Development and validation of a chemostat gut model to study both planktonic and biofilm modes of growth of Clostridium difficile and human microbiota

Copyright: 2014 Crowther et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.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.Peer reviewe

Short-term genome stability of serial Clostridium difficile ribotype 027 isolates in an experimental gut model and recurrent human disease

Copyright: © 2013 Eyre et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedClostridium 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.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).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.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.Peer reviewe

Association of Fidaxomicin with C. difficile spores: Effects of Persistence on Subsequent Spore Recovery, Outgrowth and Toxin Production.

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

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Evaluation of antimicrobial activity of ceftaroline against Clostridium difficile and propensity to induce C. difficile infection in an in vitro human gut model

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 ∼8-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.Peer reviewe

Recurrence of dual-strain Clostridium difficile infection in an in vitro human gut model

Objectives: 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 and antimicrobial activity was monitored throughout the experiment. Results: Both C. difficile strains germinated and proliferated in response to ceftriaxone instillation, with cytotoxin detection during peak vegetative growth. Vancomycin instillation resulted in a rapid decline in vegetative forms of both strains, with only spores remaining 2 days after dosing commencement. Recrudescence of both strains occurred following cessation of vancomycin, although this was observed in one strain sooner, and to a greater extent, than the other strain. Conclusions: Within a human gut model multiple C. difficile strains are able to germinate and proliferate concurrently in response to antibiotic challenge (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

Association of Fidaxomicin with <i>C</i>. <i>difficile</i> Spores: Effects of Persistence on Subsequent Spore Recovery, Outgrowth and Toxin Production - Fig 1

<p>Mean (±SE, n = 9) active antimicrobial concentration post-washing in (a) PBS and (b) FF for spores of 10 different ribotypes. Horizontal dashed line indicated limit of bioassay detection. Statistical significance was determined using two-sample t-test.</p

Growth of Control (non-antibiotic exposed washed spores), Vanc-exposed (vancomycin-exposed washed spores) and Fdx-exposed (Fidaxomicin-exposed washed spores) of strain RT078 following inoculation into BHI broth.

<p>Panel 1: Mean (n = 9) total viable counts (TVC, log₁₀ cfu/mL), spore counts (Spore, log₁₀ cfu/mL) and toxin titre (relative units) at tome points 0, 3, 6, 24 and 48 hours. Panel 2: Corresponding mean percentage vegetative cells (Av %V), phase dark spores (Av %PD), and phase bright spores (Av %PB) at time points 0, 3, 6 and 24 hours.</p