scholarly journals Novel Riboswitch-Binding Flavin Analog That Protects Mice against Clostridium difficile Infection without Inhibiting Cecal Flora

2015 ◽  
Vol 59 (9) ◽  
pp. 5736-5746 ◽  
Author(s):  
Kenneth F. Blount ◽  
Cynthia Megyola ◽  
Mark Plummer ◽  
David Osterman ◽  
Tim O'Connell ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
High Rate ◽  
Flavin Mononucleotide ◽  
Antibacterial Drug ◽  
Bacterial Gene ◽  
Content Type ◽  
Bacterial Gene Expression ◽  
Time Kill

ABSTRACTNovel mechanisms of action and new chemical scaffolds are needed to rejuvenate antibacterial drug discovery, and riboswitch regulators of bacterial gene expression are a promising class of targets for the discovery of new leads. Herein, we report the characterization of 5-(3-(4-fluorophenyl)butyl)-7,8-dimethylpyrido[3,4-b]quinoxaline-1,3(2H,5H)-dione (5FDQD)—an analog of riboflavin that was designed to bind riboswitches that naturally recognize the essential coenzyme flavin mononucleotide (FMN) and regulate FMN and riboflavin homeostasis.In vitro, 5FDQD and FMN bind to and trigger the function of an FMN riboswitch with equipotent activity. MIC and time-kill studies demonstrated that 5FDQD has potent and rapidly bactericidal activity againstClostridium difficile. In C57BL/6 mice, 5FDQD completely prevented the onset of lethal antibiotic-inducedC. difficileinfection (CDI). Against a panel of bacteria representative of healthy bowel flora, the antibacterial selectivity of 5FDQD was superior to currently marketed CDI therapeutics, with very little activity against representative strains from theBacteroides,Lactobacillus,Bifidobacterium,Actinomyces, andPrevotellagenera. Accordingly, a single oral dose of 5FDQD caused less alteration of culturable cecal flora in mice than the comparators. Collectively, these data suggest that 5FDQD or closely related analogs could potentially provide a high rate of CDI cure with a low likelihood of infection recurrence. Future studies will seek to assess the role of FMN riboswitch binding to the mechanism of 5FDQD antibacterial action. In aggregate, our results indicate that riboswitch-binding antibacterial compounds can be discovered and optimized to exhibit activity profiles that merit preclinical and clinical development as potential antibacterial therapeutic agents.

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 Endocarditis Caused by Highly Penicillin-Resistant Viridans Group Streptococci: Still Room for Vancomycin-Based Regimens

2019 ◽  
Vol 63 (8) ◽  
Author(s):  
Juan M. Pericàs ◽  
Ruvandhi Nathavitharana ◽  
Cristina Garcia-de-la-Mària ◽  
Carles Falces ◽  
Juan Ambrosioni ◽  
...  
Keyword(s):  
Bactericidal Activity ◽  
Treatment Options ◽  
Content Type ◽  
High Level ◽  
Viridans Group Streptococci ◽  
Time Kill

ABSTRACT Optimal treatment options remain unknown for infective endocarditis (IE) caused by penicillin-resistant (PEN-R) viridans group streptococcal (VGS) strains. The aims of this study were to report two cases of highly PEN-R VGS IE, perform a literature review, and evaluate various antibiotic combinations in vitro and in vivo. The following combinations were tested by time-kill studies and in the rabbit experimental endocarditis (EE) model: PEN-gentamicin, ceftriaxone-gentamicin, vancomycin-gentamicin, daptomycin-gentamicin, and daptomycin-ampicillin. Case 1 was caused by Streptococcus parasanguinis (PEN MIC, 4 μg/ml) and was treated with vancomycin plus cardiac surgery. Case 2 was caused by Streptococcus mitis (PEN MIC, 8 μg/ml) and was treated with 4 weeks of vancomycin plus gentamicin, followed by 2 weeks of vancomycin alone. Both patients were alive and relapse-free after ≥6 months follow-up. For the in vitro studies, except for daptomycin-ampicillin, all combinations demonstrated both synergy and bactericidal activity against the S. parasanguinis isolate. Only PEN-gentamicin, daptomycin-gentamicin, and daptomycin-ampicillin demonstrated both synergy and bactericidal activity against the S. mitis strain. Both strains developed high-level daptomycin resistance (HLDR) during daptomycin in vitro passage. In the EE studies, PEN alone failed to clear S. mitis from vegetations, while ceftriaxone and vancomycin were significantly more effective (P < 0.001). The combination of gentamicin with PEN or vancomycin increased bacterial eradication compared to that with the respective monotherapies. In summary, two patients with highly PEN-R VGS IE were cured using vancomycin-based therapy. In vivo, regimens of gentamicin plus either β-lactams or vancomycin were more active than their respective monotherapies. Further clinical studies are needed to confirm the role of vancomycin-based regimens for highly PEN-R VGS IE. The emergence of HLDR among these strains warrants caution in the use of daptomycin therapy for VGS IE.

scholarly journals Mutagenic Analysis of the Clostridium difficile Flagellar Proteins, FliC and FliD, and Their Contribution to Virulence in Hamsters

2011 ◽  
Vol 79 (10) ◽  
pp. 4061-4067 ◽  
Author(s):  
Tanis C. Dingle ◽  
George L. Mulvey ◽  
Glen D. Armstrong
Keyword(s):  
Clostridium Difficile ◽  
Content Type ◽  
Mutant Strains ◽  
Structural Subunit ◽  
Flagellar Proteins ◽  
Colonization Factors ◽  
Host Tissues ◽  
Better Than

ABSTRACTAlthough toxins A and B are known to be important contributors to the acute phase ofClostridium difficileinfection, the role of colonization and adherence to host tissues in the overall pathogenesis of these organisms remains unclear. Consequently, we used the recently introduced intron-based ClosTron gene interruption system to eliminate the expression of two reportedC. difficilecolonization factors, the major flagellar structural subunit (FliC) and the flagellar cap protein (FliD), to gain greater insight into how flagella and motility contribute toC. difficile's pathogenic strategy. The results demonstrate that interrupting either thefliCor thefliDgene results in a complete loss of flagella, as well as motility, inC. difficile. However, both thefliCandfliDmutant strains adhered better than the wild-type 630Δerm strain to human intestine-derived Caco-2 cells, suggesting that flagella and motility do not contribute to, or may even interfere with,C. difficileadherence to epithelial cell surfacesin vitro. Moreover, we found that the mutant strains were more virulent in hamsters, indicating either that flagella are unnecessary for virulence or that repression of motility may be a pathogenic strategy employed byC. difficilein hamsters.

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 In VitroandIn VivoAntibacterial Evaluation of Cadazolid, a New Antibiotic for Treatment of Clostridium difficile Infections

2013 ◽  
Vol 58 (2) ◽  
pp. 892-900 ◽  
Author(s):  
Hans H. Locher ◽  
Peter Seiler ◽  
Xinhua Chen ◽  
Susanne Schroeder ◽  
Philippe Pfaff ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
De Novo ◽  
Bactericidal Effect ◽  
Content Type ◽  
Growth Inhibitory ◽  
Time Kill ◽  
Full Protection ◽  
Antibacterial Evaluation

ABSTRACTClostridium difficileis a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment ofC. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report thein vitroandin vivoantibacterial evaluation of cadazolid againstC. difficile. Cadazolid showed potentin vitroactivity againstC. difficilewith a MIC range of 0.125 to 0.5 μg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect againstC. difficileisolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibitedde novotoxin A and B formation in stationary-phase cultures of toxigenicC. difficile. Cadazolid also inhibitedC. difficilespore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD.

scholarly journals Amixicile, a Novel Inhibitor of Pyruvate:Ferredoxin Oxidoreductase, Shows Efficacy against Clostridium difficile in a Mouse Infection Model

2012 ◽  
Vol 56 (8) ◽  
pp. 4103-4111 ◽  
Author(s):  
Cirle A. Warren ◽  
Edward van Opstal ◽  
T. Eric Ballard ◽  
Andrew Kennedy ◽  
Xia Wang ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Diarrheal Disease ◽  
Anaerobic Bacteria ◽  
Disease Recurrence ◽  
High Rate ◽  
Infection Model ◽  
Content Type ◽  
Mouse Infection Model ◽  
Pyruvate:Ferredoxin Oxidoreductase

ABSTRACTClostridium difficileinfection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites,Helicobacter pylori, andCampylobacter jejuni, also shows clinical efficacy against CDI. From a library of ∼250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicatedin vitroactivity in the 0.05- to 2-μg/ml range againstC. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum ofC. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.

scholarly journals Mode of Action and Bactericidal Properties of Surotomycin against Growing and Nongrowing Clostridium difficile

2015 ◽  
Vol 59 (9) ◽  
pp. 5165-5170 ◽  
Author(s):  
Mohammed Zahidul Alam ◽  
Xiaoqian Wu ◽  
Carmela Mascio ◽  
Laurent Chesnel ◽  
Julian G. Hurdle
Keyword(s):  
Membrane Potential ◽  
Clostridium Difficile ◽  
Mode Of Action ◽  
Logarithmic Phase ◽  
Macromolecular Synthesis ◽  
Content Type ◽  
Cyclic Lipopeptide ◽  
Bactericidal Properties ◽  
Time Kill

ABSTRACTSurotomycin (CB-183,315), a cyclic lipopeptide, is in phase 3 clinical development for the treatment ofClostridium difficileinfection. We report here the further characterization of thein vitromode of action of surotomycin, including its activity against growing and nongrowingC. difficile. This was assessed through time-kill kinetics, allowing a determination of the effects on the membrane potential and permeability and macromolecular synthesis inC. difficile. Against representative strains ofC. difficile, surotomycin displayed concentration-dependent killing of both logarithmic-phase and stationary-phase cultures at a concentration that was ≤16× the MIC. Exposure resulted in the inhibition of macromolecular synthesis (in DNA, RNA, proteins, and cell wall). At bactericidal concentrations, surotomycin dissipated the membrane potential ofC. difficilewithout changes to the permeability of propidium iodide. These observations are consistent with surotomycin acting as a membrane-active antibiotic, exhibiting rapid bactericidal activities against growing and nongrowingC. difficile.

scholarly journals In VitroActivity of Imipenem-Relebactam Alone or in Combination with Amikacin or Colistin againstPseudomonas aeruginosa

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Tomefa E. Asempa ◽  
David P. Nicolau ◽  
Joseph L. Kuti
Keyword(s):  
Potential Role ◽  
Multidrug Resistant ◽  
Synergistic Activity ◽  
Content Type ◽  
Class A ◽  
Susceptible Isolate ◽  
Time Kill ◽  
Lactamase Inhibitor

ABSTRACTRelebactam is a novel class A/C β-lactamase inhibitor that restores imipenemin vitroactivity against multidrug-resistant and carbapenem-nonsusceptiblePseudomonas aeruginosa. Time-kill analyses were performed to evaluate the potential role of imipenem-relebactam in combination with amikacin or colistin againstP. aeruginosa. Ten clinicalP. aeruginosaisolates (9 imipenem nonsusceptible) with imipenem-relebactam MICs ranging from 1/4 to 8/4 μg/ml were included. The isolates had varied susceptibilities to imipenem (1 to 32 μg/ml), amikacin (4 to 128 μg/ml), and colistin (0.5 to 1 μg/ml). Duplicate 24-h time-kill studies were conducted using the average steady-state concentrations (Cssavg) observed after the administration of imipenem-relebactam at 500 mg/250 mg every 6 hours (q6h) alone and in combination with theCssavgof 25 mg/kg of body weight/day amikacin and 360 mg/day colistin in humans. Imipenem-relebactam alone resulted in 24-h bacterial densities of −2.93 ± 0.38, −1.67 ± 0.29, +0.38 ± 0.96, and +0.15 ± 0.65 log10CFU/ml at imipenem-relebactam MICs of 1/4, 2/4, 4/4, and 8/4 μg/ml, respectively. No synergy was demonstrated against the single imipenem-susceptible isolate. Against the imipenem-nonsusceptible isolates (n = 9), imipenem-relebactam combined with amikacin resulted in synergy (−2.61 ± 1.50 log10CFU/ml) against all amikacin-susceptible isolates and in two of three amikacin-intermediate (i.e., MIC, 32 μg/ml) isolates (−2.06 ± 0.19 log10CFU/ml). Synergy with amikacin was not observed when the amikacin MIC was >32 μg/ml. Imipenem-relebactam combined with colistin demonstrated synergy in eight out of the nine imipenem-resistant isolates (−3.17 ± 1.00 log10CFU/ml). Against these 10 P. aeruginosaisolates, imipenem-relebactam combined with either amikacin or colistin resulted in synergistic activity against the majority of strains. Further studies evaluating combination therapy with imipenem-relebactam are warranted.

scholarly journals Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile

2014 ◽  
Vol 82 (10) ◽  
pp. 4276-4291 ◽  
Author(s):  
Adrianne N. Edwards ◽  
Kathryn L. Nawrocki ◽  
Shonna M. McBride
Keyword(s):  
Clostridium Difficile ◽  
Hamster Model ◽  
Content Type ◽  
Gastrointestinal Pathogen ◽  
Dormant Spore ◽  
Sporulation Initiation ◽  
Spore Forming Bacteria

ABSTRACTThe anaerobic gastrointestinal pathogenClostridium difficilemust form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by whichC. difficileinitiates and controls the early stages of sporulation inC. difficileare not highly conserved in otherClostridiumorBacillusspecies. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation inC. difficile. These permeases are known to positively affect sporulation inBacillusspecies through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases inC. difficileresulted in the earlier expression of early sporulation genes and increased sporulationin vitro. Furthermore, disruption ofoppandappresulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model ofC. difficileinfection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence inC. difficilethan inBacillus subtilisand suggest that nutrient availability plays a significant role in pathogenesis and sporulationin vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation inC. difficile.

scholarly journals In VitroandIn VivoCharacterization of CB-183,315, a Novel Lipopeptide Antibiotic for Treatment of Clostridium difficile

2012 ◽  
Vol 56 (10) ◽  
pp. 5023-5030 ◽  
Author(s):  
Carmela T. M. Mascio ◽  
Lawrence I. Mortin ◽  
Karen T. Howland ◽  
Andrew D. G. Van Praagh ◽  
Shuxin Zhang ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Limit Of Detection ◽  
Disease Onset ◽  
Serial Passage ◽  
Postantibiotic Effect ◽  
Hamster Model ◽  
Content Type ◽  
Lipopeptide Antibiotic ◽  
Time Kill

ABSTRACTCB-183,315 is a novel lipopeptide antibiotic structurally related to daptomycin currently in phase 3 clinical development forClostridium difficile-associated diarrhea (CDAD). We report here thein vitromechanism of action, spontaneous resistance incidence, resistance by serial passage, time-kill kinetics, postantibiotic effect, and efficacy of CB-183,315 in a hamster model of lethal infection.In vitrodata showed that CB-183,315 dissipated the membrane potential ofStaphylococcus aureuswithout inducing changes in membrane permeability to small molecules. The rate of spontaneous resistance to CB-183,315 at 8× the MIC was below the limit of detection inC. difficile. Under selective pressure by serial passage with CB-183,315 againstC. difficile, the susceptibility of the bacteria changed no more than 2-fold during 15 days of serial passages. At 16× the MIC, CB-183,315 produced a ≥3-log reduction ofC. difficilein the time-kill assay. The postantibiotic effect of CB-183,315 at 8× the MIC was 0.9 h. At 80× the MIC the postantibiotic effect was more than 6 h. In the hamster model of CDAD, CB-183,315 and vancomycin both demonstrated potent efficacy in resolving initial disease onset, even at very low doses. After the conclusion of dosing, CB-183,315 and vancomycin showed a similar dose- and time-dependent pattern with respect to rates of CDAD recurrence.

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