scholarly journals Subinhibitory Concentrations of LFF571 Reduce Toxin Production by Clostridium difficile

2014 ◽  
Vol 59 (2) ◽  
pp. 1252-1257 ◽  
Author(s):  
Meena Sachdeva ◽  
Jennifer A. Leeds
Keyword(s):  
Clostridium Difficile ◽  
Elongation Factor ◽  
Viable Cell ◽  
Toxin Production ◽  
Synthesis Inhibitor ◽  
Content Type ◽  
Cell Counts ◽  
A Cell ◽  
Subinhibitory Concentrations ◽  
Bacterial Protein Synthesis

ABSTRACTLFF571 is a novel semisynthetic thiopeptide antibacterial that is undergoing investigation for safety and efficacy in patients with moderateClostridium difficileinfections. LFF571 inhibits bacterial protein synthesis by interacting with elongation factor Tu (EF-Tu) and interrupting complex formation between EF-Tu and aminoacyl-tRNA. Given this mechanism of action, we hypothesized that concentrations of LFF571 below those necessary to inhibit bacterial growth would reduce steady-state toxin levels inC. difficilecultures. We investigatedC. difficilegrowth and toxin A and B levels in the presence of LFF571, fidaxomicin, vancomycin, and metronidazole. LFF571 led to strain-dependent effects on toxin production, including decreased toxin levels after treatment with subinhibitory concentrations, and more rapid declines in toxin production than in inhibition of colony formation. Fidaxomicin, which is an RNA synthesis inhibitor, conferred a similar pattern to LFF571 with respect to toxin levels versus viable cell counts. The incubation of two toxigenicC. difficilestrains with subinhibitory concentrations of vancomycin, a cell wall synthesis inhibitor, increased toxin levels in the supernatant over those of untreated cultures. A similar phenomenon was observed with one metronidazole-treated strain ofC. difficile. These studies indicate that LFF571 and fidaxomicin generally result in decreasedC. difficiletoxin levels in culture supernatants, whereas treatment of some strains with vancomycin or metronidazole had the potential to increase toxin levels. Although the relevance of these findings remains to be studied in patients, reducing toxin levels with sub-growth-inhibitory concentrations of an antibiotic is hypothesized to be beneficial in alleviating symptoms.

scholarly journals Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches inClostridium difficile

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Robert W. McKee ◽  
Carissa K. Harvest ◽  
Rita Tamayo
Keyword(s):  
Gene Expression ◽  
Clostridium Difficile ◽  
Biofilm Formation ◽  
Toxin Production ◽  
Intestinal Colonization ◽  
Cyclic Diguanylate ◽  
Signaling Molecule ◽  
Content Type ◽  
Surface Motility

ABSTRACTThe intracellular signaling molecule cyclic diguanylate (c-di-GMP) regulates many processes in bacteria, with a central role in controlling the switch between motile and nonmotile lifestyles. Recent work has shown that inClostridium difficile(also calledClostridioides difficile), c-di-GMP regulates swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we determined the transcriptional regulon of c-di-GMP inC. difficile,employing overexpression of a diguanylate cyclase gene to artificially manipulate intracellular c-di-GMP. Consistent with prior work, c-di-GMP regulated the expression of genes involved in swimming and surface motility. c-di-GMP also affected the expression of multiple genes encoding cell envelope proteins, several of which affected biofilm formationin vitro. A substantial proportion of the c-di-GMP regulon appears to be controlled either directly or indirectly via riboswitches. We confirmed the functionality of 11 c-di-GMP riboswitches, demonstrating their effects on downstream gene expression independent of the upstream promoters. The class I riboswitches uniformly functioned as “off” switches in response to c-di-GMP, while class II riboswitches acted as “on” switches. Transcriptional analyses of genes 3′ of c-di-GMP riboswitches over a broad range of c-di-GMP levels showed that relatively modest changes in c-di-GMP levels are capable of altering gene transcription, with concomitant effects on microbial behavior. This work expands the known c-di-GMP signaling network inC. difficileand emphasizes the role of the riboswitches in controlling known and putative virulence factors inC. difficile.IMPORTANCEInClostridium difficile, the signaling molecule c-di-GMP regulates multiple processes affecting its ability to cause disease, including swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we used RNA-seq to define the transcriptional regulon of c-di-GMP inC. difficile. Many new targets of c-di-GMP regulation were identified, including multiple putative colonization factors. Transcriptional analyses revealed a prominent role for riboswitches in c-di-GMP signaling. Only a subset of the 16 previously predicted c-di-GMP riboswitches were functionalin vivoand displayed potential variability in their response kinetics to c-di-GMP. This work underscores the importance of studying c-di-GMP riboswitches in a relevant biological context and highlights the role of the riboswitches in controlling gene expression inC. difficile.

scholarly journals The Urinary Antibiotic 5-Nitro-8-Hydroxyquinoline (Nitroxoline) Reduces the Formation and Induces the Dispersal of Pseudomonas aeruginosa Biofilms by Chelation of Iron and Zinc

2012 ◽  
Vol 56 (11) ◽  
pp. 6021-6025 ◽  
Author(s):  
A. Sobke ◽  
M. Klinger ◽  
B. Hermann ◽  
S. Sachse ◽  
S. Nietzsche ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Broad Spectrum ◽  
Viable Cell ◽  
Antibiofilm Activity ◽  
Content Type ◽  
Cell Counts ◽  
Iron And Zinc ◽  
Chelation Of Iron

ABSTRACTSince cations have been reported as essential regulators of biofilm, we investigated the potential of the broad-spectrum antimicrobial and cation-chelator nitroxoline as an antibiofilm agent. Biofilm mass synthesis was reduced by up to 80% at sub-MIC nitroxoline concentrations inPseudomonas aeruginosa, and structures formed were reticulate rather than compact. In preformed biofilms, viable cell counts were reduced by 4 logs at therapeutic concentrations. Complexation of iron and zinc was demonstrated to underlie nitroxoline's potent antibiofilm activity.

scholarly journals Differential Effects of Linezolid and Ciprofloxacin on Toxin Production by Bacillus anthracis in anIn VitroPharmacodynamic System

2011 ◽  
Vol 56 (1) ◽  
pp. 513-517 ◽  
Author(s):  
Arnold Louie ◽  
Brian D. VanScoy ◽  
Henry S. Heine ◽  
Weiguo Liu ◽  
Terry Abshire ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Bacterial Population ◽  
Total Population ◽  
Toxin Production ◽  
Pharmacodynamic Model ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Content Type ◽  
Linezolid Therapy

ABSTRACTBacillus anthraciscauses anthrax. Ciprofloxacin is a gold standard for the treatment of anthrax. Previously, using the non-toxin-producing ΔSterne strain ofB. anthracis, we demonstrated that linezolid was equivalent to ciprofloxacin for reducing the total (vegetative and spore) bacterial population. With ciprofloxacin therapy, the total population consisted of spores. With linezolid therapy, the population consisted primarily of vegetative bacteria. Linezolid is a protein synthesis inhibitor, while ciprofloxacin is not. Since toxins are produced only by vegetativeB. anthracis, the effect of linezolid and ciprofloxacin on toxin production is of interest. The effect of simulated clinical regimens of ciprofloxacin and linezolid on the vegetative and spore populations and on toxin production was examined in anin vitropharmacodynamic model over 15 days by using the toxin-producing Sterne strain ofB. anthracis. Ciprofloxacin and linezolid reduced the total Sterne population at similar rates. With ciprofloxacin therapy, the total Sterne population consisted of spores. With linezolid therapy, >90% of the population was vegetativeB. anthracis. With ciprofloxacin therapy, toxin was first detectable at 3 h and remained detectable for at least 5 h. Toxin was never detected with linezolid therapy. Ciprofloxacin and linezolid reduced the total Sterne population at similar rates. However, theB. anthracispopulation was primarily spores with ciprofloxacin therapy and was primarily vegetative bacteria with linezolid therapy. Toxin production was detected for at least 5 h with ciprofloxacin therapy but was never detected with linezolid treatment. Linezolid may have an advantage over ciprofloxacin for the treatment ofB. anthracisinfections.

scholarly journals Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

2016 ◽  
Vol 84 (10) ◽  
pp. 2871-2877 ◽  
Author(s):  
Nicole M. Chumbler ◽  
Melissa A. Farrow ◽  
Lynne A. Lapierre ◽  
Jeffrey L. Franklin ◽  
D. Borden Lacy
Keyword(s):  
Clostridium Difficile ◽  
Apoptotic Cell ◽  
Large Body ◽  
Health Care Facilities ◽  
Dependent Manner ◽  
Content Type ◽  
Culture Model ◽  
Care Facilities ◽  
Antibiotic Associated Diarrhea ◽  
A Cell

As the major cause of antibiotic-associated diarrhea,Clostridium difficileis a serious problem in health care facilities worldwide.C. difficileproduces two large toxins, TcdA and TcdB, which are the primary virulence factors in disease. The respective functions of these toxins have been difficult to discern, in part because the cytotoxicity profiles for these toxins differ with concentration and cell type. The goal of this study was to develop a cell culture model that would allow a side-by-side mechanistic comparison of the toxins. Conditionally immortalized, young adult mouse colonic (YAMC) epithelial cells demonstrate an exquisite sensitivity to both toxins with phenotypes that agree with observations in tissue explants. TcdA intoxication results in an apoptotic cell death that is dependent on the glucosyltransferase activity of the toxin. In contrast, TcdB has a bimodal mechanism; it induces apoptosis in a glucosyltransferase-dependent manner at lower concentrations and glucosyltransferase-independent necrotic death at higher concentrations. The direct comparison of the responses to TcdA and TcdB in cells and colonic explants provides the opportunity to unify a large body of observations made by many independent investigators.

scholarly journals A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Erin B. Purcell ◽  
Robert W. McKee ◽  
David S. Courson ◽  
Elizabeth M. Garrett ◽  
Shonna M. McBride ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Stationary Phase ◽  
Nutrient Availability ◽  
Biofilm Formation ◽  
Biochemical Characterization ◽  
Toxin Production ◽  
Physiological Adaptation ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Wide Range

ABSTRACT The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability.

scholarly journals Evaluating the Effects of Surotomycin Treatment on Clostridium difficile Toxin A and B Production, Immune Response, and Morphological Changes

2016 ◽  
Vol 60 (6) ◽  
pp. 3519-3523 ◽  
Author(s):  
Bradley T. Endres ◽  
Eugénie Bassères ◽  
Mohammed Khaleduzzaman ◽  
M. Jahangir Alam ◽  
Laurent Chesnel ◽  
...  
Keyword(s):  
Immune Response ◽  
Clostridium Difficile ◽  
Morphological Changes ◽  
Toxin Production ◽  
Morphological Data ◽  
Growth Media ◽  
Toxin A ◽  
Content Type ◽  
Cyclic Lipopeptide ◽  
The Impact

Surotomycin is a cyclic lipopeptide in development forClostridium difficile-associated diarrhea. This study aimed to assess the impact of surotomycin exposure onC. difficiletoxin A and B concentrations and the associated changes in immune response in comparison to vancomycin and metronidazole. Time-kill curve assays were performed using strain R20291 (BI/NAP1/027) at supra-MICs (4× and 40×) and sub-MICs (0.5×) of surotomycin and comparators. Following treatment, CFU counts, toxin A and B concentrations, and cellular morphological changes using scanning electron microscopy were examined. Inflammatory response was determined by measuring interleukin-8 (IL-8) concentrations from polarized Caco-2 cells exposed to antibiotic-treatedC. difficilegrowth media. Supra-MICs (4× and 40×) of surotomycin resulted in a reduction of vegetative cells over 72 h (4-log difference,P< 0.01) compared to controls. These results correlated with decreases of 77% and 68% in toxin A and B production at 48 h, respectively (P< 0.005, each), which resulted in a significant reduction in IL-8 concentration compared to controls. Similar results were observed with comparator antibiotics. Bacterial cell morphology showed that the cell wall was broken apart by surotomycin treatment at supra-MICs while sub-MIC studies showed a “deflated” phenotype plus a rippling effect. These results suggest that surotomycin has potent killing effects onC. difficilethat results in reduced toxin production and attenuates the immune response similar to comparator antibiotics. The morphological data also confirm observations that surotomycin is a membrane-active antibiotic.

scholarly journals Effect of Ceftobiprole Treatment on Growth of and Toxin Production by Clostridium difficile in Cecal Contents of Mice

2011 ◽  
Vol 55 (5) ◽  
pp. 2174-2177 ◽  
Author(s):  
Michelle M. Nerandzic ◽  
Curtis J. Donskey
Keyword(s):  
Clostridium Difficile ◽  
Inhibitory Activity ◽  
Toxin Production ◽  
Content Type ◽  
Anaerobic Microflora

ABSTRACTCeftobiprole and ceftobiprole medocaril did not promote growth of or toxin production byClostridium difficilein mouse cecal contents, whereas ceftazidime, cefoxitin, ceftriaxone, cefotaxime, and ertapenem did. The relatively low propensity of ceftobiprole to promoteC. difficilewas attributable to inhibitory activity againstC. difficileand sparing of anaerobic microflora.

scholarly journals Bactericidal Activity of ACH-702 against Nondividing and Biofilm Staphylococci

2012 ◽  
Vol 56 (7) ◽  
pp. 3812-3818 ◽  
Author(s):  
Steven D. Podos ◽  
Jane A. Thanassi ◽  
Melissa Leggio ◽  
Michael J. Pucci
Keyword(s):  
Protein Synthesis ◽  
Stationary Phase ◽  
Bactericidal Activity ◽  
Bacterial Infections ◽  
De Novo ◽  
Viable Cell ◽  
Bacterial Cells ◽  
Protein Synthesis Inhibitors ◽  
Content Type ◽  
Cell Counts

ABSTRACTMany bacterial infections involve slow or nondividing bacterial growth states and localized high cell densities. Antibiotics with demonstrated bactericidal activity rarely remain bactericidal at therapeutic concentrations under these conditions. The isothiazoloquinolone (ITQ) ACH-702 is a potent, bactericidal compound with activity against many antibiotic-resistant pathogens, including methicillin-resistantStaphylococcus aureus(MRSA). We evaluated its bactericidal activity under conditions where bacterial cells were not dividing and/or had slowed their growth. AgainstS. aureuscultures in stationary phase, ACH-702 showed concentration-dependent bactericidal activity and achieved a 3-log-unit reduction in viable cell counts within 6 h of treatment at ≥16× MIC values; in comparison, the bactericidal quinolone moxifloxacin and the additional comparator compounds vancomycin, linezolid, and rifampin at 16× to 32× MICs showed little or no bactericidal activity against stationary-phase cells. ACH-702 at 32× MIC retained bactericidal activity against stationary-phaseS. aureusacross a range of inoculum densities. ACH-702 did not kill cold-arrested cells yet remained bactericidal against cells arrested by protein synthesis inhibitors, suggesting that its bactericidal activity against nondividing cells requires active metabolism but notde novoprotein synthesis. ACH-702 also showed a degree of bactericidal activity at 16× MIC againstS. epidermidisbiofilm cells that was superior to that of moxifloxacin, rifampin, and vancomycin. The bactericidal activity of ACH-702 against stationary-phase staphylococci and biofilms suggests potential clinical utility in infections containing cells in these physiological states.

scholarly journals Subinhibitory Dalbavancin Attenuates Exotoxin Production from Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureusIn Vitro

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Sarah E. Hobdey ◽  
Eva J. Katahira ◽  
Pamela Dockstader ◽  
Stephen M. Davidson ◽  
Laura Bond ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Toxin Production ◽  
Glycopeptide Antibiotics ◽  
Glycopeptide Antibiotic ◽  
Beta Lactam ◽  
Methicillin Resistant ◽  
Content Type ◽  
Recent Success ◽  
Subinhibitory Concentrations

ABSTRACT This study investigated the effects of subinhibitory doses of the lipoglycopeptide antibiotic dalbavancin on Staphylococcus aureus toxin production in vitro. S. aureus toxin production levels were compared to those seen with the natural glycopeptide antibiotic vancomycin and with representative beta-lactam and oxazolidinone antibiotics. While neither dalbavancin nor vancomycin adversely affected toxin production, of these glycopeptide antibiotics, only dalbavancin significantly attenuated toxin production at subinhibitory concentrations. These findings support the recent success of dalbavancin for treatment of staphylococcal infections.

scholarly journals The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile

2016 ◽  
Vol 84 (12) ◽  
pp. 3434-3444 ◽  
Author(s):  
Kevin O. Childress ◽  
Adrianne N. Edwards ◽  
Kathryn L. Nawrocki ◽  
Sarah E. Anderson ◽  
Emily C. Woods ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Clostridium Difficile ◽  
Molecular Mechanisms ◽  
Toxin Production ◽  
Negative Regulator ◽  
Hamster Model ◽  
Content Type ◽  
Sporulation Initiation

The formation of spores is critical for the survival ofClostridium difficileoutside the host gastrointestinal tract. Persistence ofC. difficilespores greatly contributes to the spread ofC. difficileinfection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation toC. difficilepathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved inC. difficileand few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion ofCD1492resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in theCD1492mutant. Deletion of CD1492 also resulted in decreased toxin productionin vitroand in decreased virulence in the hamster model of CDI. Further, theCD1492mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lowersigDandrstAtranscription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence thatC. difficilesporulation is regulated differently from that of other endospore-forming species.

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