On the spectroastrometric separation of binary point-source fluxes

Spectroastrometry is a technique which has the potential to resolve flux distributions on scales of milliarcseconds. In this study, we examine the application of spectroastrometry to binary point sources which are spatially unresolved due to the observational point spread function convolution. The technique uses measurements with sub-pixel accuracy of the position centroid of high signal-to-noise long-slit spectrum observations. With the objects in the binary contributing fractionally more or less at different wavelengths (particularly across spectral lines), the variation of the position centroid with wavelength provides some information on the spatial distribution of the flux. We examine the width of the flux distribution in the spatial direction, and present its relation to the ratio of the fluxes of the two components of the binary. Measurement of three observables (total flux, position centroid and flux distribution width) at each wavelength allows a unique separation of the total flux into its component parts even though the angular separation of the binary is smaller than the observations' point-spread function. This is because we have three relevant observables for three unknowns (the two fluxes, and the angular separation of the binary), which therefore generates a closed problem. This is a wholly different technique than conventional deconvolution methods, which produce information on angular sizes of the sampling scale. Spectroastrometry can produce information on smaller scales than conventional deconvolution, and is successful in separating fluxes in a binary object with a separation of less than one pixel. We present an analysis of the errors involved in making binary object spectroastrometric measurements and the separation method, and highlight necessary observing methodology.Comment: 11 pages, 8 figures, accepted for publication in Astronomy and Astrophysic

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

In vitro assessment of Clostridium difficile PCR ribotype 002: the most prevalent C. difficile ribotype in the United Kingdom.

Simon Baines, Iye Ameh, Jane Freeman, W.N. Fawley, M.H. Wilcox, ‘In vitro assessment of Clostridium difficile PCR ribotype 002: the most prevalent C. difficile ribotype in the United Kingdom’, poster presented at the 25th European Congress on Clinical Microbiology and Infectious Diseases, Copenhagen, Denmark, 25-28 August, 2015.Background: Clostridium difficile infection (CDI) causes substantial morbidity and healthcare expenditure across Europe. UK prevalence of C. difficile PCR ribotype 027 (NAP1) has declined dramatically recently and other ribotypes have emerged, including ribotype 002 (CD002); now the most prevalent UK ribotype. CD002 is also responsible for CDI in many countries across Europe, including: France, Germany, Ireland, and The Netherlands. We assessed the in vitro phenotypic characteristics of CD002 from across Europe to determine traits that may contribute to its increasing clinical prevalence. Material/methods: Sixty CD002 were studied: UK isolates from 2007-2008 (geographically distinct, N=15), UK isolates from 2011-2013 (19 locations, N=22), and non-UK European isolates from 2012-2014 (N=23, 20 locations). Antimicrobial susceptibilities (13 antimicrobials) were evaluated using an agar incorporation method. Maximum specific growth rates (μmax) were calculated and cytotoxin titres (log10-relative units, RU) determined using Vero cell cytotoxicity assays. Biofilm formation was quantified using 96-well microtitre plate assays and sporulation capacities assessed in liquid culture by quantifying spore-formation over 120 h (CFU/mL). Results: All isolates were susceptible metronidazole, vancomycin, tetracycline and linezolid (MICs ≤2 mg/L). Clindamycin resistance (MIC ≥8 mg/L) was more common in non-UK CD002 (30%) than UK strains (5-13%). Resistance to erythromycin, clarithromycin, nitrofurantoin, chloramphenicol, and moxifloxacin was uncommon (5-7%). MICs for penicillin’s remained below resistance breakpoints, regardless of origin, in all but one isolate (ampicillin MIC 2 mg/L). All CD002 were resistant to trimethoprim (MICs >128 mg/L) and ciprofloxacin (MICs ≥8 mg/L). One MDR strain (UK, 2007) was observed that was macrolide, fluoroquinolone, ampicillin, and nitrofurantoin resistant. Significantly faster μmax was seen in non-UK CD002 (0.92 ±0.058 h-1) than recent/older UK strains (0.76 ±0.063/0.69 ±0.028 h-1 respectively) (P<0.001). Cytotoxin production did not differ significantly (median titres 2-3 RU) between CD002 groups. Recent UK/non-UK CD002 formed significantly greater biofilms by 3 days than asynchronous UK CD002 (P<0.001). Sporulation studies demonstrated that recent UK/non-UK CD002 sporulated more at 24 h than older UK CD002; 18.6-fold/31.2-fold respectively (P<0.05), but by 120 h sporulation did not differ. Conclusions: Recent CD002 from diverse European locations were assessed for traits that may help to explain emergence of CD002 in the UK and compared to asynchronous CD002. Previous studies demonstrated elevated CD002 μmax compared to hypervirulent ribotypes 027/078; and the present study demonstrated that recent non-UK CD002 μmax were significantly further elevated vs. UK isolates. Non-UK CD002 were more clindamycin resistant, but other antimicrobial susceptibilities were similar between CD002 groups. Recent CD002 demonstrated significantly increased sporulation capacities at 24 h and more extensive 3 day biofilm formation compared to asynchronous UK CD002, which could enhance their survival and transmission early in an episode CDI. Further phenotypic and genetic studies are required to evaluate further characteristics of CD002 that may be associated with its emergence in the UK.Peer reviewedFinal Published versio

Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria

Clostridium difficile is a Gram-positive spore-forming anaerobe and a major cause of antibiotic-associated diarrhoea. Disruption of the commensal microbiota, such as through treatment with broad-spectrum antibiotics, is a critical precursor for colonisation by C. difficile and subsequent disease. Furthermore, failure of the gut microbiota to recover colonisation resistance can result in recurrence of infection. An unusual characteristic of C. difficile among gut bacteria is its ability to produce the bacteriostatic compound para-cresol (p-cresol) through fermentation of tyrosine. Here, we demonstrate that the ability of C. difficile to produce p-cresol in vitro provides a competitive advantage over gut bacteria including Escherichia coli, Klebsiella oxytoca and Bacteroides thetaiotaomicron. Metabolic profiling of competitive co-cultures revealed that acetate, alanine, butyrate, isobutyrate, p-cresol and p-hydroxyphenylacetate were the main metabolites responsible for differentiating the parent strain C. difficile (630Δerm) from a defined mutant deficient in p-cresol production. Moreover, we show that the p-cresol mutant displays a fitness defect in a mouse relapse model of C. difficile infection (CDI). Analysis of the microbiome from this mouse model of CDI demonstrates that colonisation by the p-cresol mutant results in a distinctly altered intestinal microbiota, and metabolic profile, with a greater representation of Gammaproteobacteria, including the Pseudomonales and Enterobacteriales. We demonstrate that Gammaproteobacteria are susceptible to exogenous p-cresol in vitro and that there is a clear divide between bacterial Phyla and their susceptibility to p-cresol. In general, Gram-negative species were relatively sensitive to p-cresol, whereas Gram-positive species were more tolerant. This study demonstrates that production of p-cresol by C. difficile has an effect on the viability of intestinal bacteria as well as the major metabolites produced in vitro. These observations are upheld in a mouse model of CDI, in which p-cresol production affects the biodiversity of gut microbiota and faecal metabolite profiles, suggesting that p-cresol production contributes to C. difficile survival and pathogenesis.Peer reviewedFinal Published versio

How to Weigh a Star Using a Moon

We show that for a transiting exoplanet accompanied by a moon which also transits, the absolute masses and radii of the star, planet and moon are determinable. For a planet-star system, it is well known that the density of the star is calculable from the lightcurve by manipulation of Kepler's Third Law. In an analogous way, the planetary density is calculable for a planet-moon system which transits a star, and thus the ratio-of-densities is known. By combining this ratio with the observed ratio-of-radii and the radial velocity measurements of the system, we show that the absolute dimensions of the star and planet are determinable. This means such systems could be used as calibrators of stellar evolution. The detection of dynamical effects, such as transit timing variations, allows the absolute mass of the moon to be determined as well, which may be combined with the radius to infer the satellite's composition.Comment: 5 pages; Accepted in MNRA

High sporulation and overexpression of virulence factors in biofilms and reduced susceptibility to vancomycin and linezolid in recurrent Clostridium [Clostridioides] difficile infection isolates

Clostridium [Clostridioides] difficile infection (CDI) is one of the leading causes of diarrhea associated with medical care worldwide, and up to 60% of patients with CDI can develop a recurrent infection (R-CDI). A multi-species microbiota biofilm model of C. difficile was designed to evaluate the differences in the production of biofilms, sporulation, susceptibility to drugs, expression of sporulating (sigH, spo0A), quorum sensing (agrD1, and luxS), and adhesion-associated (slpA and cwp84) pathway genes between selected C. difficile isolates from R-CDI and non-recurrent patients (NR-CDI). We obtained 102 C. difficile isolates from 254 patients with confirmed CDI (66 from NR-CDI and 36 from R-CDI). Most of the isolates were biofilm producers, and most of the strains were ribotype 027 (81.374%, 83/102). Most C. difficile isolates were producers of biofilm (100/102), and most were strongly adherent. Sporulation was higher in the R-CDI than in the NR-CDI isolates (p = 0.015). The isolates from R-CDI patients more frequently demonstrated reduced susceptibility to vancomycin than isolates of NR-CDI patients (27.78% [10/36] and 9.09% [6/66], respectively, p = 0.013). The minimum inhibitory concentrations for vancomycin and linezolid against biofilms (BMIC) were up to 100 times and 20 times higher, respectively, than the corresponding planktonic MICs. Expression of sigH, spo0A, cwp84, and agrD1 was higher in R-CDI than in NR-CDI isolates. Most of the C. difficile isolates were producers of biofilms with no correlation with the ribotype. Sporulation was greater in R-CDI than in NR-CDI isolates in the biofilm model of C. difficile. The R-CDI isolates more frequently demonstrated reduced susceptibility to vancomycin and linezolid than the NR-CDI isolates in both planktonic cells and biofilm isolates. A higher expression of sporulating pathway (sigH, spo0A), quorum sensing (agrD1), and adhesion-associated (cwp84) genes was found in R-CDI than in NR-CDI isolates. All of these factors can have effect on the recurrence of the infection.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

Circulation of Highly Drug-Resistant Clostridium difficile Ribotypes 027 and 001 in Two Tertiary-Care Hospitals in Mexico

© 2018, Mary Ann Liebert, Inc.OBJECTIVE: To assess drug susceptibility and characterize Clostridium difficile ribotypes in isolates from two tertiary-care hospitals in Mexico. METHODS: Isolates were evaluated for genotyping, antimicrobial susceptibility testing and detection of mutations associated with drug resistance. PCR ribotyping was performed using a combination of gel-based and capillary electrophoresis-based approaches. RESULTS: MIC50 and MIC90 were ≥128 mg/L for ciprofloxacin, erythromycin, clindamycin, and rifampicin. There was no reduced susceptibility to metronidazole or tetracycline; however, reduced susceptibility to vancomycin (≥4 mg/L) and fidaxomicin (≥2 mg/L) was detected in 50 (40.3%) and 4 (3.2%) isolates, respectively. Furthermore, the rpoB Arg505Lys mutation was more frequently detected in isolates with high minimum inhibitory concentration (MIC) to rifampicin (≥32 mg/L) (OR = 52.5; 95% CI = 5.17-532.6; p < 0.000). Of the 124 C. difficile isolates recovered, 84 (66.7%) were of ribotype 027, 18 (14.5%) of ribotype 001, and the remainder were other ribotypes (353, 255, 220, 208, 176, 106, 076, 020, 019, 017, 014, 012, 003, and 002). CONCLUSION: Ribotypes 027 and 001 were the most frequent C. difficile isolates recovered in this study, and demonstrated higher MICs. Furthermore, we found four isolates with reduced susceptibility to fidaxomicin, raising a concern since this drug is currently unavailable in Mexican Hospitals.Peer reviewedFinal Accepted Versio