scholarly journals Broad Coverage of Genetically Diverse Strains of Clostridium difficile by Actoxumab and Bezlotoxumab Predicted byIn VitroNeutralization and Epitope Modeling

2014 ◽  
Vol 59 (2) ◽  
pp. 1052-1060 ◽  
Author(s):  
Lorraine D. Hernandez ◽  
Fred Racine ◽  
Li Xiao ◽  
Edward DiNunzio ◽  
Nichelle Hairston ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Standard Of Care ◽  
Content Type ◽  
Cytopathic Effects ◽  
Toxin Binding ◽  
Phase Ii Clinical Study ◽  
A Cell ◽  
Relative Differences ◽  
Hospital Acquired

ABSTRACTClostridium difficileinfections (CDIs) are the leading cause of hospital-acquired infectious diarrhea and primarily involve two exotoxins, TcdA and TcdB. Actoxumab and bezlotoxumab are human monoclonal antibodies that neutralize the cytotoxic/cytopathic effects of TcdA and TcdB, respectively. In a phase II clinical study, the actoxumab-bezlotoxumab combination reduced the rate of CDI recurrence in patients who were also treated with standard-of-care antibiotics. However, it is not known whether the antibody combination will be effective against a broad range ofC. difficilestrains. As a first step toward addressing this, we tested the ability of actoxumab and bezlotoxumab to neutralize the activities of toxins from a number of clinically relevant and geographically diverse strains ofC. difficile. Neutralization potencies, as measured in a cell growth/survival assay with purified toxins from variousC. difficilestrains, correlated well with antibody/toxin binding affinities. Actoxumab and bezlotoxumab neutralized toxins from culture supernatants of all clinical isolates tested, including multiple isolates of the BI/NAP1/027 and BK/NAP7/078 strains, at antibody concentrations well below plasma levels observed in humans. We compared the bezlotoxumab epitopes in the TcdB receptor binding domain across known TcdB sequences and found that key substitutions within the bezlotoxumab epitopes correlated with the relative differences in potencies of bezlotoxumab against TcdB of some strains, including ribotypes 027 and 078. Combined within vitroneutralization data, epitope modeling will enhance our ability to predict the coverage of new and emerging strains by actoxumab-bezlotoxumab in the clinic.

scholarly journals Using a Novel Lysin To Help Control Clostridium difficile Infections

2015 ◽  
Vol 59 (12) ◽  
pp. 7447-7457 ◽  
Author(s):  
Qiong Wang ◽  
Chad W. Euler ◽  
Aurelia Delaune ◽  
Vincent A. Fischetti
Keyword(s):  
Clostridium Difficile ◽  
Catalytic Domain ◽  
Ex Vivo ◽  
Bacterial Species ◽  
Lytic Activity ◽  
Content Type ◽  
Intestinal Pathogen ◽  
Drug Treatments ◽  
Hospital Acquired

ABSTRACTAs a consequence of excessive antibiotic therapies in hospitalized patients,Clostridium difficile, a Gram-positive anaerobic spore-forming intestinal pathogen, is the leading cause of hospital-acquired diarrhea and colitis. Drug treatments for these diseases are often complicated by antibiotic-resistant strains and a high frequency of treatment failures and relapse; therefore, novel nonantibiotic approaches may prove to be more effective. In this study, we recombinantly expressed a prophage lysin identified from aC. difficilestrain, CD630, which we named PlyCD. PlyCD was found to have lytic activity against specificC. difficilestrains. However, the recombinantly expressed catalytic domain of this protein, PlyCD1–174, displayed significantly greater lytic activity (>4-log kill) and a broader lytic spectrum againstC. difficilestrains while still retaining a high degree of specificity towardC. difficileversus commensal clostridia and other bacterial species. Our data also indicated that noneffective doses of vancomycin and PlyCD1–174when combinedin vitrocould be significantly more bactericidal againstC. difficile. In anex vivotreatment model of mouse colon infection, we found that PlyCD1–174functioned in the presence of intestinal contents, significantly decreasing colonizingC. difficilecompared to controls. Together, these data suggest that PlyCD1–174has potential as a novel therapeutic for clinical application againstC. difficileinfection, either alone or in combination with other preexisting treatments to improve their efficacy.

scholarly journals Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

2014 ◽  
Vol 83 (2) ◽  
pp. 502-513 ◽  
Author(s):  
Shan Li ◽  
Lianfa Shi ◽  
Zhiyong Yang ◽  
Yongrong Zhang ◽  
Gregorio Perez-Cordon ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Cysteine Protease ◽  
Lethal Dose ◽  
Enzymatic Activities ◽  
Target Cells ◽  
Content Type ◽  
Cytopathic Effects

TcdB is one of the key virulence factors ofClostridium difficilethat is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (VHH) library raised against TcdB, we identified two VHH antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB inin vivotoxicity using a sensitive systemic challenge model in mice. Consistent with thesein vitroresults, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.

scholarly journals A Mixture of Functionally Oligoclonal Humanized Monoclonal Antibodies That Neutralize Clostridium difficile TcdA and TcdB with High Levels ofIn VitroPotency ShowsIn VivoProtection in a Hamster Infection Model

2013 ◽  
Vol 20 (3) ◽  
pp. 377-390 ◽  
Author(s):  
Nicola L. Davies ◽  
Joanne E. Compson ◽  
Brendon MacKenzie ◽  
Victoria L. O'Dowd ◽  
Amanda K. F. Oxbrow ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clostridium Difficile ◽  
Neutralizing Antibodies ◽  
Disease Process ◽  
Care Facility ◽  
Infection Model ◽  
Hamster Model ◽  
Content Type ◽  
Toxin Binding

ABSTRACTClostridium difficileinfections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments,C. difficileinfection (CDI) is still a major cause of patient suffering, death, and substantial health care costs.Clostridium difficileexerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety ofin vitrobinding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.

scholarly journals Ferric Uptake Regulator Fur Control of Putative Iron Acquisition Systems in Clostridium difficile

2015 ◽  
Vol 197 (18) ◽  
pp. 2930-2940 ◽  
Author(s):  
Theresa D. Ho ◽  
Craig D. Ellermeier
Keyword(s):  
Clostridium Difficile ◽  
Oxidative Damage ◽  
Iron Acquisition ◽  
Infectious Diarrhea ◽  
Intracellular Iron ◽  
Common Cause ◽  
Content Type ◽  
Hospital Acquired

ABSTRACTClostridium difficileis an anaerobic, Gram-positive, spore-forming opportunistic pathogen and is the most common cause of hospital-acquired infectious diarrhea. Although iron acquisition in the host is a key to survival of bacterial pathogens, high levels of intracellular iron can increase oxidative damage. Therefore, expression of iron acquisition mechanisms is tightly controlled by transcriptional regulators. We identified aC. difficilehomologue of the master bacterial iron regulator Fur. Using targetron mutagenesis, we generated afurinsertion mutant ofC. difficile. To identify the genes regulated by Fur inC. difficile, we used microarray analysis to compare transcriptional differences between thefurmutant and the wild type when grown in high-iron medium. Thefurmutant had increased expression of greater than 70 transcriptional units. Using quantitative reverse transcriptase PCR (qRT-PCR), we analyzed several of the Fur-regulated genes identified by the microarray and verified that they are both iron and Fur regulated inC. difficile. Among those Fur- and iron-repressed genes wereC. difficilegenes encoding 7 putative cation transport systems of different classes. We found that Fur was able to bind the DNA upstream of three Fur-repressed genes in electrophoretic mobility shift assays. We also demonstrate that expression of Fur-regulated putative iron acquisition systems was increased duringC. difficileinfection using the hamster model. Our data suggest thatC. difficileexpresses multiple iron transport mechanisms in response iron depletionin vitroandin vivo.IMPORTANCEClostridium difficileis the most common cause of hospital-acquired infectious diarrhea and has been recently classified as an “urgent” antibiotic resistance threat by the CDC. To survive and cause disease, most bacterial pathogens must acquire the essential enzymatic cofactor iron. While import of adequate iron is essential for most bacterial growth, excess intracellular iron can lead to extensive oxidative damage. Thus, bacteria must regulate iron import to maintain iron homeostasis. We demonstrate here thatC. difficileregulates expression of several putative iron acquisition systems using the transcriptional regulator Fur. These import mechanisms are induced under iron-limiting conditionsin vitroand duringC. difficileinfection of the host. This suggests that during aC. difficileinfection, iron availability is limitedin vivo.

scholarly journals Characterizations of Clinical Isolates of Clostridium difficile by Toxin Genotypes and by Susceptibility to 12 Antimicrobial Agents, Including Fidaxomicin (OPT-80) and Rifaximin: a Multicenter Study in Taiwan

2012 ◽  
Vol 56 (7) ◽  
pp. 3943-3949 ◽  
Author(s):  
Chun-Hsing Liao ◽  
Wen-Chien Ko ◽  
Jang-Jih Lu ◽  
Po-Ren Hsueh
Keyword(s):  
Clostridium Difficile ◽  
Care Setting ◽  
Antimicrobial Agents ◽  
Teaching Hospitals ◽  
Binary Toxin ◽  
Content Type ◽  
Vancomycin Mics ◽  
Major Teaching ◽  
Southern Taiwan

ABSTRACTA total of 403 nonduplicate isolates ofClostridium difficilewere collected at three major teaching hospitals representing northern, central, and southern Taiwan from January 2005 to December 2010. Of these 403 isolates, 170 (42.2%) were presumed to be nontoxigenic due to the absence of genes for toxins A or B or binary toxin. The remaining 233 (57.8%) isolates carried toxin A and B genes, and 39 (16.7%) of these also had binary toxin genes. The MIC90of all isolates for fidaxomicin and rifaximin was 0.5 μg/ml (range, ≤0.015 to 0.5 μg/ml) and >128 μg/ml (range, ≤0.015 to >128 μg/ml), respectively. All isolates were susceptible to metronidazole (MIC90of 0.5 μg/ml; range, ≤0.03 to 4 μg/ml). Two isolates had reduced susceptibility to vancomycin (MICs, 4 μg/ml). Only 13.6% of isolates were susceptible to clindamycin (MIC of ≤2 μg/ml). Nonsusceptibility to moxifloxacin (n= 81, 20.1%) was accompanied by single or multiple mutations ingyrAandgyrBgenes in all but eight moxifloxacin-nonsusceptible isolates. Two previously unreportedgyrBmutations might independently confer resistance (MIC, 16 μg/ml), Ser416 to Ala and Glu466 to Lys. Moxifloxacin-resistant isolates were cross-resistant to ciprofloxacin and levofloxacin, but some moxifloxacin-nonsusceptible isolates remained susceptible to gemifloxacin or nemonoxacin at 0.5 μg/ml. This study found the diversity of toxigenic and nontoxigenic strains ofC. difficilein the health care setting in Taiwan. All isolates tested were susceptible to metronidazole and vancomycin. Fidaxomicin exhibited potentin vitroactivity against all isolates tested, while the more than 10% of Taiwanese isolates with rifaximin MICs of ≥128 μg/ml raises concerns.

scholarly journals MBX-500, a Hybrid Antibiotic withIn VitroandIn VivoEfficacy against Toxigenic Clostridium difficile

2012 ◽  
Vol 56 (9) ◽  
pp. 4786-4792 ◽  
Author(s):  
Michelle M. Butler ◽  
Dean L. Shinabarger ◽  
Diane M. Citron ◽  
Ciarán P. Kelly ◽  
Sofya Dvoskin ◽  
...  
Keyword(s):  
Weight Gain ◽  
Clostridium Difficile ◽  
Severe Disease ◽  
Normal Weight ◽  
New Drugs ◽  
Hamster Model ◽  
Resistance Development ◽  
Content Type

ABSTRACTClostridium difficileinfection (CDI) causes moderate to severe disease, resulting in diarrhea and pseudomembranous colitis. CDI is difficult to treat due to production of inflammation-inducing toxins, resistance development, and high probability of recurrence. Only two antibiotics are approved for the treatment of CDI, and the pipeline for therapeutic agents contains few new drugs. MBX-500 is a hybrid antibacterial, composed of an anilinouracil DNA polymerase inhibitor linked to a fluoroquinolone DNA gyrase/topoisomerase inhibitor, with potential as a new therapeutic for CDI treatment. Since MBX-500 inhibits three bacterial targets, it has been previously shown to be minimally susceptible to resistance development. In the present study, thein vitroandin vivoefficacies of MBX-500 were explored against the Gram-positive anaerobe,C. difficile. MBX-500 displayed potency across nearly 50 isolates, including those of the fluoroquinolone-resistant, toxin-overproducing NAP1/027 ribotype, performing as well as comparator antibiotics vancomycin and metronidazole. Furthermore, MBX-500 was a narrow-spectrum agent, displaying poor activity against many other gut anaerobes. MBX-500 was active in acute and recurrent infections in a toxigenic hamster model of CDI, exhibiting full protection against acute infections and prevention of recurrence in 70% of the animals. Hamsters treated with MBX-500 displayed significantly greater weight gain than did those treated with vancomycin. Finally, MBX-500 was efficacious in a murine model of CDI, again demonstrating a fully protective effect and permitting near-normal weight gain in the treated animals. These selective anti-CDI features support the further development of MBX 500 for the treatment of CDI.

scholarly journals Germinant Synergy FacilitatesClostridium difficileSpore Germination under Physiological Conditions

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Travis J. Kochan ◽  
Michelle S. Shoshiev ◽  
Jessica L. Hastie ◽  
Madeline J. Somers ◽  
Yael M. Plotnick ◽  
...  
Keyword(s):  
Amino Acids ◽  
Clostridium Difficile ◽  
Spore Germination ◽  
Bile Salts ◽  
Gi Tract ◽  
Infectious Diarrhea ◽  
Content Type ◽  
Increased Risk ◽  
Calcium Reabsorption

ABSTRACTClostridium difficileis a Gram-positive obligate anaerobe that forms spores in order to survive for long periods in the unfavorable environment outside a host.C. difficileis the leading cause of nosocomial infectious diarrhea worldwide.C. difficileinfection (CDI) arises after a patient treated with broad-spectrum antibiotics ingests infectious spores. The first step inC. difficilepathogenesis is the metabolic reactivation of dormant spores within the gastrointestinal (GI) tract through a process known as germination. In this work, we aim to elucidate the specific conditions and the location within the GI tract that facilitate this process. Our data suggest thatC. difficilegermination occurs through a two-step biochemical process that is regulated by pH and bile salts, amino acids, and calcium present within the GI tract. Maximal germination occurs at a pH ranging from 6.5 to 8.5 in the terminal small intestine prior to bile salt and calcium reabsorption by the host. Germination can be initiated by lower concentrations of germinants when spores are incubated with a combination of bile salts, calcium, and amino acids, and this synergy is dependent on the availability of calcium. The synergy described here allows germination to proceed in the presence of inhibitory bile salts and at physiological concentrations of germinants, effectively decreasing the concentrations of nutrients required to initiate an essential step of pathogenesis.IMPORTANCEClostridium difficileis an anaerobic spore-forming human pathogen that is the leading cause of nosocomial infectious diarrhea worldwide. Germination of infectious spores is the first step in the development of aC. difficileinfection (CDI) after ingestion and passage through the stomach. This study investigates the specific conditions that facilitateC. difficilespore germination, including the following: location within the gastrointestinal (GI) tract, pH, temperature, and germinant concentration. The germinants that have been identified in culture include combinations of bile salts and amino acids or bile salts and calcium, butin vitro, these function at concentrations that far exceed normal physiological ranges normally found in the mammalian GI tract. In this work, we describe and quantify a previously unreported synergy observed when bile salts, calcium, and amino acids are added together. These germinant cocktails improve germination efficiency by decreasing the required concentrations of germinants to physiologically relevant levels. Combinations of multiple germinant types are also able to overcome the effects of inhibitory bile salts. In addition, we propose that the acidic conditions within the GI tract regulateC. difficilespore germination and could provide a biological explanation for why patients taking proton pump inhibitors are associated with increased risk of developing a CDI.

scholarly journals In VitroandIn VivoActivities of the Novel Ketolide RBx 14255 against Clostridium difficile

2012 ◽  
Vol 56 (11) ◽  
pp. 5986-5989 ◽  
Author(s):  
Manoj Kumar ◽  
Tarun Mathur ◽  
Tarani K. Barman ◽  
G. Ramkumar ◽  
Ashish Bhati ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Time Dependent ◽  
Kinetics Study ◽  
The Novel ◽  
Promising Candidate ◽  
Hamster Model ◽  
Bacterial Killing ◽  
Content Type ◽  
Time Kill

ABSTRACTThe MIC90of RBx 14255, a novel ketolide, againstClostridium difficilewas 4 μg/ml (MIC range, 0.125 to 8 μg/ml), and this drug was found to be more potent than comparator drugs. Anin vitrotime-kill kinetics study of RBx 14255 showed time-dependent bacterial killing forC. difficile. Furthermore, in the hamster model ofC. difficileinfection, RBx 14255 demonstrated greater efficacy than metronidazole and vancomycin, making it a promising candidate forC. difficiletreatment.

scholarly journals Impact of Oral Fidaxomicin Administration on the Intestinal Microbiota and Susceptibility to Clostridium difficile Colonization in Mice

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
N. J. Ajami ◽  
J. L. Cope ◽  
M. C. Wong ◽  
J. F. Petrosino ◽  
L. Chesnel
Keyword(s):  
Clostridium Difficile ◽  
Gut Microbiota ◽  
16S Rrna Gene Sequencing ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Colonization Resistance ◽  
Content Type ◽  
Rrna Gene Sequencing ◽  
Hospital Acquired ◽  
Predetermined Time

ABSTRACT Clostridium difficile infection (CDI), a common cause of hospital-acquired infections, typically occurs after disruption of the normal gut microbiome by broad-spectrum antibiotics. Fidaxomicin is a narrow-spectrum antibiotic that demonstrates a reduced impact on the normal gut microbiota and is approved for the treatment of CDI. To further explore the benefits of this property, we used a murine model to examine the effects of fidaxomicin versus vancomycin on gut microbiota and susceptibility to C. difficile colonization while tracking microbiota recovery over time. Mice were exposed to fidaxomicin or vancomycin by oral gavage for 3 days and subsequently challenged with C. difficile spores at predetermined time points up to 21 days postexposure to antibiotics. Fecal samples were subsequently collected for analysis. Twenty-four hours postchallenge, mice were euthanized and the colon contents harvested. The microbiota was characterized using 16S rRNA gene sequencing. All fidaxomicin-exposed mice (except for one at day 8) were resistant to C. difficile colonization. However, 9 of 15 vancomycin-exposed mice were susceptible to C. difficile colonization until day 12. All vancomycin-exposed mice recovered colonization resistance by day 16. Bacterial diversity was similar prior to antibiotic exposure in both arms and decreased substantially after exposure. A shift in taxonomic structure and composition occurred after both exposures; however, the shift was greater in vancomycin-exposed than in fidaxomicin-exposed mice. In summary, compared with vancomycin, fidaxomicin exposure had less impact on microbiota composition, promoted faster microbial recovery, and had less impact on the loss of C. difficile colonization resistance.

scholarly journals Activity of Telavancin against Staphylococcus aureus Isolates, Including Those with Decreased Susceptibility to Ceftaroline, from Cystic Fibrosis Patients

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Melanie Roch ◽  
Maria Celeste Varela ◽  
Agustina Taglialegna ◽  
Warren E. Rose ◽  
Adriana E. Rosato
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Galleria Mellonella ◽  
Therapeutic Option ◽  
The United States ◽  
Infection Model ◽  
Content Type ◽  
Complicated Skin ◽  
Hospital Acquired

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) acquisition in cystic fibrosis (CF) patients confers a clinical outcome worse than that in non-CF patients with an increased rate of declined lung function. Telavancin, an approved lipoglycopeptide used to treat infections due to S. aureus, has a dual mode of action causing inhibition of peptidoglycan synthesis and membrane depolarization. MRSA infections in CF patients remain an important problem with no foreseeable decline in prevalence rates. Although telavancin is currently in clinical use for the treatment of complicated skin infections and hospital-acquired pneumonia, the activity against S. aureus infections in CF patients has not been investigated. In this work, we studied the activity of telavancin against CF patient-derived S. aureus strains collected from geographically diverse CF centers in the United States. We found that the telavancin MIC90 was 0.06 μg/ml, 8-fold lower than the ceftaroline or daptomycin MIC90 and 25-fold lower than the linezolid and vancomycin MIC90. We demonstrate that telavancin at serum free concentrations has rapid bactericidal activity, with a decrease of more than 3 log10 CFU/ml being achieved during the first 4 to 6 h of treatment, performing better in this assay than vancomycin and ceftaroline, including against S. aureus strains resistant to ceftaroline. Telavancin resistance was infrequent (0.3%), although we found that it can occur in vitro in both CF- and non-CF patient-derived S. aureus strains by progressive passages with subinhibitory concentrations. Genetic analysis of telavancin-resistant in vitro mutants showed gene polymorphisms in cell wall and virulence genes and increased survival in a Galleria mellonella infection model. Thus, we conclude that telavancin represents a promising therapeutic option for infections in CF patients with potent in vitro activity and a low resistance development potential.

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