer (prepared in D2O) to 8.3% w/v. Homogenised faecal waters were centrifuged at 16,200 x g at room temperature for 5 min. The supernatants were filter sterilised (0.2 µm) and placed in a 5 mm NMR tube. The 1H NMR spectra were recorded at 600 MHz on a Bruker Avance spectrometer (Bruker BioSpin GmbH, Germany) running Topspin 2.0 software and fitted with a cryoprobe and a 60-slot autosampler. Each 1H NMR spectrum was acquired with 1280 scans, a spectral width of 12,300 Hz, and an acquisition time of 2.67 s. The “noesypr1d” pre-saturation sequence was used to suppress the residual water signal with a low-power selective irradiation at the water frequency during the recycle delay and a mixing time of 10 ms. Spectra were transformed with a 0.3 Hz line broadening, and were manually phased, baseline corrected, and referenced by setting the TSP methyl signal to 0 ppm. The metabolites were quantified using the software Chenomx® NMR Suite 7.0TM.

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