Drivers of Clostridioides difficile hypervirulent ribotype 027 spore germination, vegetative cell growth and toxin production in vitro

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.

Download Full-text

Effects of Exposure of Clostridium difficile PCR Ribotypes 027 and 001 to Fluoroquinolones in a Human Gut Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00306-08 ◽

2008 ◽

Vol 53 (2) ◽

pp. 412-420 ◽

Cited By ~ 35

Author(s):

Katie Saxton ◽

Simon D. Baines ◽

Jane Freeman ◽

Rachael O'Connor ◽

Mark H. Wilcox

Keyword(s):

Clostridium Difficile ◽

Gut Microbiota ◽

Spore Germination ◽

Toxin Production ◽

Human Gut ◽

Ribotype 027 ◽

Resistant Mutants ◽

High Level ◽

Pcr Ribotype 027

ABSTRACT The incidence of Clostridium difficile infection is increasing, with reports implicating fluoroquinolone use. A three-stage chemostat gut model was used to study the effects of three fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin) on the gut microbiota and two epidemic C. difficile strains, strains of PCR ribotypes 027 and 001, in separate experiments. C. difficile total viable counts, spore counts, and cytotoxin titers were determined. The emergence of C. difficile isolates with reduced antibiotic susceptibility was monitored with fluoroquinolone-containing medium, and molecular analysis of the quinolone resistance-determining region was performed. C. difficile spores were quiescent in the absence of fluoroquinolones. Instillation of each fluoroquinolone led to C. difficile spore germination and high-level cytotoxin production. High-level toxin production occurred after detectable spore germination in all experiments except those with C. difficile PCR ribotype 027 and moxifloxacin, in which marked cytotoxin production preceded detectable germination, which coincided with isolate recovery on fluoroquinolone-containing medium. Three C. difficile PCR ribotype 027 isolates and one C. difficile PCR ribotype 001 isolate from fluoroquinolone-containing medium exhibited elevated MICs (80 to ≥180 mg/liter) and possessed mutations in gyrA or gyrB. These in vitro results suggest that all fluoroquinolones have the propensity to induce C. difficile infection, regardless of their antianaerobe activities. Resistant mutants were seen only following moxifloxacin exposure.

Download Full-text

In vitro antivirulence activity of baicalin against Clostridioides difficile

Journal of Medical Microbiology ◽

10.1099/jmm.0.001179 ◽

2020 ◽

Vol 69 (4) ◽

pp. 631-639

Author(s):

Abraham Joseph Pellissery ◽

Poonam Gopika Vinayamohan ◽

Kumar Venkitanarayanan

Keyword(s):

Spore Germination ◽

Type Species ◽

Brain Heart Infusion ◽

Toxin Production ◽

Strictly Anaerobic ◽

Content Type ◽

Link Type ◽

Clostridioides Difficile ◽

Spore Outgrowth

Introduction. Clostridioides difficile is an enteric pathogen that causes a serious toxin-mediated colitis in humans. Bacterial exotoxins and sporulation are critical virulence components that contribute to pathogenesis, and disease transmission and relapse, respectively. Therefore, reducing toxin production and sporulation could significantly minimize C. difficile pathogenicity and disease outcome in affected individuals. Aim. This study investigated the efficacy of a natural flavone glycoside, baicalin, in reducing toxin synthesis, sporulation and spore germination in C. difficile in vitro. Methodology. Hypervirulent C. difficile isolates BAA 1870 or 1803 were cultured in brain heart infusion broth with or without the subinhibitory concentration (SIC) of baicalin, and incubated at 37 °C for 24 h under strictly anaerobic conditions. The supernatant was harvested after 24 h for determining C. difficile toxin production by ELISA. In addition, a similar experiment was performed wherein samples were harvested for assessing total viable counts, and heat-resistant spore counts at 72 h of incubation. Furthermore, C. difficile spore germination and spore outgrowth kinetics, with or without baicalin treatment, was measured in a plate reader by recording optical density at 600 nm. Finally, the effect of baicalin on C. difficile toxin, sporulation and virulence-associated genes was investigated using real-time quantitative PCR. Results. The SIC of baicalin significantly reduced toxin synthesis, sporulation and spore outgrowth when compared to control. In addition, C. difficile genes critical for pathogenesis were significantly down-regulated in the presence of baicalin. Conclusion. Our results suggest that baicalin could potentially be used to control C. difficile , and warrant future studies in vivo.

Download Full-text

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01031-19 ◽

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.

Download Full-text

An aniline-substituted bile salt analog protects both mice and hamsters from multiple Clostridioides difficile strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01435-21 ◽

2021 ◽

Author(s):

Jacqueline R. Phan ◽

Dung M. Do ◽

Minh Chau Truong ◽

Connie Ngo ◽

Julian H. Phan ◽

...

Keyword(s):

Bile Salt ◽

Spore Germination ◽

Dependent Manner ◽

Germination Inhibitors ◽

New Methods ◽

Clostridioides Difficile ◽

Antibiotic Associated Diarrhea ◽

Careful Screening ◽

Strain Dependent

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea. The emergence of hypervirulent C. difficile strains has led to increases in both hospital- and community-acquired CDI. Furthermore, CDI relapse from hypervirulent strains can reach up to 25%. Thus, standard treatments are rendered less effective, making new methods of prevention and treatment more critical. Previously, the bile salt analog CamSA was shown to inhibit spore germination in vitro and protect mice and hamsters from C. difficile strain 630. Here, we show that CamSA was less active at preventing spore germination of other C. difficile ribotypes, including the hypervirulent strain R20291. Strain-specific in vitro germination activity of CamSA correlated with its ability to prevent CDI in mice. Additional bile salt analogs were screened for in vitro germination inhibition activity against strain R20291, and the most active compounds were tested against other strains. An aniline-substituted bile salt analog, (CaPA), was found to be a better anti-germinant than CamSA against eight different C. difficile strains. In addition, CaPA was capable of reducing, delaying, or preventing murine CDI signs in all strains tested. CaPA-treated mice showed no obvious toxicity and showed minor effects on their gut microbiome. CaPA’s efficacy was further confirmed by its ability to prevent CDI in hamsters infected with strain 630. These data suggest that C. difficile spores respond to germination inhibitors in a strain-dependent manner. However, careful screening can identify anti-germinants with broad CDI prophylaxis activity.

Download Full-text

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

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa062 ◽

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 < 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.

Download Full-text

Optimization of an Assay To Determine Colonization Resistance to Clostridioides difficile in Fecal Samples from Healthy Subjects and Those Treated with Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01401-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01401-20

Author(s):

Hannah C. Harris ◽

Emma L. Best ◽

Charmaine Normington ◽

Nathalie Saint-Lu ◽

Frédérique Sablier-Gallis ◽

...

Keyword(s):

Healthy Subjects ◽

Toxin Production ◽

Antibiotic Exposure ◽

Anaerobic Incubation ◽

Colonization Resistance ◽

Fecal Samples ◽

Content Type ◽

Clostridioides Difficile ◽

Gastrointestinal Pathogens

ABSTRACTA healthy, intact gut microbiota is often resistant to colonization by gastrointestinal pathogens. During periods of dysbiosis, however, organisms such as Clostridioides difficile can thrive. We describe an optimized in vitro colonization resistance assay for C. difficile in stool (CRACS) and demonstrate the utility of this assay by assessing changes in colonization resistance following antibiotic exposure. Fecal samples were obtained from healthy volunteers (n = 6) and from healthy subjects receiving 5 days of moxifloxacin (n = 11) or no antibiotics (n = 10). Samples were separated and either not manipulated (raw) or sterilized (autoclaved or filtered) prior to inoculation with C. difficile ribotype 027 spores and anaerobic incubation for 72 h. Different methods of storing fecal samples were also investigated in order to optimize the CRACS. In healthy, raw fecal samples, incubation with spores did not lead to increased C. difficile total viable counts (TVCs) or cytotoxin detection. In contrast, increased C. difficile TVCs and cytotoxin detection occurred in sterilized healthy fecal samples or those from antibiotic-treated individuals. The CRACS was functional with fecal samples stored at either 4°C or −80°C but not with those stored with glycerol (12% or 30% [vol/vol]). Our data show that the CRACS successfully models in vitro the loss of colonization resistance and subsequent C. difficile proliferation and toxin production. The CRACS could be used as a proxy for C. difficile infection in clinical studies or to determine if an individual is at risk of developing C. difficile infection or other potential infections occurring due to a loss of colonization resistance.

Download Full-text

Repurposing the Antiamoebic Drug Diiodohydroxyquinoline for Treatment of Clostridioides difficile Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02115-19 ◽

2020 ◽

Vol 64 (6) ◽

Cited By ~ 1

Author(s):

Nader S. Abutaleb ◽

Mohamed N. Seleem

Keyword(s):

Toxin Production ◽

New Drugs ◽

Recurrence Rates ◽

Content Type ◽

Clostridioides Difficile ◽

Key Species ◽

Bacterial Inoculum ◽

Antiparasitic Drugs ◽

Time Kill

ABSTRACT Clostridioides difficile, the leading cause of nosocomial infections, is an urgent health threat worldwide. The increased incidence and severity of disease, the high recurrence rates, and the dearth of effective anticlostridial drugs have created an urgent need for new therapeutic agents. In an effort to discover new drugs for the treatment of Clostridioides difficile infections (CDIs), we investigated a panel of FDA-approved antiparasitic drugs against C. difficile and identified diiodohydroxyquinoline (DIHQ), an FDA-approved oral antiamoebic drug. DIHQ exhibited potent activity against 39 C. difficile isolates, inhibiting growth of 50% and 90% of these isolates at concentrations of 0.5 μg/ml and 2 μg/ml, respectively. In a time-kill assay, DIHQ was superior to vancomycin and metronidazole, reducing a high bacterial inoculum by 3 log10 within 6 h. Furthermore, DIHQ reacted synergistically with vancomycin and metronidazole against C. difficile in vitro. Moreover, at subinhibitory concentrations, DIHQ was superior to vancomycin and metronidazole in inhibiting two key virulence factors of C. difficile, toxin production and spore formation. Additionally, DIHQ did not inhibit the growth of key species that compose the host intestinal microbiota, such as Bacteroides, Bifidobacterium, and Lactobacillus spp. Collectively, our results indicate that DIHQ is a promising anticlostridial drug that warrants further investigation as a new therapeutic for CDIs.

Download Full-text

Effect ofCymbopogon martinii, Foeniculum vulgare, andTrachyspermum ammiEssential Oils on the Growth and Mycotoxins Production byAspergillusSpecies

International Journal of Food Science ◽

10.1155/2014/874135 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Negero Gemeda ◽

Yimtubezinash Woldeamanuel ◽

Daniel Asrat ◽

Asfaw Debella

Keyword(s):

Essential Oils ◽

Spore Germination ◽

Tween 80 ◽

Toxin Production ◽

Natural Food ◽

Foeniculum Vulgare ◽

Plant Materials ◽

Toxigenic Strains ◽

Mammalian Toxicity

This study was performed to investigate effect of essential oils onAspergillusspore germination, growth, and mycotoxin production.In vitroantifungal and antiaflatoxigenic activities ofCymbopogon martinii, Foeniculum vulgare,andTrachyspermum ammiessential oils were carried out on toxigenic strains ofAspergillusspecies. Plant materials were hydrodistilled for 4-5 h in Clevenger apparatus. 0.25 μL/mL, 0.5 μL/mL, 1 μL/mL, 2 μL/mL, and 4 μL/mL concentrations of each essential oil were prepared in 0.1% Tween 80 (V/V).T. ammioil showed highest antifungal activity. Absolute mycelial inhibition was recorded at 1 μL/mL by essential oils ofT. ammi. The oil also showed complete inhibition of spore germination at a concentration of 2 μL/mL. In addition,T. ammioil showed significant antiaflatoxigenic potency by totally inhibiting toxin production fromA. nigerandA. flavusat 0.5 and 0.75 μL/mL, respectively.C. martinii, F. vulgare,andT. ammioils as antifungals were found superior over synthetic preservative. Moreover, a concentration of 5336.297 μL/kg body weight was recorded for LC50 on mice indicating the low mammalian toxicity. In conclusion, the essential oils fromT. ammican be a potential source of safe natural food preservative for food commodities contamination byAspergillusspecies.

Download Full-text

Cationic Homopolymers Inhibit Spore and Vegetative Cell Growth of Clostridioides difficile

ACS Infectious Diseases ◽

10.1021/acsinfecdis.0c00843 ◽

2021 ◽

Author(s):

Joshua B. Jones ◽

Lei Liu ◽

Leslie A. Rank ◽

Daniela Wetzel ◽

Emily C. Woods ◽

...

Keyword(s):

Cell Growth ◽

Vegetative Cell ◽

Clostridioides Difficile

Download Full-text