RESUMO
PURPOSE OF REVIEW: As a significant cause of global morbidity and mortality, Clostridioides difficile infections (CDIs) are listed by the Centres for Disease Control and prevention as one of the top 5 urgent threats in the USA. CDI occurs from gut microbiome dysbiosis, typically through antibiotic-mediated disruption; however, antibiotics are the treatment of choice, which can result in recurrent infections. Here, we highlight new treatments available and provide a perspective on different classes of future treatments. RECENT FINDINGS: Due to the reduced risk of disease recurrence, the microbiome-sparing antibiotic Fidaxomicin has been recommended as the first-line treatment for C. difficile infection. Based on the success of faecal microbiota transplantations (FMT) in treating CDI recurrence, defined microbiome biotherapeutics offer a safer and more tightly controlled alterative as an adjunct to antibiotic therapy. Given the association between antibiotic-mediated dysbiosis of the intestinal microbiota and the recurrence of CDI, future prospective therapies aim to reduce the dependence on antibiotics for the treatment of CDI. SUMMARY: With current first-in-line antibiotic therapy options associated with high levels of recurrent CDI, the availability of new generation targeted therapeutics can really impact treatment success. There are still unknowns about the long-term implications of these new CDI therapeutics, but efforts to expand the CDI treatment toolbox can offer multiple solutions for clinicians to treat this multifaceted infectious disease to reduce patient suffering.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Humanos , Disbiose/terapia , Antibacterianos/uso terapêutico , Infecções por Clostridium/tratamento farmacológico , Fidaxomicina/uso terapêutico , Transplante de Microbiota FecalRESUMO
A 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.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Antibacterianos/farmacologia , Clostridioides , Infecções por Clostridium/tratamento farmacológico , Voluntários Saudáveis , HumanosRESUMO
BACKGROUND: Clostridioides difficile infection (CDI) has a high recurrent infection rate. Faecal microbiota transplantation (FMT) has been used successfully to treat recurrent CDI, but much remains unknown about the human gut microbiota response to replacement therapies. In this study, antibiotic-mediated dysbiosis of gut microbiota and bacterial growth dynamics were investigated by two quantitative methods: real-time quantitative PCR (qPCR) and direct culture enumeration, in triple-stage chemostat models of the human colon. Three in vitro models were exposed to clindamycin to induce simulated CDI. All models were treated with vancomycin, and two received an FMT. Populations of total bacteria, Bacteroides spp., Lactobacillus spp., Enterococcus spp., Bifidobacterium spp., C. difficile, and Enterobacteriaceae were monitored using both methods. Total clostridia were monitored by selective culture. Using qPCR analysis, we additionally monitored populations of Prevotella spp., Clostridium coccoides group, and Clostridium leptum group. RESULTS: Both methods showed an exacerbation of disruption of the colonic microbiota following vancomycin (and earlier clindamycin) exposure, and a quicker recovery (within 4 days) of the bacterial populations in the models that received the FMT. C. difficile proliferation, consistent with CDI, was also observed by both qPCR and culture. Pearson correlation coefficient showed an association between results varying from 98% for Bacteroides spp., to 62% for Enterobacteriaceae. CONCLUSIONS: Generally, a good correlation was observed between qPCR and bacterial culture. Overall, the molecular assays offer results in real-time, important for treatment efficacy, and allow the monitoring of additional microbiota groups. However, individual quantification of some genera (e.g. clostridia) might not be possible without selective culture.
Assuntos
Antibacterianos/efeitos adversos , Bactérias/classificação , Técnicas Bacteriológicas/métodos , Clostridioides difficile/patogenicidade , Infecções por Clostridium/terapia , Transplante de Microbiota Fecal/efeitos adversos , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Clindamicina/efeitos adversos , Infecções por Clostridium/induzido quimicamente , Colo/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Viabilidade Microbiana , Modelos Biológicos , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Vancomicina/efeitos adversosRESUMO
A clinically reflective model of the human colon was used to investigate the effects of the broad-spectrum antibiotic omadacycline on the gut microbiome and the subsequent potential to induce simulated Clostridium difficile infection (CDI). Triple-stage chemostat gut models were inoculated with pooled human fecal slurry from healthy volunteers (age, ≥60 years). Models were challenged twice with 107 CFU C. difficile spores (PCR ribotype 027). Omadacycline effects were assessed in a single gut model. Observations were confirmed in a parallel study with omadacycline and moxifloxacin. Antibiotic instillation was performed once daily for 7 days. The models were observed for 3 weeks postantibiotic challenge. Gut microbiota populations and C. difficile total viable and spore counts were enumerated daily by culture. Cytotoxin titers and antibiotic concentrations were also measured. Gut microbiota populations were stable before antibiotic challenge. Moxifloxacin instillation caused an â¼4 log10 CFU/ml decline in enterococci and Bacteroides fragilis group populations and an â¼3 log10 CFU/ml decline in bifidobacteria and lactobacilli, followed by simulated CDI (vegetative cell proliferation and detectable toxin). In both models, omadacycline instillation decreased populations of bifidobacteria (â¼8 log10 CFU/ml), B. fragilis group populations (7 to 8 log10 CFU/ml), lactobacilli (2 to 6 log10 CFU/ml), and enterococci (4 to 6 log10 CFU/ml). Despite these microbial shifts, there was no evidence of C. difficile bacteria germination or toxin production. In contrast to moxifloxacin, omadacycline exposure did not facilitate simulated CDI, suggesting this antibiotic may have a low propensity to induce CDI in the clinical setting.
Assuntos
Clostridioides difficile/efeitos dos fármacos , Colo/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Tetraciclinas/farmacologia , Bacteroides fragilis/efeitos dos fármacos , Bacteroides fragilis/patogenicidade , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidade , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Enterococcus/efeitos dos fármacos , Enterococcus/patogenicidade , Humanos , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Moxifloxacina/uso terapêutico , Tetraciclinas/uso terapêuticoRESUMO
OBJECTIVES: Effects of two vancomycin extended-dosing regimens on microbiota populations within an in vitro gut model of simulated Clostridium difficile infection (CDI) were evaluated. METHODS: Two chemostat gut models were inoculated with faecal emulsion and clindamycin instilled to induce CDI. Simulated CDI was treated with vancomycin (125 mg/L four times daily, 7 days) followed by different vancomycin dosing extensions totalling 7 g (lower dose) or 9.5 g (higher dose) over 6 weeks in Model A and Model B, respectively. Microbiota populations, C. difficile vegetative cells and spores, cytotoxin, antimicrobial concentrations and vancomycin-tolerant enterococci (VTE) were measured every 1-2 days. RESULTS: In both models, vancomycin instillation caused a rapid decline in vegetative cells and cytotoxin, and declines in the Bacteroides fragilis group, bifidobacteria and clostridia populations to the lower limit of detection. Bifidobacteria failed to recover for the remainder of the experiment. B. fragilis group populations recovered to pre-dosing levels during the dosing extension in Model A and after dosing ceased in Model B. Recurrent CDI was observed on the penultimate day of Model B, but not Model A. VTE were observed throughout the experiment in both models, but populations increased during vancomycin instillation and post-vancomycin instillation. CONCLUSIONS: The two vancomycin extended-dosing regimens were efficacious in initial treatment of simulated CDI. Both had a prolonged deleterious effect on the indigenous gut microbiota, a factor that may contribute to recurrence; recurrence was observed only in Model B, although the potential for vegetative regrowth within Model A cannot be excluded. Vancomycin exposure appeared to select for VTE populations.
Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Clostridioides difficile , Enterocolite Pseudomembranosa/tratamento farmacológico , Trato Gastrointestinal/microbiologia , Vancomicina/administração & dosagem , Vancomicina/uso terapêutico , Carga Bacteriana , Bacteroides fragilis/efeitos dos fármacos , Farmacorresistência Bacteriana , Enterocolite Pseudomembranosa/microbiologia , Fezes/microbiologia , Humanos , Microbiota , Modelos Biológicos , RecidivaRESUMO
BACKGROUND: Clostridium difficile infection (CDI) is still a major challenge to healthcare facilities. The detection of multiple C. difficile strains has been reported in some patient samples during initial and recurrent CDI episodes. However, the behaviour of individual strains and their contribution to symptomatic disease is unclear. METHODS: An in vitro human gut model was used to investigate the germination and proliferation of two distinct C. difficile strains during initial and recurrent simulated CDI, as well as their response to vancomycin treatment. The gut model was inoculated with a pooled human faecal emulsion and indigenous gut microbiota, C. difficile populations (vegetative and spore forms), cytotoxin levels and antimicrobial activity were monitored throughout the experiment. RESULTS: Both C. difficile strains germinated and proliferated in response to ceftriaxone instillation, with cytotoxin detected during the peak vegetative growth. Vancomycin instillation resulted in a rapid decline in the vegetative forms of both strains, with only spores remaining 2 days after the start of dosing. A recrudescence of both strains occurred following the cessation of vancomycin installation, although this was observed more quickly, and to a greater extent, in one strain than the other. CONCLUSIONS: Within a human gut model, multiple C. difficile strains are able to germinate and proliferate concurrently in response to antibiotic challenge (the onset of simulated CDI). Similarly, more than one strain can proliferate during simulated recurrent CDI, although with differences in germination and growth rate and timing. It appears probable that multiple strains can contribute to CDI within an individual patient, with possible implications for management and bacterial transmission.
Assuntos
Antibacterianos/administração & dosagem , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/microbiologia , Trato Gastrointestinal/microbiologia , Vancomicina/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Clostridioides difficile/classificação , Humanos , Pessoa de Meia-Idade , Modelos Teóricos , Recidiva , Resultado do TratamentoRESUMO
BACKGROUND: Biofilms are characteristic of some chronic or recurrent infections and this mode of growth tends to reduce treatment efficacy. Clostridium difficile infection (CDI) is associated with a high rate of recurrent symptomatic disease. The presence and behaviour of C. difficile within intestinal biofilms remains largely unexplored, but may factor in recurrent infection. METHODS: A triple-stage chemostat gut model designed to facilitate the formation of intestinal biofilm was inoculated with a pooled human faecal emulsion. Bacterial populations were allowed to equilibrate before simulated CDI was induced by clindamycin (33.9 mg/L, four times daily, 7 days) and subsequently treated with vancomycin (125 mg/L, four times daily, 7 days). Indigenous gut microbiota, C. difficile total viable counts, spores, cytotoxin and antimicrobial activity in planktonic and biofilm communities were monitored during the 10 week experimental period. RESULTS: Vancomycin successfully treated the initial episode of simulated CDI, but â¼18 days after therapy cessation, recurrent infection occurred. Germination, proliferation and toxin production were evident within planktonic communities in both initial and recurrent CDI. In contrast, sessile C. difficile remained in dormant spore form for the duration of the experiment. The effects of and recovery from clindamycin and vancomycin exposure for sessile populations was delayed compared with responses for planktonic bacteria. CONCLUSIONS: Intestinal biofilms provide a potential reservoir for C. difficile spore persistence, possibly facilitating their dispersal into the gut lumen after therapeutic intervention, leading to recurrent infection. Therapeutic options for CDI could have increased efficacy if they are more effective against sessile C. difficile.
Assuntos
Biofilmes/crescimento & desenvolvimento , Clostridioides difficile/crescimento & desenvolvimento , Intestinos/microbiologia , Plâncton/crescimento & desenvolvimento , Antibacterianos/farmacologia , Clindamicina/farmacologia , Fezes/microbiologia , Microbiota/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimento , Vancomicina/farmacologiaRESUMO
OBJECTIVES: First-line treatment options for Clostridium difficile infection (CDI) are limited. NVB302 is a novel type B lantibiotic under evaluation for the treatment of CDI. We compared the responses to NVB302 and vancomycin when used to treat simulated CDI in an in vitro gut model. METHODS: We used ceftriaxone to elicit simulated CDI in an in vitro gut model primed with human faeces. Vancomycin and NVB302 were instilled into separate gut models and the indigenous gut microbiota and C. difficile total viable counts, spores and toxin levels were monitored throughout. RESULTS: Ceftriaxone instillation promoted C. difficile germination and high-level toxin production. Commencement of NVB302 and vancomycin instillation reduced C. difficile total viable counts rapidly with only C. difficile spores remaining within 3 and 4 days, respectively. Cytotoxin was reduced to undetectable levels 5 and 7 days after vancomycin and NVB302 instillation commenced in vessel 2 and 3, respectively, and remained undetectable for the remainder of the experiments. C. difficile spores were unaffected by the presence of vancomycin or NVB302. NVB302 treatment was associated with faster resolution of Bacteroides fragilis group. CONCLUSIONS: Both NVB302 and vancomycin were effective in treating simulated CDI in an in vitro gut model. C. difficile spore recrudescence was not observed following successful treatment with either NVB302 or vancomycin. NVB302 displayed non-inferiority to vancomycin in the treatment of simulated CDI, and had less deleterious effects against B. fragilis group. NVB302 warrants further clinical investigation as a potentially novel antimicrobial agent for the treatment of CDI.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Clostridioides difficile/efeitos dos fármacos , Infecções por Clostridium/tratamento farmacológico , Trato Gastrointestinal/efeitos dos fármacos , Vancomicina/farmacologia , Idoso , Antibacterianos/uso terapêutico , Bacteriocinas/uso terapêutico , Infecções por Clostridium/fisiopatologia , Avaliação Pré-Clínica de Medicamentos , Fezes/microbiologia , Trato Gastrointestinal/microbiologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Vancomicina/uso terapêuticoRESUMO
OBJECTIVES: To examine the effects of exposure to ceftaroline or ceftriaxone on the epidemic Clostridium difficile strain PCR ribotype 027 and the indigenous gut microflora in an in vitro human gut model. Additionally, the MICs of ceftriaxone and ceftaroline for 60 C. difficile isolates were determined. METHODS: Two triple-stage chemostat gut models were primed with human faeces and exposed to ceftaroline (10 mg/L, twice daily, 7 days) or ceftriaxone (150 mg/L, once daily, 7 days). Populations of indigenous gut microorganisms, C. difficile total viable counts, spore counts, cytotoxin titres and antimicrobial concentrations were monitored throughout. MICs were determined by a standard agar incorporation method. RESULTS: In the gut model, both ceftaroline and ceftriaxone induced C. difficile spore germination, proliferation and toxin production, although germination occurred 5 days later in the ceftaroline-exposed model. Toxin detection was sustained until the end of the experimental period in both models. No active antimicrobial was detected in vessel 3 of either model, although inhibitory effects on microflora populations were observed. Ceftaroline was â¼8-fold more active against C. difficile than ceftriaxone (geometric mean MICs, 3.38 versus 28.18 mg/L; MIC90s, 4 versus 64 mg/L; and MIC ranges, 0.125-16 versus 8-128 mg/L). CONCLUSIONS: Ceftaroline, like ceftriaxone, can induce simulated C. difficile infection in a human gut model. However, low in vivo gut concentrations of ceftaroline and increased activity against C. difficile in comparison with ceftriaxone mean that the true propensity of this novel cephalosporin to induce C. difficile infection remains unclear.
Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/crescimento & desenvolvimento , Infecções por Clostridium/microbiologia , Trato Gastrointestinal/microbiologia , Idoso , Idoso de 80 Anos ou mais , Carga Bacteriana , Toxinas Bacterianas/análise , Ceftriaxona/farmacologia , Clostridioides difficile/isolamento & purificação , Voluntários Saudáveis , Atividades Humanas , Humanos , Testes de Sensibilidade Microbiana , Modelos Teóricos , CeftarolinaRESUMO
OBJECTIVES: Clostridium difficile infection (CDI) is still a major clinical challenge. Previous studies have demonstrated multiple distinct C. difficile strains in the faeces of patients with CDI; yet whether true mixed CDI occurs in vivo is unclear. In this study we evaluated whether two distinct C. difficile strains could co-germinate and co-proliferate in an in vitro human gut model. METHODS: An in vitro triple-stage chemostat was used to study the responses of two PCR ribotype 001 C. difficile strains following exposure to ceftriaxone at concentrations observed in vivo (7 days). C. difficile viable counts (vegetative and spore forms), cytotoxin titres and indigenous microflora viable counts were monitored throughout the experiment. RESULTS: Both C. difficile strains germinated and proliferated following exposure to ceftriaxone. Cytotoxin production was detected in the gut model following C. difficile spore germination and proliferation. Ceftriaxone elicited reduced viable counts of Bifidobacterium spp. and elevated viable counts of Enterococcus spp. CONCLUSIONS: These data suggest that multiple C. difficile strains are able to proliferate concurrently in an in vitro model reflective of the human colon. Previous studies in the gut model have reflected clinical observations so clinicians should be mindful of the possibility that multiple C. difficile strains may infect patients. These observations augment recent human epidemiological studies in this area.
Assuntos
Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/crescimento & desenvolvimento , Trato Gastrointestinal/microbiologia , Antibacterianos/metabolismo , Carga Bacteriana , Bifidobacterium/efeitos dos fármacos , Ceftriaxona/metabolismo , Enterococcus/efeitos dos fármacos , Humanos , Viabilidade Microbiana , Modelos Teóricos , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimentoRESUMO
OBJECTIVES: Co-amoxiclav is widely prescribed in hospitals. Although reports have suggested it may be linked to onset of Clostridium difficile infection (CDI), data on the risk of CDI associated with specific antibiotics is difficult to obtain, due to confounding clinical factors. We have examined the propensity of co-amoxiclav to induce CDI using a human gut model. METHODS: We used a triple-stage chemostat human gut model to study the effects of co-amoxiclav on indigenous gut microorganisms and C. difficile PCR ribotype 027. C. difficile viable counts and spores were evaluated, and cytotoxin titres were assayed. Co-amoxiclav concentrations were measured using a large plate bioassay. RESULTS: Co-amoxiclav induced rapid C. difficile germination and high toxin production in the gut model, from 5 days after commencement of instillation. Cell proliferation and toxin production were prolonged and continued throughout the duration of the experiment. Only very low levels of co-amoxiclav antimicrobial activity could be detected within the gut model, despite having a marked effect on gut flora microorganisms. CONCLUSIONS: Co-amoxiclav induced CDI within the gut model, supporting clinical observations linking co-amoxiclav treatment with CDI onset. This reinforces the value of the gut model as a clinically relevant means of studying CDI. Caution should be exercised in the prescription of co-amoxiclav to patients in high CDI risk settings.
Assuntos
Combinação Amoxicilina e Clavulanato de Potássio/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/biossíntese , Toxinas Bacterianas/biossíntese , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/crescimento & desenvolvimento , Trato Gastrointestinal/microbiologia , Idoso , Carga Bacteriana , Clostridioides difficile/classificação , Clostridioides difficile/genética , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Modelos Teóricos , Ribotipagem , Virulência/efeitos dos fármacosRESUMO
OBJECTIVES: To evaluate the relative propensities of oritavancin and vancomycin to induce Clostridium difficile infection (CDI) in hamster and in vitro human gut models. METHODS: Hamsters received clindamycin (100 mg/kg orally or subcutaneously), oritavancin (50 mg/kg orally) or vancomycin (50 mg/kg orally). C. difficile spores were administered orally the next day. Control hamsters received vehicle only (polyethylene glycol 400) plus spores or clindamycin but no spores. Hamsters were monitored for clinical signs for 20 days. Caecal contents were analysed for C. difficile cells, spores and the presence of (cyto)toxin. Oritavancin and vancomycin were instilled over 7 days into separate in vitro gut models primed with pooled human faeces and inoculated with C. difficile ribotype 027 spores. Gut flora, C. difficile total viable and spore counts, toxin titres and antimicrobial concentrations were determined. RESULTS: All hamsters treated with oritavancin survived up to 20 days, with no evidence of C. difficile spores, vegetative cells or toxin in their caeca. No hamsters treated with clindamycin or vancomycin survived >6 days after spore administration. Death was associated with high C. difficile counts and toxin in caecal contents. In the gut model, oritavancin dosing elicited a rapid, marked decrease in total viable C. difficile and spore counts to below the limit of detection. Vancomycin did not elicit germination or toxin production in the gut model, but C. difficile remained present as spores throughout. CONCLUSIONS: Oritavancin exposure, unlike exposure to vancomycin or clindamycin, did not lead to CDI in hamsters. In both models, oritavancin reduced C. difficile total counts and spores to below detectable limits. The data indicate the potential of oritavancin for CDI treatment, since exposure did not induce C. difficile germination and toxin production, which are known to exacerbate the disease state.
Assuntos
Antibacterianos/metabolismo , Toxinas Bacterianas/biossíntese , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/crescimento & desenvolvimento , Glicopeptídeos/metabolismo , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimento , Idoso , Idoso de 80 Anos ou mais , Animais , Carga Bacteriana , Ceco/química , Ceco/microbiologia , Infecções por Clostridium/microbiologia , Infecções por Clostridium/patologia , Cricetinae , Modelos Animais de Doenças , Fezes/química , Fezes/microbiologia , Humanos , Lipoglicopeptídeos , Vancomicina/metabolismoRESUMO
The in vitro gut model is an invaluable research tool to study indigenous gut microbiota communities, the behavior of pathogenic organisms, and the therapeutic and adverse effect of antimicrobial administration on these communities. The model has been validated against the intestinal contents of sudden death victims to reflect the physicochemical and microbiological conditions of the proximal to distal colon, and has been extensively used to investigate the interplay between gut microbiota populations, antibiotic exposure, and Clostridium difficile infection. More recently the gut model has been adapted to additionally model intestinal biofilm. Here we describe the structure, assembly, and application of the biofilm gut model.
Assuntos
Biofilmes/efeitos dos fármacos , Clostridioides difficile/efeitos dos fármacos , Colo/microbiologia , Microbioma Gastrointestinal/fisiologia , Modelos Biológicos , Anaerobiose , Antibacterianos/farmacologia , Bacillus/efeitos dos fármacos , Bacillus/crescimento & desenvolvimento , Bifidobacterium/efeitos dos fármacos , Bifidobacterium/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Reatores Biológicos , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/patogenicidade , Infecções por Clostridium/microbiologia , Infecções por Clostridium/mortalidade , Infecções por Clostridium/patologia , Colo/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , Morte Súbita , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/crescimento & desenvolvimento , Fezes/microbiologia , Fermentação , Humanos , Lactobacillales/efeitos dos fármacos , Lactobacillales/crescimento & desenvolvimentoRESUMO
BACKGROUND: We have previously shown that fidaxomicin instillation prevents spore recovery in an in-vitro gut model, whereas vancomycin does not. The reasons for this are unclear. Here, we have investigated persistence of fidaxomicin and vancomycin on C. difficile spores, and examined post-antibiotic exposure spore recovery, outgrowth and toxin production. METHODS: Prevalent UK C. difficile ribotypes (n = 10) were incubated with 200mg/L fidaxomicin, vancomycin or a non-antimicrobial containing control for 1 h in faecal filtrate or Phosphate Buffered Saline. Spores were washed three times with faecal filtrate or phosphate buffered saline, and residual spore-associated antimicrobial activity was determined by bioassay. For three ribotypes (027, 078, 015), antimicrobial-exposed, faecal filtrate-washed spores and controls were inoculated into broth. Viable vegetative and spore counts were enumerated on CCEYL agar. Percentage phase bright spores, phase dark spores and vegetative cells were enumerated by phase contrast microscopy at 0, 3, 6, 24 and 48 h post-inoculation. Toxin levels (24 and 48h) were determined by cell cytotoxicity assay. RESULTS: Fidaxomicin, but not vancomycin persisted on spores of all ribotypes following washing in saline (mean = 10.1mg/L; range = 4.0-14mg/L) and faecal filtrate (mean = 17.4mg/L; 8.4-22.1mg/L). Outgrowth and proliferation rates of vancomycin-exposed spores were similar to controls, whereas fidaxomicin-exposed spores showed no vegetative cell growth after 24 and 48 h. At 48h, toxin levels averaged 3.7 and 3.3 relative units (RU) in control and vancomycin-exposed samples, respectively, but were undetectable in fidaxomicin-exposed samples. CONCLUSION: Fidaxomicin persists on C. difficile spores, whereas vancomycin does not. This persistence prevents subsequent growth and toxin production in vitro. This may have implications on spore viability, thereby impacting CDI recurrence and transmission rates.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Toxinas Bacterianas/biossíntese , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/fisiologia , Infecções por Clostridium/microbiologia , Esporos Bacterianos/efeitos dos fármacos , Clostridioides difficile/isolamento & purificação , Fezes/microbiologia , Fidaxomicina , Humanos , Testes de Sensibilidade MicrobianaRESUMO
In vivo and in vitro models are widely used to simulate Clostridium difficile infection (CDI). They have made considerable contributions in the study of C difficile pathogenesis, antibiotic predisposition to CDI, and population dynamics as well as the evaluation of new antimicrobial and immunologic therapeutics. Although CDI models have greatly increased understanding of this complicated pathogen, all have limitations in reproducing human disease, notably their inability to generate a truly reflective immune response. This review summarizes the most commonly used models of CDI and discusses their pros and cons and their predictive values in terms of clinical outcomes.
Assuntos
Antibacterianos/uso terapêutico , Clostridioides difficile , Infecções por Clostridium/tratamento farmacológico , Enteropatias/tratamento farmacológico , Animais , Ensaios Clínicos como Assunto , Infecções por Clostridium/microbiologia , Modelos Animais de Doenças , Trato Gastrointestinal/microbiologia , Humanos , Enteropatias/microbiologia , Valor Preditivo dos TestesRESUMO
The human gastrointestinal tract harbours a complex microbial community which exist in planktonic and sessile form. The degree to which composition and function of faecal and mucosal microbiota differ remains unclear. We describe the development and characterisation of an in vitro human gut model, which can be used to facilitate the formation and longitudinal analysis of mature mixed species biofilms. This enables the investigation of the role of biofilms in Clostridium difficile infection (CDI). A well established and validated human gut model of simulated CDI was adapted to incorporate glass rods that create a solid-gaseous-liquid interface for biofilm formation. The continuous chemostat model was inoculated with a pooled human faecal emulsion and controlled to mimic colonic conditions in vivo. Planktonic and sessile bacterial populations were enumerated for up to 46 days. Biofilm consistently formed macroscopic structures on all glass rods over extended periods of time, providing a framework to sample and analyse biofilm structures independently. Whilst variation in biofilm biomass is evident between rods, populations of sessile bacterial groups (log10 cfu/g of biofilm) remain relatively consistent between rods at each sampling point. All bacterial groups enumerated within the planktonic communities were also present within biofilm structures. The planktonic mode of growth of C. difficile and gut microbiota closely reflected observations within the original gut model. However, distinct differences were observed in the behaviour of sessile and planktonic C. difficile populations, with C. difficile spores preferentially persisting within biofilm structures. The redesigned biofilm chemostat model has been validated for reproducible and consistent formation of mixed species intestinal biofilms. This model can be utilised for the analysis of sessile mixed species communities longitudinally, potentially providing information of the role of biofilms in CDI.
Assuntos
Biofilmes/crescimento & desenvolvimento , Clostridioides difficile/crescimento & desenvolvimento , Trato Gastrointestinal/microbiologia , Microbiota , Modelos Biológicos , Plâncton/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/isolamento & purificação , Meios de Cultura/farmacologia , Trato Gastrointestinal/efeitos dos fármacos , Humanos , Microbiota/efeitos dos fármacos , Plâncton/efeitos dos fármacos , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Clostridium difficile whole genome sequencing has the potential to identify related isolates, even among otherwise indistinguishable strains, but interpretation depends on understanding genomic variation within isolates and individuals. METHODS: Serial isolates from two scenarios were whole genome sequenced. Firstly, 62 isolates from 29 timepoints from three in vitro gut models, inoculated with a NAP1/027 strain. Secondly, 122 isolates from 44 patients (2-8 samples/patient) with mostly recurrent/on-going symptomatic NAP-1/027 C. difficile infection. Reference-based mapping was used to identify single nucleotide variants (SNVs). RESULTS: Across three gut model inductions, two with antibiotic treatment, total 137 days, only two new SNVs became established. Pre-existing minority SNVs became dominant in two models. Several SNVs were detected, only present in the minority of colonies at one/two timepoints. The median (inter-quartile range) [range] time between patients' first and last samples was 60 (29.5-118.5) [0-561] days. Within-patient C. difficile evolution was 0.45 SNVs/called genome/year (95%CI 0.00-1.28) and within-host diversity was 0.28 SNVs/called genome (0.05-0.53). 26/28 gut model and patient SNVs were non-synonymous, affecting a range of gene targets. CONCLUSIONS: The consistency of whole genome sequencing data from gut model C. difficile isolates, and the high stability of genomic sequences in isolates from patients, supports the use of whole genome sequencing in detailed transmission investigations.