Showing papers by "Emma Allen-Vercoe published in 2020"

A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity.

Abstract: Our knowledge about the gut microbiota of pigs is still scarce, despite the importance of these animals for biomedical research and agriculture. Here, we present a collection of cultured bacteria from the pig gut, including 110 species across 40 families and nine phyla. We provide taxonomic descriptions for 22 novel species and 16 genera. Meta-analysis of 16S rRNA amplicon sequence data and metagenome-assembled genomes reveal prevalent and pig-specific species within Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium, and several new genera described in this study. Potentially interesting functions discovered in these organisms include a fucosyltransferase encoded in the genome of the novel species Clostridium porci, and prevalent gene clusters for biosynthesis of sactipeptide-like peptides. Many strains deconjugate primary bile acids in in vitro assays, and a Clostridium scindens strain produces secondary bile acids via dehydroxylation. In addition, cells of the novel species Bullifex porci are coccoidal or spherical under the culture conditions tested, in contrast with the usual helical shape of other members of the family Spirochaetaceae. The strain collection, called 'Pig intestinal bacterial collection' (PiBAC), is publicly available at www.dsmz.de/pibac and opens new avenues for functional studies of the pig gut microbiota.

Protease-dependent excitation of nodose ganglion neurons by commensal gut bacteria.

Abstract: Key points The vagus nerve has been implicated in mediating behavioural effects of the gut microbiota on the central nervous system. This study examined whether the secretory products of commensal gut bacteria can modulate the excitability of vagal afferent neurons with cell bodies in nodose ganglia. Cysteine proteases from commensal bacteria increased the excitability of vagal afferent neurons via activation of protease-activated receptor 2 and modulation of the voltage dependence of Na+ conductance activation. Lipopolysaccharide, a component of the cell wall of gram-negative bacteria, increased the excitability of nodose ganglia neurons via TLR4-dependent activation of nuclear factor kappa B. Our study identified potential mechanisms by which gut microbiota influences the activity of vagal afferent pathways, which may in turn impact on autonomic reflexes and behaviour. Abstract Behavioural studies have implicated vagal afferent neurons as an important component of the microbiota-gut-brain axis. However, the mechanisms underlying the ability of the gut microbiota to affect vagal afferent pathways are unclear. We examined the effect of supernatant from a community of 33 commensal gastrointestinal bacterial derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) on the excitability of mouse vagal afferent neurons. Perforated patch clamp electrophysiology was used to measure the excitability of dissociated nodose ganglion (NG) neurons. NG neuronal excitability was assayed by measuring the amount of current required to elicit an action potential, the rheobase. MET-1 supernatant increased the excitability of NG neurons by hyperpolarizing the voltage dependence of activation of Na+ conductance. The increase in excitability elicited by MET-1 supernatant was blocked by the cysteine protease inhibitor E-64 (30 nm). The protease activated receptor-2 (PAR2 ) antagonist (GB 83, 10 μm) also blocked the effect of MET-1 supernatant on NG neurons. Supernatant from Lactobacillus paracasei 6MRS, a component of MET-1, recapitulated the effect of MET-1 supernatant on NG neurons. Lastly, we compared the effects of MET-1 supernatant and lipopolysaccharide (LPS) from Escherichia coli 05:B5 on NG neuron excitability. LPS increased the excitability of NG neurons in a toll-like receptor 4 (TLR4 )-dependent and PAR2 -independent manner, whereas the excitatory effects of MET-1 supernatant were independent of TLR4 activation. Together, our findings suggest that cysteine proteases from commensal bacteria increase the excitability of vagal afferent neurons by the activation of PAR2 .

A survey of Fusobacterium nucleatum genes modulated by host cell infection.

Abstract: Here, we report comprehensive transcriptomic profiles from Fusobacterium nucleatum under conditions that mimic the first stages of bacterial infection in a highly differentiated adenocarcinoma epithelial cell line. Our transcriptomic in vitro adenocarcinoma approach allows us to measure the expression dynamics and regulation of bacterial virulence and response factors in real time, and is a novel strategy for clarifying the role of F. nucleatum infection in colorectal cancer (CRC) progression. Our data show that: (i) infection alters metabolic and functional pathways in F. nucleatum, allowing the bacterium to adapt to the host-imposed milieu; (ii) infection also stimulates the expression of genes required to help induce and promote a hypoxic and inflammatory microenvironment in the host; and (iii) F. nucleatum invasion occurs by a haematogenous route of infection. Our study identifies novel gene targets from F. nucleatum that are activated during invasion and which may aid in determining how this species invades and promotes disease within the human gastrointestinal tract. These invasion-specific genes may be useful as biomarkers for CRC progression in a host and could also assist in the development of new diagnostic tools and treatments (such as vaccines or small molecule drug targets), which will be able to combat infection and inflammation in the host while circumventing the potential problem of F. nucleatum tolerization.

Effects of Antibiotic Pretreatment of an Ulcerative Colitis-Derived Fecal Microbial Community on the Integration of Therapeutic Bacteria In Vitro.

Abstract: Fecal microbiota transplantation (FMT) is a proposedly useful strategy for the treatment of gastrointestinal (GI) disorders through remediation of the patient gut microbiota. However, its therapeutic success has been variable, necessitating research to uncover mechanisms that improve patient response. Antibiotic pretreatment has been proposed as one method to enhance the success rate by increasing niche availability for introduced species. Several limitations hinder exploring this hypothesis in clinical studies, such as deleterious side effects and the development of antimicrobial resistance in patients. Thus, the purpose of this study was to evaluate the use of an in vitro, bioreactor-based, colonic ecosystem model as a form of preclinical testing by determining how pretreatment with the antibiotic rifaximin influenced engraftment of bacterial strains sourced from a healthy donor into an ulcerative colitis-derived defined microbial community. Distinct species integrated under the pretreated and untreated conditions, with the relative rifaximin resistance of the microbial strains being an important influencer. However, both conditions resulted in the integration of taxa from Clostridium clusters IV and XIVa, a concomitant reduction of Proteobacteria, and similar decreases in metabolites associated with poor health status. Our results agree with the findings of similar research in the clinic by others, which observed no difference in primary patient outcomes whether or not patients were given rifaximin prior to FMT. We therefore conclude that our model is useful for screening for antibiotics that could improve efficacy of FMT when used as a pretreatment.IMPORTANCE Patients with gastrointestinal disorders often exhibit derangements in their gut microbiota, which can exacerbate their symptoms. Replenishing these ecosystems with beneficial bacteria through fecal microbiota transplantation is thus a proposedly useful therapeutic; however, clinical success has varied, necessitating research into strategies to improve outcomes. Antibiotic pretreatment has been suggested as one such approach, but concerns over harmful side effects have hindered testing this hypothesis clinically. Here, we evaluate the use of bioreactors supporting defined microbial communities derived from human fecal samples as models of the colonic microbiota in determining the effectiveness of antibiotic pretreatment. We found that relative antimicrobial resistance was a key determinant of successful microbial engraftment with rifaximin (broad-spectrum antibiotic) pretreatment, despite careful timing of the application of the therapeutic agents, resulting in distinct species profiles from those of the control but with similar overall outcomes. Our model had results comparable to the clinical findings and thus can be used to screen for useful antibiotics.

Function is what counts: how microbial community complexity affects species, proteome and pathway coverage in metaproteomics.

Abstract: Introduction: Metaproteomics is an established method to obtain a comprehensive taxonomic and functional view of microbial communities. After more than a decade, we are now able to describe the promise, reality, and perspectives of metaproteomics and provide useful information about the choice of method, applications, and potential improvement strategies.Areas covered: In this article, we will discuss current challenges of species and proteome coverage, and also highlight functional aspects of metaproteomics analysis of microbial communities with different levels of complexity. To do this, we re-analyzed data from microbial communities with low to high complexity (8, 72, 200 and >300 species). High species diversity leads to a reduced number of protein group identifications in a complex community, and thus the number of species resolved is underestimated. Ultimately, low abundance species remain undiscovered in complex communities. However, we observed that the main functional categories were better represented within complex microbiomes when compared to species coverage.Expert opinion: Our findings showed that even with low species coverage, metaproteomics has the potential to reveal habitat-specific functional features. Finally, we exploit this information to highlight future research avenues that are urgently needed to enhance our understanding of taxonomic composition and functions of complex microbiomes.

1D 1 H NMR as a Tool for Fecal Metabolomics.

Abstract: Metabolomic studies allow a deeper understanding of the processes of a given ecological community than nucleic acid-based surveys alone. In the case of the gut microbiota, a metabolic profile of, for example, a fecal sample provides details about the function and interactions within the distal region of the gastrointestinal tract, and such a profile can be generated in a number of different ways. This unit elaborates on the use of 1D 1 H NMR spectroscopy as a commonly used method to characterize small-molecule metabolites of the fecal metabonome (meta-metabolome). We describe a set of protocols for the preparation of fecal water extraction, storage, scanning, measurement of pH, and spectral processing and analysis. We also compare the effects of various sample storage conditions for processed and unprocessed samples to provide a framework for comprehensive analysis of small molecules from stool-derived samples. © 2020 Wiley Periodicals LLC Basic Protocol 1: Extracting fecal water from crude fecal samples Alternate Protocol 1: Extracting fecal water from small crude fecal samples Basic Protocol 2: Acquiring NMR spectra of metabolite samples Alternate Protocol 2: Acquiring NMR spectra of metabolite samples using Bruker spectrometer running TopSpin 3.x Alternate Protocol 3: Acquiring NMR spectra of metabolite samples by semiautomated process Basic Protocol 3: Measuring sample pH Support Protocol 1: Cleaning NMR tubes Basic Protocol 4: Processing raw spectra data Basic Protocol 5: Profiling spectra Support Protocol 2: Spectral profiling of sugars and other complex metabolites.

Fusobacteria: physiology, form, and function

Abstract: Members of the enigmatic Fusobacteria phylum, once an obscure group, including pathogens primarily of dental and veterinary interest, have recently emerged as contributing features in a range of important chronic diseases in humans, including colorectal cancer (CRC). The fastidious and anaerobic Fusobacterium genus and, in particular, its member species, F. nucleatum, have received the most research attention, which has revealed complex interactions between other member species of the human microbiota, as well as human host cells. Such interactions, particularly in the context of CRC, are of paramount interest to the fields of infectious microbiology and oncology. However, foundational understanding of the basic microbiology, physiology, and function of Fusobacteria is critical, as is an appreciation for the species and strain variability within many genera of this phylum. F. nucleatum, for example, is highly heterogenous and different strains can display quite disparate characteristics. In this chapter, we attempt to summarize the main physiological, phylogenetic, and genotypic attributes of the Fusobacteria, with special emphasis on F. nucleatum.

Invasion by Fusobacterium nucleatum Causes Upregulation of dll4 and klf4 Expression in Caco-2 Cells

Abstract: Fusobacterium nucleatum is an emerging microbe of importance in the pathogenesis of colorectal cancer. Strains of this enigmatic bacterial species vary in their capacity to invade human epithelial cells, a virulence determinant which has important implications in disease. Here, we infected human colorectal epithelial (Caco-2) cells in vitro with a known, highly invasive strain of F. nucleatum isolated from a Crohn’s Disease patient, as well as a further invasive isolate of F. nucleatum derived from a colorectal cancer tumour. We used transcriptional profiling to determine the human genes upregulated during the invasion process compared to exposure to a non-invasive E.coli control strain. Infection with F. nucleatum strains resulted in the upregulation of several host genes, including two associated with tumorigenesis: dll4 and klf4.

Manipulating the gut microbiota

Abstract: A number of pediatric illnesses, both enteric and systemic, have been linked to an abnormal gut microbiota, although causality has yet to be confirmed. Several approaches have been advanced for the manipulation of the gut microbiota in order to treat or prevent microbiota-related disease conditions. These include established methods, including the use of pre-, pro-, and synbiotics, as well as more experimental techniques, including fecal microbiota transplantation and microbial ecosystem-derived therapeutics. This chapter reviews research surrounding the safety, efficacy, and sustained effects of these methods in a number of indications, with a focus on the pediatric population.