Intestinal pathogenic Escherichia coli: identification and characterisation of their virulence determinants

The human microbiota consists of over 100 trillion microbial cells, primarily, bacteria that reside in the gastrointestinal tract (Turnbaugh et al., 2007). The relationship between these bacteria and the host is typically symbiotic, as many microbiota-derived species play an important role in host physiology. Thus, maintaining the balance of the intestinal flora, and controlling the overgrowth of potentially pathogenic species, is important in preventing disease. Indeed, specific changes to the bacterial composition of the gut can initiate chronic inflammation of the intestine and result in the onset of gastrointestinal disorders such as inflammatory bowel disease (IBD) (Baldelli et al., 2021). The rapid advancement of sequencing methods and analytical techniques has enabled researchers to understand the microbiome and to ask the question, how do we define the microbiome and its inhabitants? There are many factors that can influence one’s microbiome, including the host genotype, lifestyle and environment (Turnbaugh et al., 2007). Therefore, it is not surprising that the gut microbiome can be up to 80-90% different between individuals, a stark contrast to the human genome, where individual humans are approximately 99.9% identical (Ursell et al., 2012). The composition of the gut microbiota is highly variable between geographical locations, particularly between humans living in markedly different socio-economic settings (Yatsunenko et al., 2012). Moreover, in certain low-income countries (LICs), many enteric pathogens are endemic throughout these regions and are frequently isolated from stool of healthy individuals, suggesting that organisms can be carried asymptomatically. Enteropathogenic E. coli (EPEC) is typically associated with diarrhoeal disease in infants, however, EPEC is frequently isolated from healthy individuals as well as from those with gastroenteritis. Indeed, it remains unknown how these ‘pathogens’ colonise individuals without any symptoms, and begs the question whether these can truly be defined as pathogens at all? Therefore, the primary aim of this thesis was to isolate and characterise the virulence determinants of asymptomatic EPEC isolates. And secondly, to identify a novel strategy for clearing these strains without compromising the composition of the host microbiota. Unfortunately, due to ceased contact with my industrial collaborator, no asymptomatic EPEC strains were received and thus these investigations did not produce any conclusive findings. In consequence, the focus of this project was redirected towards investigating virulence in a different subset of E. coli, specifically strains belonging to the B2 phylogroup which harbour the pks island, a genomic island encoding the biosynthesis genes for a genotoxin termed colibactin. In eukaryotic cells, colibactin induces DNA damage resulting in chromosomal instability and cell cycle arrest. Strikingly, E. coli strains harbouring the pks island are often overrepresented in biopsies from IBD patients and furthermore, the production of colibactin has been implicated in the promotion and development of colorectal cancer in these individuals (Arthur et al., 2012). Thus, as the composition of the human diet has been identified as a key factor in governing intestinal homeostasis, the effect of dietary amino acids on the expression of pks encoded genes was explored using RT-qPCR. Several D-amino acids exhibited the ability to inhibit expression of clbB with D-Serine exerting the strongest repressing activity in two pks+ E. coli strains CFT073 and Nissle 1917. The effect of D-Serine on the colibactin induced cellular response was also observed during infection of HeLa cells with live pks+ strains. Levels of g-H2AX (a marker of DNA double strand breaks) were significantly reduced in cells infected with D-Serine treatment. Moreover, exposure of pks+ E. coli to D-Serine during infection caused a reduction in cellular senescence that was observable at 72 hours post infection. These findings have revealed the potential of D-amino acids in reducing colibactin expression in distinct pks+ E. coli strains. Furthermore, D-Serine, and other D-amino acids are key components of our diet and therefore present opportunities for dietary supplementation that might yield important health benefits by modulating gene expression of E. coli pks+ strains present in the microbiota

Designing and implementing online assessment in the clinical workplace

Peer reviewe

Digital assessment in the clinical workplace : Design, implementation, opportunities, challenges

Peer reviewe

How do students want their workplace-based feedback visualized in order to support self-regulated learning? Initial results & reflections from a co-design study in medical education

Developing good self-regulated learning (SRL) skills is highly important for medical students, not only to help them to navigate and succeed at their current study, but to support their continuing professional development and lifelong learning once they enter the workplace. A key component of SRL is the ability to reflect on feedback and to use this to spot gaps in knowledge/skills, identify learning opportunities and plan new learning goals and activities. Technology can help by providing students with tools that scaffold their development of these skills. This paper reports on the co-design of myPAL, a student-facing learning analytics system. Within co-design workshops, we worked with students to improve myPAL. These hands-on, creative workshops involved students in discussion of their current and desired use of feedback, practical interface/visualisation design and prototype use and adaptation. Using this participative approach we have identified one key visualisation and a set of functions/features that students want to be available to help them to review and act on their feedback. In this paper we report and reflect on the co-design approach that has been used, including the observed benefits of taking such an approach as well as its limitations. We also outline the further work that is planned to develop & evaluate the required improvements to myPAL

The Bacteroidetes Aequorivita sp. and Kaistella jeonii Produce Promiscuous Esterases With PET-Hydrolyzing Activity

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation

Model-observation and reanalyses comparison at key locations for heat transport to the Arctic: Assessment of key lower latitude influences on the Arctic and their simulation

Blue-Action Work Package 2 (WP2) focuses on lower latitude drivers of Arctic change, with a focus on the influence of the Atlantic Ocean and atmosphere on the Arctic. In particular, warm water travels from the Atlantic, across the Greenland-Scotland ridge, through the Norwegian Sea towards the Arctic. A large proportion of the heat transported northwards by the ocean is released to the atmosphere and carried eastward towards Europe by the prevailing westerly winds. This is an important contribution to northwestern Europe's mild climate. The remaining heat travels north into the Arctic. Variations in the amount of heat transported into the Arctic will influence the long term climate of the Northern Hemisphere. Here we assess how well the state of the art coupled climate models estimate this northwards transport of heat in the ocean, and how the atmospheric heat transport varies with changes in the ocean heat transport. We seek to improve the ocean monitoring systems that are in place by introducing measurements from ocean gliders, Argo floats and satellites. These state of the art computer simulations are evaluated by comparison with key trans-Atlantic observations. In addition to the coupled models ‘ocean-only’ evaluations are made. In general the coupled model simulations have too much heat going into the Arctic region and the transports have too much variability. The models generally reproduce the variability of the Atlantic Meridional Ocean Circulation (AMOC) well. All models in this study have a too strong southwards transport of freshwater at 26°N in the North Atlantic, but the divergence between 26°N and Bering Straits is generally reproduced really well in all the models. Altimetry from satellites have been used to reconstruct the ocean circulation 26°N in the Atlantic, over the Greenland Scotland Ridge and alongside ship based observations along the GO-SHIP OVIDE Section. Although it is still a challenge to estimate the ocean circulation at 26°N without using the RAPID 26°N array, satellites can be used to reconstruct the longer term ocean signal. The OSNAP project measures the oceanic transport of heat across a section which stretches from Canada to the UK, via Greenland. The project has used ocean gliders to great success to measure the transport on the eastern side of the array. Every 10 days up to 4000 Argo floats measure temperature and salinity in the top 2000m of the ocean, away from ocean boundaries, and report back the measurements via satellite. These data are employed at 26°N in the Atlantic to enable the calculation of the heat and freshwater transports. As explained above, both ocean and atmosphere carry vast amounts of heat poleward in the Atlantic. In the long term average the Atlantic ocean releases large amounts of heat to the atmosphere between the subtropical and subpolar regions, heat which is then carried by the atmosphere to western Europe and the Arctic. On shorter timescales, interannual to decadal, the amounts of heat carried by ocean and atmosphere vary considerably. An important question is whether the total amount of heat transported, atmosphere plus ocean, remains roughly constant, whether significant amounts of heat are gained or lost from space and how the relative amount transported by the atmosphere and ocean change with time. This is an important distinction because the same amount of anomalous heat transport will have very different effects depending on whether it is transported by ocean or the atmosphere. For example the effects on Arctic sea ice will depend very much on whether the surface of the ice experiences anomalous warming by the atmosphere versus the base of the ice experiencing anomalous warming from the ocean. In Blue-Action we investigated the relationship between atmospheric and oceanic heat transports at key locations corresponding to the positions of observational arrays (RAPID at 26°N, OSNAP at ~55N, and the Denmark Strait, Iceland-Scotland Ridge and Davis Strait at ~67N) in a number of cutting edge high resolution coupled ocean-atmosphere simulations. We split the analysis into two different timescales, interannual to decadal (1-10 years) and multidecadal (greater than 10 years). In the 1-10 year case, the relationship between ocean and atmosphere transports is complex, but a robust result is that although there is little local correlation between oceanic and atmospheric heat transports, Correlations do occur at different latitudes. Thus increased oceanic heat transport at 26°N is accompanied by reduced heat transport at ~50N and a longitudinal shift in the location of atmospheric flow of heat into the Arctic. Conversely, on longer timescales, there appears to be a much stronger local compensation between oceanic and atmospheric heat transport i.e. Bjerknes compensation

Aquarium Nitrification Revisited: Thaumarchaeota Are the Dominant Ammonia Oxidizers in Freshwater Aquarium Biofilters

Ammonia-oxidizing archaea (AOA) outnumber ammonia-oxidizing bacteria (AOB) in many terrestrial and aquatic environments. Although nitrification is the primary function of aquarium biofilters, very few studies have investigated the microorganisms responsible for this process in aquaria. This study used quantitative real-time PCR (qPCR) to quantify the ammonia monooxygenase (amoA) and 16S rRNA genes of Bacteria and Thaumarchaeota in freshwater aquarium biofilters, in addition to assessing the diversity of AOA amoA genes by denaturing gradient gel electrophoresis (DGGE) and clone libraries. AOA were numerically dominant in 23 of 27 freshwater biofilters, and in 12 of these biofilters AOA contributed all detectable amoA genes. Eight saltwater aquaria and two commercial aquarium nitrifier supplements were included for comparison. Both thaumarchaeal and bacterial amoA genes were detected in all saltwater samples, with AOA genes outnumbering AOB genes in five of eight biofilters. Bacterial amoA genes were abundant in both supplements, but thaumarchaeal amoA and 16S rRNA genes could not be detected. For freshwater aquaria, the proportion of amoA genes from AOA relative to AOB was inversely correlated with ammonium concentration. DGGE of AOA amoA genes revealed variable diversity across samples, with nonmetric multidimensional scaling (NMDS) indicating separation of freshwater and saltwater fingerprints. Composite clone libraries of AOA amoA genes revealed distinct freshwater and saltwater clusters, as well as mixed clusters containing both freshwater and saltwater amoA gene sequences. These results reveal insight into commonplace residential biofilters and suggest that aquarium biofilters may represent valuable biofilm microcosms for future studies of AOA ecology

A communal catalogue reveals Earth's multiscale microbial diversity

Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth's microbial diversity.Peer reviewe

A communal catalogue reveals Earth’s multiscale microbial diversity

Our growing awareness of the microbial world’s importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth’s microbial diversity