Mutations Associated with Reduced Surotomycin Susceptibility in Clostridium difficile and Enterococcus Species

ABSTRACTClostridium difficileinfection (CDI) is an urgent public health concern causing considerable clinical and economic burdens. CDI can be treated with antibiotics, but recurrence of the disease following successful treatment of the initial episode often occurs. Surotomycin is a rapidly bactericidal cyclic lipopeptide antibiotic that is in clinical trials for CDI treatment and that has demonstrated superiority over vancomycin in preventing CDI relapse. Surotomycin is a structural analogue of the membrane-active antibiotic daptomycin. Previously, we utilizedin vitroserial passage experiments to deriveC. difficilestrains with reduced surotomycin susceptibilities. The parent strains used included ATCC 700057 and clinical isolates from the restriction endonuclease analysis (REA) groups BI and K. Serial passage experiments were also performed with vancomycin-resistant and vancomycin-susceptibleEnterococcus faeciumandEnterococcus faecalis. The goal of this study is to identify mutations associated with reduced surotomycin susceptibility inC. difficileand enterococci. Illumina sequence data generated for the parent strains and serial passage isolates were compared. We identified nonsynonymous mutations in genes coding for cardiolipin synthase inC. difficileATCC 700057, enoyl-(acyl carrier protein) reductase II (FabK) and cell division protein FtsH2 inC. difficileREA type BI, and a PadR family transcriptional regulator inC. difficileREA type K. Among the 4 enterococcal strain pairs, 20 mutations were identified, and those mutations overlap those associated with daptomycin resistance. These data give insight into the mechanism of action of surotomycin againstC. difficile, possible mechanisms for resistance emergence during clinical use, and the potential impacts of surotomycin therapy on intestinal enterococci.

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Epidemic Clostridium difficile Strains Demonstrate Increased Competitive Fitness Compared to Nonepidemic Isolates

Infection and Immunity ◽

10.1128/iai.01524-14 ◽

2014 ◽

Vol 82 (7) ◽

pp. 2815-2825 ◽

Cited By ~ 39

Author(s):

Catherine D. Robinson ◽

Jennifer M. Auchtung ◽

James Collins ◽

Robert A. Britton

Keyword(s):

Clostridium Difficile ◽

Restriction Endonuclease Analysis ◽

Clinical Isolates ◽

Subsequent Work ◽

Competitive Fitness ◽

Content Type ◽

Ribotype 027 ◽

Pulsed Field Electrophoresis ◽

Significant Health

ABSTRACTClostridium difficileinfection is the most common cause of severe cases of antibiotic-associated diarrhea (AAD) and is a significant health burden. Recent increases in the rate ofC. difficileinfection have paralleled the emergence of a specific phylogenetic clade ofC. difficilestrains (ribotype 027; North American pulsed-field electrophoresis 1 [NAP1]; restriction endonuclease analysis [REA] group BI). Initial reports indicated that ribotype 027 strains were associated with increased morbidity and mortality and might be hypervirulent. Although subsequent work has raised some doubt as to whether ribotype 027 strains are hypervirulent, the strains are considered epidemic isolates that have caused severe outbreaks across the globe. We hypothesized that one factor that could lead to the increased prevalence of ribotype 027 strains would be if these strains had increased competitive fitness compared to strains of other ribotypes. We developed a moderate-throughputin vitromodel ofC. difficileinfection and used it to test competition between four ribotype 027 clinical isolates and clinical isolates of four other ribotypes (001, 002, 014, and 053). We found that ribotype 027 strains outcompeted the strains of other ribotypes. A similar competitive advantage was observed when two ribotype pairs were competed in a mouse model ofC. difficileinfection. Based upon these results, we conclude that one possible mechanism through which ribotype 027 strains have caused outbreaks worldwide is their increased ability to compete in the presence of a complex microbiota.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01087-15 ◽

2015 ◽

Vol 59 (9) ◽

pp. 5165-5170 ◽

Cited By ~ 27

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00057-12 ◽

2012 ◽

Vol 56 (10) ◽

pp. 5023-5030 ◽

Cited By ~ 62

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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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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00191-12 ◽

2012 ◽

Vol 56 (7) ◽

pp. 3943-3949 ◽

Cited By ~ 34

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00508-12 ◽

2012 ◽

Vol 56 (9) ◽

pp. 4786-4792 ◽

Cited By ~ 16

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.

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Germinant Synergy FacilitatesClostridium difficileSpore Germination under Physiological Conditions

mSphere ◽

10.1128/msphere.00335-18 ◽

2018 ◽

Vol 3 (5) ◽

Cited By ~ 13

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00015-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5986-5989 ◽

Cited By ~ 5

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.

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Nontoxigenic Clostridium difficile Protects Hamsters against Challenge with Historic and Epidemic Strains of Toxigenic BI/NAP1/027 C. difficile

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00580-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5266-5270 ◽

Cited By ~ 50

Author(s):

Kristin J. Nagaro ◽

S. Tyler Phillips ◽

Adam K. Cheknis ◽

Susan P. Sambol ◽

Walter E. Zukowski ◽

...

Keyword(s):

Clostridium Difficile ◽

Restriction Endonuclease ◽

Restriction Endonuclease Analysis ◽

Epidemic Strain ◽

Hamster Model ◽

Content Type ◽

Analysis Group ◽

Endonuclease Analysis

ABSTRACTNontoxigenicClostridium difficile(NTCD) has been shown to prevent fatalC. difficileinfection in the hamster model when hamsters are challenged with standard toxigenicC. difficilestrains. The purpose of this study was to determine if NTCD can preventC. difficileinfection in the hamster model when hamsters are challenged with restriction endonuclease analysis group BIC. difficilestrains. Groups of 10 hamsters were given oral clindamycin, followed on day 2 by 106CFU of spores of NTCD strain M3 or T7, and were challenged on day 5 with 100 CFU of spores of BI1 or BI6. To conserve animals, results for control hamsters challenged with BI1 or BI6 from the present study and controls from previous identical experiments were combined for statistical comparisons. NTCD strains M3 and T7 achieved 100% colonization and were 100% protective against challenge with BI1 (P≤ 0.001). M3 colonized 9/10 hamsters and protected against BI6 challenge in the colonized hamsters (P= 0.0003). T7 colonized 10/10 hamsters, but following BI6 challenge, cocolonization occurred in 5 hamsters, 4 of which died, for protection of 6/10 animals (P= 0.02). NTCD colonization provides protection against challenge with toxigenic BI group strains. M3 is more effective than T7 in preventingC. difficileinfection caused by the BI6 epidemic strain. Prevention ofC. difficileinfection caused by the epidemic BI6 strain may be more challenging than that of infections caused by historic BI1 and non-BIC. difficilestrains.

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Evidence ofIn VivoProphage Induction during Clostridium difficile Infection

Applied and Environmental Microbiology ◽

10.1128/aem.02275-12 ◽

2012 ◽

Vol 78 (21) ◽

pp. 7662-7670 ◽

Cited By ~ 58

Author(s):

Mathieu Meessen-Pinard ◽

Ognjen Sekulovic ◽

Louis-Charles Fortier

Keyword(s):

Clostridium Difficile ◽

Bioinformatics Analyses ◽

Content Type ◽

Unique Character ◽

Viral Particles ◽

Potential Impact ◽

First Time ◽

Culture Supernatants

ABSTRACTProphages contribute to the evolution and virulence of most bacterial pathogens, but their role inClostridium difficileis unclear. Here we describe the isolation of fourMyoviridaephages, ϕMMP01, ϕMMP02, ϕMMP03, and ϕMMP04, that were recovered as free viral particles in the filter-sterilized stool supernatants of patients suffering fromC. difficileinfection (CDI). Furthermore, identical prophages were found in the chromosomes ofC. difficileisolated from the corresponding fecal samples. We therefore provide, for the first time, evidence ofin vivoprophage induction during CDI. We completely sequenced the genomes of ϕMMP02 and ϕMMP04, and bioinformatics analyses did not reveal the presence of virulence factors but underlined the unique character of ϕMMP04. We also studied the mobility of ϕMMP02 and ϕMMP04 prophagesin vitro. Both prophages were spontaneously induced, with 4 to 5 log PFU/ml detected in the culture supernatants of the corresponding lysogens. When lysogens were grown in the presence of subinhibitory concentrations of ciprofloxacin, moxifloxacin, levofloxacin, or mitomycin C, the phage titers further increased, reaching 8 to 9 log PFU/ml in the case of ϕMMP04. In summary, our study highlights the extensive genetic diversity and mobility ofC. difficileprophages. Moreover, antibiotics known to represent risk factors for CDI, such as quinolones, can stimulate prophage mobilityin vitroand probablyin vivoas well, which underscores their potential impact on phage-mediated horizontal gene transfer events and the evolution ofC. difficile.

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The HtrA-Like Protease CD3284 Modulates Virulence of Clostridium difficile

Infection and Immunity ◽

10.1128/iai.02336-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4222-4232 ◽

Cited By ~ 14

Author(s):

Dennis Bakker ◽

Anthony M. Buckley ◽

Anne de Jong ◽

Vincent J. C. van Winden ◽

Joost P. A. Verhoeks ◽

...

Keyword(s):

Clostridium Difficile ◽

Virulence Factors ◽

Pathogenic Bacteria ◽

Microarray Data Analysis ◽

Toxin A ◽

Hamster Model ◽

Golden Syrian Hamster ◽

Content Type

ABSTRACTIn the past decade,Clostridium difficilehas emerged as an important gut pathogen. Symptoms ofC. difficileinfection range from mild diarrhea to pseudomembranous colitis. Besides the two main virulence factors toxin A and toxin B, other virulence factors are likely to play a role in the pathogenesis of the disease. In other Gram-positive and Gram-negative pathogenic bacteria, conserved high-temperature requirement A (HtrA)-like proteases have been shown to have a role in protein homeostasis and quality control. This affects the functionality of virulence factors and the resistance of bacteria to (host-induced) environmental stresses. We found that theC. difficile630 genome encodes a single HtrA-like protease (CD3284; HtrA) and have analyzed its rolein vivoandin vitrothrough the creation of an isogenic ClosTron-basedhtrAmutant ofC. difficilestrain 630Δerm(wild type). In contrast to the attenuated phenotype seen withhtrAdeletion in other pathogens, this mutant showed enhanced virulence in the Golden Syrian hamster model of acuteC. difficileinfection. Microarray data analysis showed a pleiotropic effect ofhtrAon the transcriptome ofC. difficile, including upregulation of the toxin A gene. In addition,the htrAmutant showed reduced spore formation and adherence to colonic cells. Together, our data show thathtrAcan modulate virulence inC. difficile.

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