- Volume 1, Issue 7, 2019

The gut microbiota has an important role in maintaining intestinal health and protecting against enteric infections (colonisation resistance). Nevertheless the majority of these interactions haven't been explored, largely due to a lack of experimental model systems that can culture oxygen-sensitive commensals alongside intestinal cells. In this project, we have established a novel in vitro model system of the human intestinal epithelium (Vertical Diffusion Chamber, VDC) which also supports growth of strictly anaerobic bacteria. We have applied this system to investigate the interactions of gut symbiont Ruminococcus gnavus with a functioning mucus-producing epithelium, established using T84 and goblet-like LS174T cell lines, and its effect on infection with foodborne pathogen enteropathogenic E. coli (EPEC).

Initial work focused on identifying a culture medium that supports R. gnavus and EPEC growth whilst maintaining epithelial integrity and barrier function. This has been achieved by establishing bacterial growth curves in different media and assessing epithelial barrier function by transepithelial electrical resistance and immunofluorescence staining (IFS) of tight-junction protein occludin. Further IFS demonstrated that introduction of LS174T cells to the epithelium causes mucin secretion and facilitates colonisation by R. gnavus. Co-culture of EPEC with R. gnavus reduces numbers of viable and adherent EPEC, but only when LS174T are present.

These data indicate potential colonisation resistance activity by R. gnavus, discovered by utilising a model system that supports anaerobic culture and a functioning epithelium side-by-side. Future work will investigate colonisation resistance activity for a panel of R. gnavus strains and attempt to elucidate mechanisms behind this activity.

Members of the class Coriobacteriia are little studied, important fastidious anaerobes within the human gut microbiota. Collinsella aerofaciens is a core member of the gut microbiota that can present several different fermentation profiles within individuals, while Eggerthella lenta is implicated in xenobiotic metabolism. Recent ‘culturomics’ studies have greatly increased the number of Coriobacteriia recovered from human-associated samples, with the names of nine novel genera comprising 12 species published to date. However, the ecological range and genomic diversity of the class Coriobacteriia are poorly understood. Taxonomic assignments within the class Coriobacteriia are unclear, limiting the ways in which data from 16S rRNA gene-based diversity studies in humans and rodent models are interpreted.

Whole-genome and 16S rRNA gene sequences of members of the class Coriobacteriia were analysed and assigned to the families Atopobiaceae, Coriobacteriaceae and Eggerthellaceae. Inconsistencies between 16S rRNA gene sequence and whole genome sequence data deposited in public databases were identified. Newly annotated data searched against 85,000 16S rRNA gene sequence datasets included in the IMNGS (Integrated Microbial Next Generation Sequencing) database demonstrates humans and rodents harbour distinct Coriobacteriia populations. Members of the family Coriobacteriaceae predominate in the human gut, while Eggerthellaceae are more representative of the rodent gut. Metabolic capabilities of the three families of Coriobacteriia vary greatly, with Eggerthellaceae asaccharolytic compared with Coriobacteriaceae and Atopobiaceae. Correct annotation of Coriobacteriia in 16S rRNA gene (and by extension shotgun metagenomic) datasets is required to determine the contributions of these increasingly important bacteria to different ecosystems.

Our intestines play home to over one thousand bacterial species, often referred to as the gut microbiota. This microbiota profoundly affects our bodies, providing an array of benefits. Disturbances to this community are associated with pathogenic infection and diseases such as obesity, diabetes and inflammatory bowel disease. Our aim is to engineer a synthetic microbiota, with an emphasis on tracking individual species. Introducing this community into our in vitro gut epithelial culture system will provide insights into pathogen infection strategies, and mechanisms by which the microbiota generates resistance. This tool can be used for the screening of drug/probiotic candidates, potentially reducing the need for animals. Using PMA-qPCR we are able to track up to ten single species in a mixed biofilm. Clostridium difficile an opportunistic pathogen targets the gut during times of depleted microbiota. We have begun using our tracking technology to see how a representative microbiota reacts to a C. difficile invasion. Alongside this, we investigated the effect of the gut commensal - Bacteroides dorei on C. difficile utilizing classical quantification. In both a standard biofilm and in our in vitro model, we see a reduced number of C. difficile when in mixed culture.

This study investigates how English-language news sources represented faecal microbiota transplants (FMT) between 2003 and 2017. In the context of this study, FMT is understood to be the process of transferring stool from a healthy donor to a recipient with a dysfunctional intestinal flora in order to repopulate their gut microbiome. A corpus of news articles on FMT, was produced by searching for ‘fa(e)cal microbial’, ‘microbiota transplant‘ and ‘stool transplant’ on the Nexis® UK news database, generating a corpus suitable for qualitative analysis (n = 504 articles). In order to uncover emerging social representations, we investigated press coverage of stool transplants, as well as broader themes associated with health and the gut microbiome. Our findings show that print media focused particularly on creating novel, mainly hopeful, social representations of faeces through wordplay and punning, side-lining issues of risk and fear. We also identify changing metaphorical framings of microbes and bacteria from ‘enemies’ to ‘friends’. Additionally, we found readers are familiarised with FMT through the depiction of the process as being both mundane and highly medicalised. We argue emerging media representations have the potential to shape more positive social representations of FMT in the general population, paving the way for FMT to become a more socially acceptable and effective medical procedure. Future research can build on this baseline in order to study how social representations circulate in the wider media and public sphere, and how they may change over time and differ between countries as research into FMT progresses.

https://www.tandfonline.com/doi/full/10.1080/14636778.2019.1637721

Up to 95% of the human microbiome consists of anaerobic bacteria. However, they are not as well studied as the aerobic bacteria. Among all anaerobes, the Bacteroides group is the best known and most studied. There has been an increase in carbapenem resistance in Bacteroidales strains without a known cause.

We isolated a Parabacteroides timonensis strain, related to Bacteroides, resistant for amoxicillin, clindamycin and meropenem.

The strain was sequenced using the Miseq (Illumina, San Diego, CA), followed by a de novo assembly using CLC workbench to create a draft genome. Resistance genes were detected with ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/). Genome annotation was performed by RAST (http://rast.nmpdr.org/). Protein modeling was performed, on genes of interest, using Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) and 3DLigandSite (http://www.sbg.bio.ic.ac.uk/3dligandsite/). FatCat (http://fatcat.burnham.org/) was used to do a pairwise structure alignment to compare protein structures.

The draft genome had a size of 6,356,323 bp, present on 95 contigs. Using Resfinder 1 resistance gene was found, tetQ. A possible novel resistance gene for a metallo β-lactamase related to cfiA was found using RAST. The protein model showed similarities with the model for cfiA and includes several Zn2+ ions, used by metallo β-lactamase to hydrolyze carbapenems.

Our results indicate the presence of a not yet described metallo β-lactamase. There are also indications that a conjugative transposon is present, enabling horizontal gene transfer. No erm genes, encoding for clindamycin resistance, were detected in the draft genome.

The National Collection of Type Cultures (NCTC) is the world’s oldest repository of medically-relevant bacteria. NCTC contains 5,500 bacterial strains, 500 of which are anaerobes, with the collection regularly receiving new Type strains and recent clinical isolates.

Fastidious anaerobes pose a unique challenge during the authentication process. NCTC must ensure that the strains are free from contamination and that the organism survives the lyophilisation process. Species-level identification of anaerobic bacterial strains is achieved using a combination of the both MALDI-TOF MS and VITEK2 instruments.

This study evaluates the use of MALDI-TOF MS (Bruker) and VITEK2 (BioMerieux) to identify the anaerobic strains in the NCTC collection. 176 NCTC strains were tested on the MALDI-TOF platform and 60 strains were identified using VITEK2.

MALDI-TOF was able to identify 79% anaerobes to genus-level and 64% to species-level. In comparison the VITEK2 identified 68% to genus and 46% to species-level. The main limiting factor for both these platforms is the database. This may be due to novel anaerobe species NCTC receives not being present on the databases. In the event of no identification, 16S ribosomal RNA sequencing is employed. Furthermore, detection of specific characteristics is carried out by specialist reference laboratories in Cardiff and Colindale, ensuring a robust collection of anaerobic bacteria for use in research and as control strains.

Oxygen and reactive oxygen species (ROS) can react with multiple cellular components, leading to the inactivation of a plethora of metabolic pathways. Therefore, organisms have systems to sense and eliminate O2 and ROS, contributing to their survival in the adverse host environment.

Clostridium difficile P28 is an anaerobic pathogenic bacterium that contains in its genome a flavodiiron protein (FDP) and a rubrerythrin (Rbr), that are putatively involved in the detoxification pathways used by this organism.

Flavodiiron proteins are widespread in all life domains, with a crucial role in O2 detoxification, through its reduction directly to water. FDPs are cytoplasmic enzymes with a minimal structural unit composed by two main domains, a metallo-β-lactamase domain, containing the catalytic diiron site, and a flavodoxin domain having a flavin mononucleotide. Rubrerythrins are generally considered to act as NADH-linked hydrogen peroxide reductases, thus eliminating this ROS, and are composed by two iron sites: a diiron center and a rubredoxin-like FeCys4 center.

In this work with characterized biochemically, spectroscopically, structurally and kinetically the FDP and Rbr and their two putative redox partners, a High Molecular Weight Rubredoxin (HRb) and a Rubredoxin (Rd). We confirmed the existence of direct electron transfer between HRb, Rd and FDP and also between HRb, Rd and Rbr. In addition, we also established the reaction rates for the reduction, by FDP, of O2(0.43s-1) and H2O2 (0.06s-1) and for the reduction of H2O2by Rbr (1.53s-1). The enzymatic activity of Rbr towards O2 was also investigated.

Romão, C.V., et al, 2016.JBIC., 21:39-52.

Martins, M.C., et al., 2019. FRBM, in press.

Clostridium difficile is the most prevalent pathogen among all healthcare-associated infections. This anaerobic bacterium can colonize the human gut, typically following agents that disrupt the normal gut microbiota, like antibiotics. In the gut, C. difficile is subjected to oxygen, which it has to eliminate for survival. Its genome encodes for two flavodiiron proteins, capable to reduce oxygen to water. Besides, some FDPs also reduce NO to N2O, an important feature as a resistance mechanism towards the human innate immune system [1,2]. Flavodiiron enzymes are constituted by a minimal core of two domains: a metallo-β-lactamase-like one, harboring the catalytic center, followed by a short-chain flavodoxin [1,2]. More complex FDPs exist, with multiple extra domains and redox centers [1,2]. C. difficile contains a ‘classical’ FDP, and a very complex one, with an extra short-type rubredoxin domain followed by an NADH:rubredoxin oxidoreductase-like one [3]. The biochemical, redox and spectroscopic studies demonstrated that this enzyme receives electrons directly from NADH, reducing its substrates, precluding the need for extra partners. This FDP is selective for O2 (16s-1), almost 10x higher than with NO. The reactivity towards hydrogen peroxide, as an H2O2 reductase with formation of water, with a non-negligible turnover (2s-1) is a novelty in the field of FDPs. Site directed mutants revealed that the rubredoxin-like center is essential for electron transfer from NADH to the catalytic center.

[1] Martins et al, Free Rad. Biol. Med., in press, 2019.

[2] Romão et al, J. Biol.Inorg. Chem., 21,39-52, 2016.

[3] Folgosa et al, Sci.Rep., 8, 10164, 2018.

Ethylene (ET), salicylic acid (SA) and indole-3-acetic acid (IAA) are important phytohormones regulating plant growth and development, as well as plant-microbe interactions. Plant growth-promoting bacteria (PGPB) naturally associate with plants and facilitate plant growth through a variety of mechanisms, including the ability to modulate the concentrations of these phytohormones in planta. Importantly, the wide presence of phytohormone degradation mechanisms amongst symbiotic and other soil- and plant-associated bacteria indicates that the ability to modulate phytohormone concentrations plays an important role in bacterial colonization and plant-growth promotion abilities. Obtaining phytohormone-degrading bacteria is therefore key for the development of novel solutions aiming to increase plant growth and protection. In this paper, we report an optimized targeted methodology and the consequent isolation of novel soil- and plant-associated bacteria, including rhizospheric, endophytic and phyllospheric strains, with the ability to degrade the phytohormones, SA and IAA, as well as the ET precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). By using an optimized targeted methodology, we rapidly isolated diverse soil- and plant-associated bacteria presenting phytohormone-degrading abilities from several plants, plant tissues and environments, without the need for prior extensive and laborious isolation and maintenance of large numbers of isolates. The developed methodology facilitates PGPB research, especially in developing countries. Here, we also report, for the first time, the isolation of bacterial strains able to concomitantly catabolize three phytohormones (SA, IAA and ACC). Ultimately, the described targeted methodology and the novel phytohormone-degrading bacteria obtained in this work may be useful tools for future plant-microbe interaction studies, and in the development of new inoculant formulations for agriculture and biotechnology.

Automated, high-throughput technologies are becoming increasingly common in microbiome studies to decrease costs and increase efficiency. However, in microbiome studies, small differences in methodology – including storage conditions, wet lab methods, sequencing platforms and data analysis – can influence the reproducibility and comparability of data across studies. There has been limited testing of the effects of high-throughput methods, including microfluidic PCR technologies. In this paper, we compare two extraction methods (the QIAamp DNA Stool Mini Kit and the MoBio PowerSoil DNA Isolation kit), two taq polymerase enzymes (MyTaq HS Red Mix and Accustart II PCR ToughMix), two primer sets (V3–V4 and V4–V5) and two amplification methods (a common two-step PCR protocol and amplicon library preparation on the Fluidigm Access Array system that allows automated multiplexing of primers). Gut microbial community profiles were significantly affected by all variables. While there were no significant differences in alpha diversity measured between the two extraction methods, there was an effect of extraction method on community composition measured by unweighted UniFrac distances. Both amplification method and primers had a significant effect on both alpha diversity and community composition. The relative abundance of Actinobacteria was significantly lower when using the MoBio kit or Fluidigm amplification method, and the relative abundance of Firmicutes was lower when using the Qiagen kit. Microbial community profiles based on Fluidigm-generated amplicon libraries were not comparable to those generated with more commonly used methods. Researchers should carefully consider the limitations and biases that different extraction and amplification methods can introduce into their results. Additionally, more thorough benchmarking of automated and multiplexing methods is necessary to determine the magnitude of the potential trade-off between the quality and the quantity of data.