- Volume 1, Issue 1A, 2019

Laboratory experiments were conducted to determine the effect of temperature (20 °C and 30 °C) on the rate of tissue decomposition in organic compost for a period of 28 days. Porcine tissue was used as a substitute for human body parts in burial microenvironment. Measurements of the decay include porcine tissue mass loss, soil pH, the metabolic activity of soil microbes, viable count and Gram stain of associated microflora. Incubation temperature had a significant impact on the post-mortem decay rates and stages at 30 °C in comparison to the samples incubated at 20 °C. Bacterial enumeration demonstrated microbial burden to be higher at higher temperatures, suggesting accelerated decomposition. Gram staining identified mostly Gram-negative bacteria, decomposers that might have originated from the organic meat as opposed to soil bacteria. There was a significant difference in soil pH levels during harvest times and at the end of the experiments in both microenvironments. It is an indication that measured pH of the depository may reveal whether the body parts were recently buried. A fluorescein diacetate (FDA) method that measured metabolic activity in compost demonstrated the highest release of fluorescein during harvests at 30 °C. A non-parametric Friedman test of differences among repeated measures rendered significant results (α=0.00) showing differences in microbial activity according to the temperature level. The results suggest that depositional microenvironment would be significantly modified by the decaying organic matter with the rise of temperature. This way, the temperature would make an impact on the decay of buried remains.

Poison frogs secrete alkaloid toxins in their skin as defence mechanisms against predators. Numerous studies have shown that the origin of alkaloid toxins in the skin is through ‘sequestration from diet’, i.e. uptake and storage of toxins or their chemical precursors, mostly from consumed arthropods. There exists the intriguing possibility that the gut microbiome of these frogs may play a role in this process. We address this question by looking at the organism together with its associated microbial communities, an effective symbiotic relationship between host and microbiome that could have allowed phenotypic adaptation of the host to a toxic diet. We sequenced the Bacterial and Archaeal 16S rRNA regions of the gut microbiome of 7 Poison frog species and 9 outgroup frog species caught in the rainforest of Eastern Peru. Frog species were selected based on sharing similar microhabitats and comparable individual sizes. A comparative analysis of the microbiome composition across all our samples allowed us to identify a core group of abundant symbiotic microbes unique to poison frogs in spite of intrinsic variation within species. We speculate this group could be associated to their ability to sequester toxins and we carried further metagenomic sequencing to allow us to determine possible functions that may be involved in toxin processing in these frogs.

As biogas from anaerobic digestion becomes an increasingly attractive biofuel, the need to improve the quality of biogas has come to the fore. Biological upgrading focuses on adding enough hydrogen to an anaerobic biogas reactor to allow methanation of the remaining carbon dioxide by methanogenic Archaea (in situ upgrading). Alternatively, biogas and hydrogen can be mixed in the absence of feedstock, in a reactor operated exclusively to facilitate methanogenesis (ex situ upgrading). This novel technology can encounter inhibition at high loading rates of hydrogen: however, in contrast to anaerobic digestion, the dynamics of this thermophilic functional microbial community are sparsely characterised. High-resolution 16S rDNA community profiles from four anaerobic biogas upgrading reactors were constructed to determine how feedstock, hydrogen, and CO₂ influence biomethanation. Presence/absence of a feedstock led to large differences between in situ and ex situ communities, determining the dominant methanogen genera, and encouraging distinct populations of hydrolysing and fermenting Firmicutes. Although high hydrogen flow rates (∼37 L/day) caused a collapse in methanogenic Methanothermobacter populations in situ, ex situ hydrogen rates greatly exceeded these levels (∼400 L/day) without collapse of Methanobacterium, despite some observed instability and proliferation of likely homoacetogens. Subsequent reduction of hydrogen rates ex situ (259 L/day) appeared to create a niche for hydrogen production, indicated by increased abundance of various syntrophic fermenters known to supply biogenic hydrogen. In either upgrading setup, instability due to increased hydrogen levels manifested as a disruption of fermenting and hydrolysing populations prior to disruption of methanogens.

Recently scientific and industrial interest has grown in relation to antimicrobial surfaces. This interest is mainly due to the persistent and prevalent microbial contamination of industrial and medical surfaces. A critical issue is the dissemination of bacterial colonies across biotic and abiotic surfaces, from surface contact-contact interaction. This ability to colonise materials presents a major problem for cross-contamination and pathogenic bacterial proliferation, resulting in wide-spread distribution and mutation, presenting increased risk to human health. There are two approaches to prevent bacterial spread – disinfection and antimicrobial surfaces. The use of disinfectants presents pollution to the surrounding environment, and the increased development of resistant microbial strains. The beneficial design of antimicrobial polymers enables contact-killing, without the release of biocides into the environment. This project aimed at synthesising sulfur polymeric materials through inverse vulcanisation, with the aim of producing functional antimicrobial materials. As of yet, there is no publication evaluating the antimicrobial effect of diisopropenyl benzene (DIB) and dicyclopentadiene (DCPD) co-polymers as bulk solids, even though elemental sulfur is known to exhibit antimicrobial effects. There is growing demand for materials with antimicrobial capabilities, especially in medical environments, where the epidemiology of hospital acquired infections is of great research interest. The aim of this study was to evaluate the antimicrobial properties of both S-DIB and S. DCPD using Escherichia coli (DSM 1576) and Staphylococcus aureus (DSM 346) against an internationally recognised standard (ISO 22196). To gain further in-depth analysis, confocal microscopy was employed to access the surface impact on bacterial cells.

It is acknowledged that the environment facilitates antibiotic resistance development and spread but not much research has been undertaken to understand the mechanisms involved. This study focuses on antibiotic resistance transfer by enterococci, normal inhabitants of the mammalian gut and important healthcare associated pathogens, under a variety of environmental conditions. These are novel environmental strains, isolated from a farm in Monaghan, Ireland. The primary aim of this project is to quantify factors controlling the horizontal gene transfer (HGT) of antibiotic resistance genes via pheromone-induced conjugation. The main objectives include; identifying enterococcal isolates capable of transferring antibiotic resistance genes under laboratory conditions; using whole genome sequencing (WGS) to characterise genetic events involved in conjugal transfer; developing markers of transfer efficiency; and developing environmentally relevant models to measure enterococcal HGT. The transfer of vancomycin resistance genes from a donor to a recipient cell have been demonstrated and transfer efficiencies range from 1.09×10−1 to 9.74×10−5. This data shows that some donors are better at donating vancomycin resistance genes than other donors and some recipients are better than other recipients at receiving them. Preliminary data also shows the transfer of trimethoprim, tetracycline and erythromycin and we can see that within a localised environment Enterococcus faecalis can spread resistance genes very differently. In conclusion this project hopes to further our knowledge of the mechanism of pheromone-induced conjugation in environmental Enterococci and to show that environmental enterococcal strains can become multi-drug resistant just as easily as clinical isolates.

Antibiotics are extensively fed to beef cattle as they act as rumen modulators, improving animal efficiency and decreasing methane emissions. However, current recommendations by health agencies to limit/ban antibiotic use in animal production call for alternatives such as natural additives. In this study, we aimed to evaluate the efficacy of natural additives in methane mitigation. Bulls (½Angus and ½Nellore) 16±2.2 months old, with average body weight of 385±20.7 kg were fed a basal diet (70 % concentrate, 30 % corn silage) offered ad libitum for 62 days in a feedlot and randomised on five treatments (8 bulls/treatment): control treatment, and addition of 1.5, 3.0, 4.5, or 6.0 g/day/animal of a blend of natural additives containing 37.5 % each of clove essential oil, the commercial blend containing vanillin, eugenol and thymol, 12.5 % and 12.5 % of castor and cashew oils). Methane production from rumen fluid was estimated based on the theoretical fermentation balance for observed molar distribution of VFAs in the rumen. DNA extracted from rumen fluid were sequenced and analysed for methane genes within the MG-RAST database. The natural additives linearly reduced methane production (76 %, P<0.02). Evaluation of Archaeaabundance showed a reduction (79 %, P<0.05) in the major methane producing genera: Halorhabdus, Ferroplasma, Methanoplanus, Picrophilus. A reduction of Fibrobacter and Lactobacillus (71 %), the greatest producers of acetate releasing hydrogen for methane formation was also observed. Our findings suggest that natural additives such as essential oils may be useful in the mitigation of greenhouse gases such as methane in animal production.

Conventional microbial sampling of air in hospitals is usually carried out using settle plates or impaction air samples. This provides little information about intermittent contamination events and is unhelpful for source attribution. Direct continuous bioaerosol sampling is an established technology used to characterise ambient external air. Portable instruments such as the Wideband Integrated Bioaerosol Sensor (WIBS) combine laser particle size and shape detection with signals of biological origin (fluorescence from amino acids and NAD(P)H) characteristic of viable bioaerosols. Monitoring is continuous for weeks at a time and data collected remotely over the internet. We present evidence of the utility of WIBS analysis in characterising air in hospitals in three different environments: operating theatres (plenum ventilated and ultraclean), a respiratory ward, and a specialist cystic fibrosis outpatients. The airborne particle profile was quantitatively and qualitatively different in each environment. Plumes of biologically-relevant airborne particles were detected and source investigation of failing conventional counts in an operating theatre aided by the continuous record. Nebulised drugs contributed a detectable effect on airborne particles which lasted for several hours on the ward despite air changes. A significant effect of plasma air treatment on airborne particles in the ward was detected by WIBS and not conventional cultures. Continuous monitoring may in future allow objective standard setting for airborne particles in different hospital environments and facilitate rapid detection of airborne infection risks.

The Polar Regions have excellent potential for bioprospecting because the microorganisms that live there have adapted to extreme environmental conditions; and these adaptations can be harnessed in a variety of biotechnological applications. In this study, we describe the community structure of a range of different cryospheric habitats to identify environments appropriate for specific bioprospecting activities. Samples were collected from Midtre Lovénbreen glacier and surrounds in Spitzbergen, Svalbard in late June to early July 2017. Many cryospheric environment types were sampled including air, snow, slush, meltwater, cryoconite, proglacial water, soil and seawater. DNA was extracted and prepared for 16S amplicon sequencing. Amplicon sequence variants (ASVs) were assigned using DADA2 and contaminants were removed using the Decontam package in R. We searched the scientific literature for biotechnological applications of abundant community members in each environment. Each environment type displayed a unique community structure, with some physically linked environment types, like glacial snow, slush and meltwater showing continuity through space, and synchronised changes over time. The snow, slush and meltwater habitats were low in biomass and dominated by Gammaproteobacteria, with Cyanobacteria increasing in abundance as melt progressed. Cryoconite was dominated by Cyanobacteria, while sea water was dominated by Bacteriodetes, Gammaproteobacteria and Cyanobacteria. Soil was by far the most biodiverse habitat, with many phyla represented, and a large number of Actinobacteria suitable for antimicrobial discovery. Many of the current EPS, cold-active enzymes, fatty acids, antioxidants and antifreeze proteins are sourced from Proteobacteria and Cyanobacteria, the most abundant phyla in this study.

The conditions of the deep subsurface, combined by perturbations caused by geological disposal of radioactive waste create multiple extreme conditions (limited space availability, high pH, high temperature etc.) for microbial communities. Microbial activity has the potential to cause corrosion of steel and alteration of bentonite clays used in geological disposal facilities. To understand the limits on microbial growth, and the potential for microbial activity to affect the swelling behaviour of the bentonite and metal corrosion, a suite of laboratory experiments is being conducted. In situ repository conditions have been replicated in the MIND (Horizon2020) project. Preliminary results show evidence of corrosion in all experiments, an increase in the basal spacings of smectites in the zone immediately surrounding the steel and inoculated samples had evidence of calcite crystal formation, accompanied by differences in the iron phases. These experiments simulate the in situ conditions well, but the complex nature of this experimental design (high pressure and flow) reduces the practicality of varying the environmental conditions. To complement these investigations, a low-tech solution has been implemented with unpressurised, hydrated bentonite batch experiments. The simpler nature of this set-up allows for investigation of more parameters. Microcosms with artificial groundwater used in the MIND set-up are being compared to the MIND groundwater composition, modelled to represent permafrost conditions. The effect of incubation temperature is also being investigated. Combined, these experiments will help to understand the influence of microbes under the extremes of geological disposal facilities and how their behaviour may change by external parameters.

The Cheshire Salt District (UK) is home to a wide range of largely unexplored inland brine springs, whose increased salinity originates from subterranean Triassic salt rock deposits. Our study focused on the Anderton Brine Spring System, a set of pools of varying salinity which are subjected to regular salinity fluctuations depending on drainage and evaporation (recorded salinities have ranged from 1.2% to 13% NaCl). Preliminary investigation of samples obtained from one of these pools (with 4.2 % NaCl) revealed a wealth of novel isolates which were analysed by 16S rRNA gene sequencing. Within these isolated strains, we have detected that strain MKS20 had a 97 % similarity to its closest relative, Motilimonas eburnea, which is currently the only characterised species in a newly discovered genus of the order Alteromonadales. All reported strains within this genus have been isolated from marine environments, namely marine-sediments and the gut of a sea cucumber. MKS20 is the first strain in the genus to be reported from an inland brine spring and thus significantly extends the ecological range of the genus Motilimonas. The distinctiveness of our strain is further supported by preliminary results from polyphasic taxonomic characterisation (e.g. metabolic and enzymatic profiling). Based on these, strain MKS20 represents a putative new species within the genus for which we propose the name Motilimonas cestriensis, referring to Cheshire, the place of isolation.

There is evidence that water may exist on Mars as brines in the subsurface. The chemistries of these brines will be greatly influenced by the local lithologies, which would impact on the organisms that could potentially live within them. There are multiple metabolisms that are theoretically viable under martian chemical conditions. In order to better establish which of these are capable of supporting persistent growth under martian conditions, we performed a series of enrichments using four geological simulants for Mars: a global composition, an early and unaltered basaltic composition, a sulfur-rich composition, and a haematite-rich composition. Enrichments were inoculated with sediment from Pyefleet mudflats in the Colne estuary (Essex, UK). Mudflat sediment was added to the simulant materials and a brine based on the chemistry of Rocknest. Enrichments were supplied with a H₂/CO₂ headspace at 1 bar pressure and incubated for 20 days. The enrichments were repeatedly subcultured into fresh simulant material and brines in order to effectively select for a community actively growing in this chemical environment. The enriched community was characterised through the isolation and identification of microbes, microscopy and the amplicon sequencing of 16S rRNA genes amplified from DNA extracted from each stage of the enrichment. Biotic and abiotic experiments were conducted to identify geochemical changes that occur due to the presence of microbial activity. We will present details on the communities enriched on the different martian simulants, metabolisms identified as present within the actively growing community and geochemical changes that were identified.

Saltmarshes are threatened coastal ecosystems, which are subject to cyclic variation in conditions which create a gradient-rich environment (e.g. salinity, pH). They support very diverse communities of plants and animals, protect the coast from erosion, and play an important role in element cycling and bio-remediation. The importance of saltmarshes has only been recently recognised, which has led to significant efforts in restoring sites that had been previously converted to Agriculture. Such restoration attempts have had limited success in returning these sites to their original biodiversity and biological structure. While not yet fully understood, it is thought that several factors are at play, including persisting differences in soil structure and quality. Coastal environments have been previously shown to harbour significant microbial populations capable of producing CaCO₃ biominerals. On the other side, CaCO₃ biomineral production is known to affect the texture and overall properties of soil, a property currently used in the improvement of soils for Agriculture. We suggest that these factors could be linked, and that the limitations of saltmarsh restoration efforts might result from differences in biomineral production. Our results provide an overview on differences between samples that we’ve collected in natural and restored sites, based on cultivation and screening efforts. Our insights might provide an important step forward in saltmarsh restoration and contribute to more successful approaches to protect these vital biotopes and increase biodiversity.

The Cabo Verde Islands constitute a biodiversity hotspot, encompassing a wide range of different biotopes. Thus far, most surveys and programmes have focused exclusively on the flora and/or fauna of the islands and no effort has been made to systematically assess and preserve locally existing microbial biodiversity. The absence of such studies is especially troubling regarding their traditional salterns, as many of them have been abandoned, and increase of construction in coastal areas has already started to indelibly change some of these unique ecosystems and will likely obliterate some of them. Here we provide an overview of the preliminary data recently collected from salterns in the islands of Sal, Maio, and Boavista and include details on their previously unreported physical-chemical characteristics (salinity, pH, temperature and ionic composition), as well as first results on microbial metabolic profiling. Our data has shown a wide diversity of environmental niches (particularly noticeable in Maio), and showcased differences in substrate use both between and within different salterns. These are the first step of our ongoing efforts in assisting in the survey of several threatened extreme environments in Cabo Verde, characterising their geochemistry and microbiology, and identifying their potential biotechnological applications.

Cell-cell communication through the production of autoinducer molecules has been widely studied in bacteria and found to play a pivotal role in biofilm formation and gene regulation. Quorum Sensing (QS) within the domain Archaea is understudied compared with their bacterial counterparts. The aim of this study was to determine whether archaea are capable of cross-kingdom signalling through the production of QS inducing compounds and QS inhibitory activities. A combination of culture-dependent (crude extracts from archaeal isolates) and culture-independent (genomic mining) techniques were employed to investigate the production of compounds capable of modulating bacterial QS. Crude archaeal extracts were screened for activity using the bioreporter strains Agrobacterium tumefaciens ATCC BAA-2240, Escherichia coli JM109 pSB536, pSB401 and pSB1142, Chromobacterium violaceum CV026 and Pseudomonas aeruginosa MW-1. Active strains were further characterised using initial bio-assay guided fractionation. Preliminary results revealed different strains were capable of eliciting a QS response in the bacterial bio-reporters. Initial characterisation using LC-MS, TLC-overlays, and other biochemical tests, suggests the possible production of Butyryl Homoserine Lactones or homologs of this molecule by at least one of the strains. Conversely, other halophilic archaea were capable of inhibiting the production of QS-controlled pathways, as demonstrated by the reduction in virulence factor production by P. aeruginosa. Further characterisation of these compounds will prove to be an invaluable insight into the poorly understood mechanisms behind archaeal QS and equally will reveal the role of these compounds in cross-kingdom signalling.

Comparisons of pulmonary microbiotas associated with healthy individuals and cystic fibrosis (CF) patients identified a less diverse microbiota among CF patients comprising numerous bacterial species not found among healthy controls. This research aimed to identify if coadhesion occurs between respiratory pathogens P. aeruginosa and S. aureus and some commensal species associated with the microbiota of CF. This was conducted in vitro by spectrophotometric co-aggregation analysis and multi-species biofilm assays in the presence and absence of an inhibitor of lectin-mediated coaggregation, testing the ability of eleven commensal representative species of the CF pulmonary microbiota. Results show that all commensal species selected to represent genera that are associated with the CF pulmonary microbiota are capable of coaggregation in vitro with respiratory pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. S. aureus co-aggregated with a higher affinity to commensal species than P. aeruginosa with a median coaggregation percentage of 62.9 % after 24 h incubation, compared to 41.9 % in P. aeruginosa. When commensal and pathogenic species were cultured in the presence of lactose, biofilm inhibition was observed in 5 of 12 cultures with P. aeruginosa and 7 of 12 with S. aureus. These results demonstrate that some commensals associated with CF participate in coaggregation with respiratory pathogens in vitro which has the ability to alter biofilm production. Identifying coadhesion between commensal and pathogenic species in vitro suggests that coadhesion could be occurring in vivo potentially causing increased susceptibility of the host by enhancing adhesion to the epithelial surface of the respiratory tract, aiding colonization and therefore increased susceptibility to infections.

One of the most severe health threats to both humans and animals is Antimicrobial resistance development in microorganisms AMR is a significant and growing challenge and to combat this issue new antimicrobials are urgently needed Fifteen microbial strains isolated from Arabian Sea were screened for their capacity to produce antimicrobial metabolites of pharmaceutical interest. These strains were associated to the brown seaweed Pelvetia canaliculata (Linnaeus) attached to the rocks of Sonmiani Beach (Karachi, Pakistan). Bacterial strains were isolated from sea weeds attached to rocks of Baluchistan coast line using marine agar 2216 and screened for antibacterial activity by agar well diffusion method and crude extract was made and antimicrobial metabolites were purified using silica gel column and structure of pure compound was elucidated using spectroscopic techniques. Crude extract filtrates from CMG 2180 strain, grew on ZMA medium, showed the most remarkable antimicrobial activity, and thus was chosen for further examination. The identification of CMG 2180 as a probable new type strain of the Actinobacterium Kocuria marina was based on phenotypic aspects and biochemical characteristics (e.g. halotolerant Gram-positive micrococcoid) as well as on the nucleotide sequence analysis of its full-length 16S rRNA gene showing the highest similarity with the type strain KMM 3905 (GenBank accession number EU073966). Interestingly, a unique UV-bioactive compound, for which the name of kocumarin was proposed, was isolated and purified from CMG 2180 strain’s crude extracts by flash silica gel column chromatography and TLC/HPTLC. Using routine methods, kocumarin demonstrated prominent and rapid activities against all tested fungi and pathogenic bacteria including MRSA. Its chemical structure was unraveled by 1D and 2D-NMR spectroscopy as 4-[(Z)−2 phenyl ethenyl] benzoic acid.

Antimicrobial resistance (AMR) is a growing threat to healthcare. Many AMR-encoding genes are carried on accessory genetic elements like plasmids, which spread between phylogenetically distant members of bacterial communities. CRISPR-Cas based technologies may help combat the spread of AMR genes by removal of such plasmids. To optimise this technology, implementation of a delivery method which can reach a diverse range of bacteria is needed. Therefore, we engineered broad host-range conjugative plasmid pKJK5 to express Cas9. pKJK5::Cas[GmR] encodes a guide RNA which targets Gentamicin resistance gene aacC1, while pKJK5::Cas[nt] encodes a non-targeting guide RNA. After confirming its Ca9 activity by electroporation of targeted and untargeted plasmids, we set up a proof-of-concept experiment to conjugatively deliver pKJK5::Cas from a donor Escherichia coli strain DH5α to unrelated recipient E. coli K12, and to remove Gentamicin-resistance encoding target plasmid pHERD30T. For these means, we mixed donors and recipients in liquid media and incubated them overnight; proportions of the total population were enumerated by differential plating. We found that Gentamicin-resistant recipients were reduced by 33 % when treated with pKJK5::Cas[GmR] compared to treatment with pKJK5::Cas[nt], indicating that pKJK5::Cas[GmR] has the ability to remove AMR plasmids from recipient cells. This proof-of-concept experiment shows how an engineered broad host-range conjugative plasmid is an effective means of removing AMR-encoding plasmids and may be a viable approach to remove resistance genes from complex bacterial communities. To make this method more effective, community experiments as well as optimisation of Cas9 activity are needed.

In healthy adults, the gastrointestinal tract harbours a diverse community of microbes that play critical roles in health and wellbeing. However, we are not born with this microbiome. Over the first months and years of life the gut microbiome gradually develops, undergoing a process akin to classic primary succession. We here develop ecological theory in order to study the factors that drive these assembly processes. We find that interactions between species can enforce order on microbiome development, with interspecies dependencies driving the predictability of succession. We combine this theory with novel sample processing techniques to interrogate both bacterial and fungal microbiome development in premature infants. We utilize machine learning to infer how members of the microbiome are affected by both one another and clinical interventions. Preliminary results identify specific inter-microbial interactions that may in part drive community dynamics, and identify the impact of antibiotic interventions in perturbing healthy microbiome development. Taken together, these results highlight the importance of disentangling microbial interactions if we are to understand and ultimately manipulate our microbiome communities.

The emergence of antibiotic resistant, hypervirulent strains of the nosocomial pathogen Clostridioides difficile, coupled with increased concerns over the physiological impact of traditional broad-spectrum antibiotics on human health has led to an increased demand for alternative therapies. Bacteriocins are proteinaceous toxins produced by bacteria that kill other bacteria and their antimicrobial activity makes them an attractive potential alternative therapy to antibiotics. This research aims to isolate environmental bacteria from porcine faeces that produce novel bacteriocins with inhibitory activity against C. difficile. At present in this study two distinct organisms with protein mediated inhibitory activity against C. difficile have been isolated and it has been shown that these strains encode various bacteriocins. The current focus of this research aims to attribute the inhibitory activity to one of the encoded bacteriocins or potentially a novel antimicrobial peptide, which could then be exploited as an alternative therapy against C. difficile.