scholarly journals Omadacycline compared to vancomycin when combined with germinants to disrupt the life cycle of Clostridioides difficile

2021 ◽  
Author(s):  
Noah Budi ◽  
Jared J. Godfrey ◽  
Nasia Safdar ◽  
Sanjay K. Shukla ◽  
Warren E. Rose
Keyword(s):  
Limit Of Detection ◽  
Disease Relapse ◽  
Vegetative Cells ◽  
Spore Form ◽  
Safety And Efficacy ◽  
Healthcare Environment ◽  
Clostridioides Difficile ◽  
Spore Shedding ◽  
Dormant Spore

Clostridioides difficile (C. difficile) infections (CDI) are commonly treated with antibiotics that do not impact the dormant spore form of the pathogen. CDI-directed antibiotics, such as vancomycin and metronidazole, can destroy the vegetative form of C. difficile and protective microbiota. After treatment, spores can germinate into vegetative cells causing clinical disease relapse and further spore shedding. This in vitro study compares the combination of germinants with vancomycin or omadacycline to antibiotics alone in eradicating C. difficile spores and vegetative cells. Among the four strains in this study, omadacycline minimum inhibitory concentrations (0.031-0.125 mg/L) were lower than vancomycin (1-4 mg/L). Omadacycline nor vancomycin in media alone reduced spore counts. In three of the four strains, including the epidemic ribotype 027, spore eradication with germinants was 94.8-97.4% with vancomycin and 99.4-99.8% with omadacycline (p<0.005). In ribotype 012, either antibiotic combined with germinants resulted in 100% spore eradication at 24 hours. The addition of germinants with either antibiotic did not result in significant toxin A or B production, which were below the limit of detection (<1.25 ng/mL) by 48 hours. Limiting the number of spores present in patient GI tracts at the end of therapy may be effective at preventing recurrent CDI and limiting spore shedding in the healthcare environment. These results with germinants warrant safety and efficacy evaluations in animal models.

scholarly journals How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O2 Tensions

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Nicolas Kint ◽  
Carolina Alves Feliciano ◽  
Maria C. Martins ◽  
Claire Morvan ◽  
Susana F. Fernandes ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Anaerobic Bacteria ◽  
Growth Defect ◽  
Strictly Anaerobic ◽  
Low Oxygen ◽  
Air Exposure ◽  
Vegetative Cells ◽  
Content Type ◽  
Clostridioides Difficile

ABSTRACT Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2. O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon. IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile. This enteropathogen has developed efficient strategies to detoxify O2. In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile. These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

scholarly journals Investigation of the effect of the adsorbent DAV131A on the propensity of moxifloxacin to induce simulated Clostridioides (Clostridium) difficile infection (CDI) in an in vitro human gut model

2020 ◽  
Vol 75 (6) ◽  
pp. 1458-1465
Author(s):  
C H Chilton ◽  
G S Crowther ◽  
C Miossec ◽  
J de Gunzburg ◽  
A Andremont ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Toxin Production ◽  
Human Gut ◽  
Antibiotic Resistant ◽  
Delayed Onset ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection ◽  
Clinical Benefits ◽  
Bacteroides Fragilis Group

Abstract Background Clostridioides difficile infection (CDI) remains a high burden worldwide. DAV131A, a novel adsorbent, reduces residual gut antimicrobial levels, reducing CDI risk in animal models. Objectives We used a validated human gut model to investigate the efficacy of DAV131A in preventing moxifloxacin-induced CDI. Methods C. difficile (CD) spores were inoculated into two models populated with pooled human faeces. Moxifloxacin was instilled (43 mg/L, once daily, 7 days) alongside DAV131A (5 g in 18 mL PBS, three times daily, 14 days, Model A), or PBS (18 mL, three times daily, 14 days, Model B). Selected gut microbiota populations, CD total counts, spore counts, cytotoxin titre and antimicrobial concentrations (HPLC) were monitored daily. We monitored for reduced susceptibility of CD to moxifloxacin. Growth of CD in faecal filtrate and medium in the presence/absence of DAV131A, or in medium pre-treated with DAV131A, was also investigated. Results DAV131A instillation reduced active moxifloxacin levels to below the limit of detection (50 ng/mL), and prevented microbiota disruption, excepting Bacteroides fragilis group populations, which declined by ∼3 log10 cfu/mL. DAV131A delayed onset of simulated CDI by ∼2 weeks, but did not prevent CD germination and toxin production. DAV131A prevented emergence of reduced susceptibility of CD to moxifloxacin. In batch culture, DAV131A had minor effects on CD vegetative growth, but significantly reduced toxin/spores (P &lt; 0.005). Conclusions DAV131A reduced moxifloxacin-induced microbiota disruption and emergence of antibiotic-resistant CD. Delayed onset of CD germination and toxin production indicates further investigations are warranted to understand the clinical benefits of DAV131A in CDI prevention.

scholarly journals Eravacycline, a novel tetracycline derivative, does not induce Clostridioides difficile infection in an in vitro human gut model

2020 ◽  
Vol 76 (1) ◽  
pp. 171-178
Author(s):  
Anthony M Buckley ◽  
James Altringham ◽  
Emma Clark ◽  
Karen Bently ◽  
William Spittal ◽  
...  
Keyword(s):  
Spore Germination ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Clinical Trial Data ◽  
Human Colon ◽  
Toxin Production ◽  
Clostridioides Difficile ◽  
New Antibiotics ◽  
Abdominal Infections

Abstract Objectives The approval of new antibiotics is essential to combat infections caused by antimicrobial-resistant pathogens; however, such agents should be tested to determine their effect on the resident microbiota and propensity to select for opportunistic pathogens, such as Clostridioides difficile. Eravacycline is a new antibiotic for the treatment of complicated intra-abdominal infections. Here, we determined the effects of eravacycline compared with moxifloxacin on the microbiota and if these were conducive to induction of C. difficile infection (CDI). Methods We seeded in vitro chemostat models, which simulate the physiological conditions of the human colon, with a human faecal slurry and instilled gut-reflective concentrations of either eravacycline or moxifloxacin. Results Eravacycline instillation was associated with decreased Bifidobacterium, Lactobacillus and Clostridium species, which recovered 1 week after exposure. However, Bacteroides spp. levels decreased to below the limit of detection and did not recover prior to the end of the experiment. Post-eravacycline, a bloom of aerobic bacterial species occurred, including Enterobacteriaceae, compared with pre-antibiotic, which remained high for the duration of the experiment. These changes in microbiota were not associated with induction of CDI, as we observed a lack of C. difficile spore germination and thus no toxin was detected. Moxifloxacin exposure sufficiently disrupted the microbiota to induce simulated CDI, where C. difficile spore germination, outgrowth and toxin production were seen. Conclusions These model data suggest that, despite the initial impact of eravacycline on the intestinal microbiota, similar to clinical trial data, this novel tetracycline has a low propensity to induce CDI.

scholarly journals The Bacillus subtilis HBsu Protein Modifies the Effects of α/β-Type, Small Acid-Soluble Spore Proteins on DNA

2000 ◽  
Vol 182 (7) ◽  
pp. 1942-1948 ◽  
Author(s):  
Margery A. Ross ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Persistence Length ◽  
Dna Supercoiling ◽  
Chromosomal Protein ◽  
Vegetative Cells ◽  
Chromosomal Proteins ◽  
Hu Proteins ◽  
Dormant Spore ◽  
Positive Supercoiling

ABSTRACT HBsu, the Bacillus subtilis homolog of theEscherichia coli HU proteins and the major chromosomal protein in vegetative cells of B. subtilis, is present at similar levels in vegetative cells and spores (∼5 × 104 monomers/genome). The level of HBsu in spores was unaffected by the presence or absence of the α/β-type, small acid-soluble proteins (SASP), which are the major chromosomal proteins in spores. In developing forespores, HBsu colocalized with α/β-type SASP on the nucleoid, suggesting that HBsu could modulate α/β-type SASP-mediated properties of spore DNA. Indeed, in vitro studies showed that HBsu altered α/β-type SASP protection of pUC19 from DNase digestion, induced negative DNA supercoiling opposing α/β-type SASP-mediated positive supercoiling, and greatly ameliorated the α/β-type SASP-mediated increase in DNA persistence length. However, HBsu did not significantly interfere with the α/β-type SASP-mediated changes in the UV photochemistry of DNA that explain the heightened resistance of spores to UV radiation. These data strongly support a role for HBsu in modulating the effects of α/β-type SASP on the properties of DNA in the developing and dormant spore.

scholarly journals Ambush of Clostridium difficile Spores by Ramoplanin: Activity in anIn VitroModel

2015 ◽  
Vol 59 (5) ◽  
pp. 2525-2530 ◽  
Author(s):  
Carl N. Kraus ◽  
Matthew W. Lyerly ◽  
Robert J. Carman
Keyword(s):  
Clostridium Difficile ◽  
Bactericidal Activity ◽  
Gastrointestinal Disease ◽  
Deionized Water ◽  
Disease Relapse ◽  
Spore Form ◽  
Content Type ◽  
Prevention Of Relapse

ABSTRACTClostridium difficileinfection (CDI) is a gastrointestinal disease caused byC. difficile, a spore-forming bacterium that in its spore form is tolerant to standard antimicrobials. Ramoplanin is a glycolipodepsipeptide antibiotic that is active againstC. difficilewith MICs ranging from 0.25 to 0.50 μg/ml. The activity of ramoplanin against the spores ofC. difficilehas not been well characterized; such activity, however, may hold promise, since posttreatment residual intraluminal spores are likely elements of disease relapse, which can impact more than 20% of patients who are successfully treated.C. difficilespores were found to be stable in deionized water for 6 days.In vitrospore counts were consistently below the level of detection for 28 days after even brief (30-min) exposure to ramoplanin at concentrations found in feces (300 μg/ml). In contrast, suppression of spore counts was not observed for metronidazole or vancomycin at human fecal concentrations during treatment (10 μg/ml and 500 μg/ml, respectively). Removal of theC. difficileexosporium resulted in an increase in spore counts after exposure to 300 μg/ml of ramoplanin. Therefore, we propose that rather than being directly sporicidal, ramoplanin adheres to the exosporium for a prolonged period, during which time it is available to attack germinating cells. This action, in conjunction with its already established bactericidal activity against vegetativeC. difficileforms, supports further evaluation of ramoplanin for the prevention of relapse afterC. difficileinfection in patients.

scholarly journals The CspC pseudoprotease regulates germination of Clostridioides difficile spores in response to multiple environmental signals

2018 ◽  
Author(s):  
Amy E. Rohlfing ◽  
Brian E. Eckenroth ◽  
Emily R. Forster ◽  
Yuzo Kevorkian ◽  
M. Lauren Donnelly ◽  
...  
Keyword(s):  
Bile Salt ◽  
Spore Form ◽  
Environmental Signals ◽  
Clostridioides Difficile ◽  
Gastrointestinal Pathogen ◽  
Increased Sensitivity ◽  
Dormant Spore ◽  
Hyper Sensitivity ◽  
Spore Forming Bacteria ◽  
Unique Mechanism

AbstractThe 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 uses the soluble pseudoprotease, CspC, to detect bile salt 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 salt germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile salt-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since the mechanism by which C. difficile spores sense co-germinants is unknown, our study provides the first evidence that CspC senses distinct classes of co-germinants in addition to bile salts. Since we observed that specific residues control CspC’s responsiveness to these different signals, CspC is critical for regulating C. difficile germination in response to multiple environmental signals.

scholarly journals Activity of Hospital Disinfectants against Vegetative Cells and Spores of Clostridioides difficile Embedded in Biofilms

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Tasnuva Rashid ◽  
Farnoosh Haghighi ◽  
Irtiza Hasan ◽  
Eugénie Bassères ◽  
M. Jahangir Alam ◽  
...  
Keyword(s):  
Cell Growth ◽  
Vegetative Cell ◽  
Growth Time ◽  
Future Research ◽  
Primary Analysis ◽  
Vegetative Cells ◽  
Biofilm Growth ◽  
Content Type ◽  
Clostridioides Difficile

ABSTRACT Clostridioides difficile spores can survive in the environment in either mono- or mixed-species biofilms. However, no previous studies have investigated chemical disinfection of C. difficile spores embedded in biofilms. Thus, the purpose of this study was to assess the in vitro effectiveness of hospital disinfectants against C. difficile spores embedded within biofilms. Five unique C. difficile strains embedded in three different biofilm types grown for 72 or 120 h were exposed to seven different hospital disinfectants. C. difficile abundance [as log(number of CFU/milliliter)] was calculated after manufacturer-determined contact times along with biofilm biomass and microscopy. The primary analysis compared differences between C. difficile vegetative cell and spore counts as well as amounts of biomass after exposure to disinfectants. C. difficile vegetative cells and spores were recovered from biofilms regardless of the type of biofilm growth or biofilm growth time. No disinfectant was able to completely eliminate C. difficile from the biofilms. Overall, Clorox, ortho-phthalaldehyde (OPA), and Virex were most effective at killing C. difficile spores regardless of biofilm age, ribotype, or wash conditions (whether biofilms are washed or unwashed) (P = 0.001, each). Clorox and OPA were also effective at killing total vegetative cell growth (P = 0.001, each), but Virex was found to be ineffective against vegetative cell growth in biofilms (P = 0.77). Clorox and Virex were most effective in reducing biomass, followed by Nixall, OPA, and Vital Oxide. No disinfectant was able to completely eliminate C. difficile embedded within biofilms although differences among disinfectants were noted. Future research will be required to determine methods to eradicate this persister reservoir.

Effect of carbon source on Phenobarbital metabolism by Rhizopus stolonifer

2010 ◽  
Vol 3 (2) ◽  
pp. 1022-1027
Author(s):  
Kavitha K ◽  
Asha S ◽  
Hima Bindu T.V.L ◽  
Vidyavathi M
Keyword(s):  
Carbon Source ◽  
High Performance ◽  
Carbon Sources ◽  
In Vitro Models ◽  
Rhizopus Stolonifer ◽  
Safety And Efficacy ◽  
Alternative Systems ◽  
Toxicological Studies ◽  
Microbial Model

The safety and efficacy of a drug is based on its metabolism or metabolite formed. The metabolism of drugs can be studied by different in vitro models, among which microbial model became popular. In the present study, eight microbes were screened for their ability to metabolize phenobarbital in a manner comparable to humans with a model to develop alternative systems to study human drug metabolism. Among the different microbes screened, a filamentous fungi Rhizopus stolonifer metabolized phenobarbital to its metabolite which is used for further pharmacological and toxicological studies. The transformation of phenobarbital was identified by high- performance liquid chromatography (HPLC). Interestingly, Rhizopus stolonifer sample showed an extra metabolite peak at 3.11min. compared to its controls. The influence of different carbon sources in media used for growth of fungus, on metabolite production was studied, to find its effect in production of metabolite as the carbon source may influence the growth of the cell.

scholarly journals Computational Design of a Molecularly Imprinted Polymer for the Biomonitoring of the Organophosphorous Metabolite Chlorferron

Biosensors ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 192
Author(s):  
Bakhtiyar Qader ◽  
Issam Hussain ◽  
Mark Baron ◽  
Rebeca Jiménez-Pérez ◽  
Guzmán Gil-Ramírez ◽  
...  
Keyword(s):  
Biological Samples ◽  
Limit Of Detection ◽  
Computational Design ◽  
Signal Change ◽  
Highly Sensitive ◽  
Limit Of Quantitation ◽  
Molecularly Imprinting Polymers ◽  
Mip Sensor ◽  
Parasitic Mites

Coumaphos is an organophosphorus compound used as insecticide and frequently used by beekeepers for the management of parasitic mites. The most important metabolite, chlorferron (CFN), has been identified in biological samples and foodstuff. The need to quickly identify the presence of typical metabolites, as an indication of interaction with coumaphos has driven the need to produce a highly sensitive electrochemical method for chlorferron analysis, based on molecularly imprinting polymers (MIP) technology. It showed irreversible behaviour with mixed diffusion/adsorption-controlled reactions at the electrode surface. A monoelectronic mechanism of reaction for oxidation has also been suggested. The linear range observed was from 0.158 to 75 µM. Median precision in terms of %RSD around 3% was also observed. For DPV, the limit of detection (LOD) and the limit of quantitation (LOQ) for the CFN-MIP were 0.158 µM and 0.48 µM, respectively. The obtained median % recovery was around 98%. The results were also validated to reference values obtained using GC-MS. Urine and human synthetic plasma spiked with CFN were used to demonstrate the usability of the method in biological samples, showing the potential for biomonitoring. The developed imprinted sensor showed maximum signal change less than 16.8% when related metabolites or pesticide were added to the mix, suggesting high selectivity of the MIP sensor toward CFN molecules. The results from in vitro metabolism of CMP analysed also demonstrates the potential for detection and quantification of CFN in environmental samples. The newly developed CFN-MIP sensor offers similar LoDs than chromatographic methods with shorter analysis time.

scholarly journals 1211. Response to Fecal Microbiota Transplant (FMT) in Refractory Clostridioides difficile Infection (CDI) is Modest Compared to Recurrent CDI in Hospitalized Patients

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S627-S627
Author(s):  
Jae Hyun Shin ◽  
R Ann Hays ◽  
Cirle Warren
Keyword(s):  
Health System ◽  
Complete Resolution ◽  
Fecal Microbiota ◽  
Inpatient Setting ◽  
University Of Virginia ◽  
Cure Rate ◽  
Fecal Microbiota Transplant ◽  
Safety And Efficacy ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection

Abstract Background There are limited options for Clostridioides difficile infection (CDI) refractory to conventional antibiotic therapy (metronidazole, vancomycin, or fidaxomicin). Fecal microbiota transplant (FMT) is considered a safe and effective treatment for recurrent CDI but has not been widely utilized for refractory CDI due to concerns about safety. Even when included in studies, refractory CDI has not been analyzed separately from recurrent CDI. We reviewed cases of FMT performed in the inpatient setting for CDI to evaluate its safety and efficacy for refractory CDI. Methods Patients who received FMT inpatient at University of Virginia Health System for recurrent or refractory CDI after Infectious Diseases and Gastroenterology consultation signed informed consent acknowledging that FMT was considered investigational use in CDI not responding to standard of care as per 2014 FDA guidance. Charts were reviewed as part of quality improvement efforts to evaluate safety and efficacy of FMT in inpatient setting. Results Starting in July 2014, 13 patients received FMT for CDI as inpatients. Six received FMT for recurrent CDI, with four having complete resolution, one had recurrent CDI, and one had persistent C. difficile-negative diarrhea, for cure rate of 83%, comparable to published studies. Seven patients received FMT for refractory CDI, with three resulting in complete resolution. One responded to FMT but refused further care, one died from multiorgan failure after initial response to FMT that was possibly related to CDI, strongyloides, and/or CMV. Two patients had ongoing diarrhea suggestive of post-infectious irritable bowel syndrome, one was C. difficile-negative and one was not tested. The cure rate was 57%, lower than that of the recurrent CDI, but without any clear evidence of microbiologic failure. Outcome of patients undergoing FMT for CDI in the inpatient setting at University of Virginia Health System Conclusion Cure rate for FMT for refractory CDI was lower than recurrent CDI, but review of the cases of treatment failures did not reveal any microbiologic evidence of failure. FMT should be considered an alternative option when treating refractory CDI. Disclosures All Authors: No reported disclosures

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