- Volume 2, Issue 7A, 2020

Starting from a yeast suppressor screening platform, we have identified the SKI complex as a potential broad-spectrum antiviral target. We found that the NS1 protein of influenza A virus (IAV) and the ORF4a protein of Middle East respiratory syndrome coronavirus (MERS-CoV), which both function to bind double-strand RNA and inhibit cellular interferon responses, cause a slow growth phenotype when expressed in yeast. Knockout of the components of the yeast SKI complex caused a loss of this slow growth phenotype, suggesting a functional link between the viral proteins and the SKI complex. The SKI complex is a helicase that unwinds double-strand RNA and sends it to the RNA exosome for degradation. We next investigated whether the highly conserved human SKI complex was important for replication of IAV and MERS-CoV. RNAi based experiments showed that both viruses were inhibited when the SKI complex was removed, suggesting the complex has a proviral role in replication. Through in silico modelling using the published crystal structure of the SKI complex, we looked for potential binding pockets for chemical compounds. We screened a selection of these compounds for antiviral activity and have found four different chemicals capable of inhibiting IAV infection. Our most studied of these also inhibits not only MERS-CoV, but also Ebolavirus Makona. Our data suggests the SKI complex may be a target for broad-spectrum antiviral therapy and we have multiple chemical structures from which to work to develop therapeutic approaches.

This study describes the analysis of DNA from heat-killed (boilate) isolates of Mycobacterium tuberculosis from two UK outbreaks where DNA was of sub-optimal quality for the standard methodologies routinely used in microbial genomics. A blunt-end Ligation based DNA library construction method, developed for sequencing ancient degraded DNA, was successfully used to obtain whole genome sequences from the sub-optimal samples. Thus, allowing analysis of the outbreak by WGS gene-by-gene MLST, SNP mapping and phylogenetics to be achieved. All cases were spoligotyped to the same Haarlem H1 sub-lineage. This is the first described application of ancient DNA library construction protocols to allow whole genome sequencing of a clinical tuberculosis outbreak. Using this method it is possible to obtain epidemiologically meaningful data even when DNA is of insufficient quality for standard methods.

Keywords: Mycobacterium tuberculosis, whole genome sequencing, outbreak investigation, ancient DNA, library construction, palaeogenomics.

Viral genomes are characterised by having high gene density and complex transcription strategies. One of the most complex is adenovirus which has a double stranded DNA genome and is the archetypal viral system in which splicing was first discovered. Understanding the transcriptional landscape using conventional mRNA cloning or more recent Illumina-based deep sequencing methods offers insight but also has limitations, including the potential for reverse transcription or PCR amplification artefacts and bias. Here we used direct RNA long read length sequencing on an Oxford Nanopore MinION device to gain a quantitative system-wide overview of transcription and splicing as it dynamically changes during a human adenovirus type 5 infection. This global overview revealed an extensive and hitherto unappreciated complexity of alternative splicing and secondary initiating codon usage. Allied to this, analysis of viral polyadenylation patterns over time showed that most viral transcripts tended to shorter polyadenylation lengths as the infection progressed. Moreover, development and use of an ORF-centric bioinformatics pipeline for analysis of sequenced mRNA, provided both a quantitative and deeper qualitative understanding of the genetic potential of this virus. The data strikingly illustrated that across the viral genome adenovirus made multiple distinctly spliced transcripts that coded for the same ORF. Indeed, as many as 11,000 different splicing patterns were recorded across the viral genome over the three time points analysed. This constitutive low level use of alternative splicing patterns and secondary ORFs potentially enables the virus to maximise its coding potential over evolutionary timescales.

Tuberculosis co-infection with other bacterial pathogens is one of the major health problems especially in tuberculosis endemic region. Bacterial pathogens that localized in the lower respiratory tract are could sometimes preclude Mycobacterium tuberculosis or manifest as pulmonary symptoms that could mislead clinicians. Therefore this study was planned to examine suspected TB patients in Kano, Nigeria for other pathogenic bacteria with view to establishing frequency of occurrence towards facilitating better outcome of therapeutic cover. A total of 170 non duplicated sputum samples were collected from patients with suspected pulmonary tuberculosis. All the samples were processed according to standard bacteriological procedures including macroscopic examination, culture and microscopic examination using both Acid Fast Bacilli (AFB) staining and Gram staining methods. Isolated organisms were subjected to appropriate biochemical testes. High incidences were observed among age group 10 – 29 and 30 – 49 years with male and urban people been the predominant patients. Overall 20.6% of samples were AFB positive indicated TB co-infection while 62.4% were positive for other bacterial growth. Nalidixic acid and Nitrofurantoin found to have highest resistant among Gram negative organisms isolated following by Levofloxacin, Ceftriaxone and Ceftazidime while the Ofloxacin and Ciprofloxacin have the least resistant. Among Gram positive Ceftazidime shown to has the highest resistant and Ofloxacin found to has the least resistant. The study indicated predominance of Streptococcus pneumoniae (47.2%) and Staphylococcus species (32.1%) with an evidence of 15.1% and 2.8% co-infection with TB respectively. x

Keywords: Mycobacterium tuberculosis, Co-infection, Bacterial pathogens, pulmonary tuberculosis.

Multiple sclerosis (MS) is an extremely debilitating auto-immune disease of people characterised by demyelination of the central nervous system and progressive neurological dysfunction. The etiology of the disease is complex (including factors such as genetics, sex hormones and vitamin D levels). The involvement of viral infections as a risk factor in triggering disease has long been suspected and two classes of viruses in particular, the herpesvirus Epstein-Barr virus (EBV) and the Human endogenous retrovirus “W” family (HERV-W) have been the focus of much recent research. Despite near ubiquitious infection (EBV), or integration into the genome as a repetitive element no longer able to function as a virus (HERV-W) a picture is gradually emerging of how these are involved in MS pathogenesis. In the case of EBV, having had infectious mononucleosis (clinical disease in patients infected post-puberty) increases the risk of developing MS. For HERV-W our recent work has confirmed that RNA is over-expressed in MS patients compared with healthy controls (though basal levels vary with ethnic background) and that EBV infection of B cells triggers expression of HERV-W RNA and proteins. There is a growing body of evidence that expression of HERV-W can trigger innate immune system inflammatory responses. There is also increasing evidence for molecular mimicry between epitopes of the HERV-W env protein, the EBV EBNA1 protein and peptides from brain proteins implicated in MS pathogenesis. Crucially these peptides are also able to bind the HLA-DR2b locus that is the strongest genetic risk factor for MS development.

Gene transfer agents (GTAs) are genetic elements that have the potential to carry out high frequency horizontal gene transfer. GTAs are similar to small bacteriophages in many ways but, instead of prioritizing the spread of their own genes, they package and disseminate the entire genome of their bacterial host. Indiscriminate transfer of bacterial genes could clearly have a major impact on bacterial evolution, fitness and antimicrobial resistance. Over the past decade, numerous pleiotropic systems have been shown to influence GTA production (e.g. nutrient stress, quorum sensing, SOS response etc.) but all act indirectly and so the mechanism of activation has remained elusive. I will present recent work that identified the missing link that couples GTA production to host regulatory pathways. I will also offer insights into the mechanism of random DNA packaging, the evolutionary role of GTAs in the environment and their potential ecological niche.

Accessory genes, such as antibiotic resistance genes (ARGs) can spread horizontally by mobile genetic elements, including plasmids and (temperate) bacteriophages leading to increased bacterial fitness in particular environments. In contrast to plasmids, temperate phages i.e. viruses that can incorporate their own genetic material into their host bacteria (then called lysogen) can additionally increase their hosts’ fitness through their ability to kill phage-susceptible competitors. However, in contrast to ARG-transfer by plasmids (conjugation), which has been extensively studied, ARG-transfer by phages (lysogenization) has received far less attention. Here we combined experiments using E. coli, phage lambda and the Rp4 plasmid (phage and plasmid both with an ampicillin resistance gene) and mathematical models to test which mechanism (lysogenization or conjugation) dominates under which conditions.

In the absence of selection and in two-species experiments (Donor & Recipient), conjugation was significantly more common than lysogenization. However, in three-species experiments (DonorPlasmid, DonorPhage& Recipient) lysogenization rates increased by several orders of magnitude. By using phage-friendly environments that favour phage adsorption we were able to shift the ratio between lysogens and transconjugants even further towards lysogens and in extreme-events to the extinction of transconjugants. Mathematical models additionally allowed us to investigate how starting population sizes of both donors and the recipient influence conjugation and lysogenization dynamics. Taken together, our results suggest that plasmids and temperate phages can influence each other’s transfer rates which, in the present study system, seems to be largely driven by population size effects.

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. A particular controversy has existed around the mechanism of ammonium exchange by the ubiquitous Amt/Mep/Rh transporter family, an essential process in all kingdoms of life. Here, using a combination of SSME electrophysiology, yeast functional complementation, and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+transport in two archetypal members of the family. The pathway underpins a mechanism by which charged H+and neutral NH3 are carried separately across the membrane after NH4+deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.

As the global population grows there is an urgent need for increased, yet sustainable civil infrastructure. The ability to harness biological processes in order to improve ground stability; as well as creating construction materials without adding to climate damage is necessary. In almost every environment on earth, microorganisms and microbially mediated mineralisation (biomineralisation) processes are active. It is well documented that microbes present in soil can induce the precipitation of calcium carbonate (CaCO3) in both the laboratory and the natural setting through microbial induced calcium carbonate precipitation (MICCP). MICCP utilises microorganisms as a result of their active metabolism, to precipitate CaCO3, strengthening the surrounding matrix. MICCP is used in a variety of different applications such as carbon sequestration, environmental remediation and improving construction materials.

The enzyme urease catalyzes the hydrolysis of urea to ammonia and CO2, and is acknowledged to be instrumental in MICCP. Bacillus subtilis is a model, gram positive, spore-forming soil bacterium that produces a functionally active urease, but with low efficiency, and the activation is not completely understood. Sporosarcina pasteurii is one of the most commonly used MICCP microbes as its urease operon has been well studied and the bacterium has proven to produce ecologically stable bioconstruction materials. The ability to clone the urease operon of S. pasteurii into the model B. subtilis would create an engineered ureolytic organism whose urease activity could be controlled. This control would enable the CaCO3 morphology and material properties to be tailored and would create a truly responsive biomaterial.

Despite their well‐known importance to the holobiont homeostasis, carbohydrate‐active enzymes (CAZymes) from the sponge microbiome have to date been largely understudied from an industrial perspective. Enzymes that degrade marine polysaccharides (MPs) are receiving increasing attention as alternatives in the biorefinery sector and for use in the generation of high‐value‐added hydrolysis products. Screening of the agarolytic Pseudoalteromonas sp. strain PA2MD11, isolated from the Brazilian sponge Plakina cyanorosea, on selective media indicated that the strain degrades agarose, κ‐carrageenan and sodium alginate. Subsequently in silico analysis of the strain's genome indicated that around 3 % of the total genome encoded for CAZymes. These consisted mostly of glycoside hydrolases (GH) and glycosyltransferases (GT). Four agarase genes were also present, three from the family GH50 and one from the family GH16, with sequences from other free‐living and psychrophile Pseudoalteromonas genomes as its closest relatives. These agarases are structurally related to exo‐(GH50) and endo‐β‐agarases (GH16), and appear to encode for a complete agarolytic pathway. The two alginate lyases were classified as polysaccharide lyases (PL) from the families 6 and 17, with chondroitin‐ and heparin‐like domains, respectively, and shared common homologues sequences with an estuarine Pseudoalteromonas lipolytica strain. Data on the cloning and heterologous expression of these genes will be presented. To our knowledge, this is the first time that a “genome mining” based approach has been undertaken to identify these groups of MPs‐degrading CAZymes from cultivable members of the sponge microbiome.

Cells generate phenotypic diversity both during development and in response to stressful and changing environments, aiding survival. Functionally vital cell fate decisions from a range of phenotypic choices are made by regulatory networks, the dynamics of which rely on gene expression and hence depend on the cellular energy budget (and particularly ATP levels). However, despite pronounced cell-to-cell ATP differences observed across biological systems, the influence of energy availability on regulatory network dynamics is often overlooked as a cellular decision-making modulator, limiting our knowledge of how energy budgets affect cell behaviour. Here, we consider a mathematical model of a highly generalisable, ATP-dependent, decision-making regulatory network, and show that cell-to-cell ATP variability changes the sets of decisions a cell can make. Our model shows that increasing intracellular energy levels can increase the number of supported stable phenotypes, corresponding to increased decision-making capacity. Model cells with sub-threshold intracellular energy are limited to a singular phenotype, forcing the adoption of a specific cell fate. We suggest that energetic differences between cells may be an important consideration to help explain observed variability in cellular decision-making across a broad range of biological systems, including bacteria and the blood stem cell system.

Rinderpest, a once much feared livestock disease, was declared eradicated in 2011, however virus-containing material is still held in laboratories worldwide. Prior to the destruction of our institute’s stocks, we determined the full genome sequence of the distinct samples of rinderpest virus (RPV) in our repository. This data would decipher the historical epidemiology of RPV and allow for recovery of the virus should the need arise.

For each sample (n=123), sequencing libraries were prepared using either transposon-based fragmentation of cDNA (Nextera XT DNA Library Prep kit) or single primer isothermal amplification (Trio RNA-Seq kit) and sequenced on the Illumina MiSeq. Regions of low or no coverage were re-sequenced using a Sanger sequencing approach.

Examination of the sequences of RPV isolates has shown that the African isolates form a single disparate clade, rather than two separate clades as was previously believed. We have also identified two groups of goat-passaged viruses which have acquired an extra 6 bases in the long untranslated region between the matrix and fusion protein coding sequences, and a group of African isolates where translation of the fusion protein begins from a non-standard start codon (AUA). In addition, the viruses that were force-passaged through alternate hosts such as rabbits or goats, appear to diverge from the clades that represent viruses which were maintained in the wild.

Our unique set of sequence data will be invaluable for forensic epidemiology investigations in the event of an unforeseen outbreak and aid in the understanding of the evolution of related morbilliviruses.

The flavivirus dengue virus (DENV) is the most significant arthropod borne virus (arbovirus) of humans, causing serious morbidity and mortality, with nearly half of the world’s population at risk of infection. Due to a lack of antivirals and limited vaccine options, vector control remains a vital defence against dengue disease. The mosquito Aedes aegypti is the major vector for DENV, and understanding mosquito immune responses and how DENV may evade them is critical. We have shown that DENV-2 can inhibit the exogenous induction of immune deficiency (IMD) signalling by classical immune stimuli. Therefore, we aimed to identify DENV antagonists of the IMD pathway, and define the molecular virus and host determinants of IMD antagonism in a well characterised Ae. aegypti derived cell line, Aag2. Each DENV protein was expressed individually in Aag2 cells and tested for their ability to block IMD signalling induced by exogenous stimuli. This screen identified NS4A as a potential antagonist of the IMD pathway. Further, we have found that the N-terminus of NS4A is responsible for this inhibition. The antagonism of IMD signalling is specific to flaviviruses transmitted by a mosquito vector, illustrating the importance of both the IMD pathway for mosquito immunity and the antagonism of this pathway by DENV. By enhancing our understanding of how DENV evades the mosquito immune response at a molecular level, we will gain insight into virus-host interactions constraining arbovirus transmission and emergence, which may be exploited for developing transmission-incompetent vectors to reduce the burden of dengue disease.

The widespread use and misuse of antibiotics has led to the global spread of antimicrobial resistance. It is increasingly evident that very low concentrations of antibiotics, well below the MIC of sensitive strains, can select for antimicrobial resistance. However, it is less clear how social interactions within bacterial communities can alter sub-MIC selection dynamics, potentially confounding the outcomes of antibiotic treatments. Here we explore how antimicrobial resistances that inactivate antibiotics can be socially exploited by sensitive members of the microbial community at the inter and intra species level. We first show that a beta-lactamase encoded multi-drug resistance plasmid provides high levels of protection to plasmid-free antibiotic-sensitive cells within single-species populations in a frequency-dependent manner. Second, a similar protection can also occur between different species during polymicrobial infections. Using model Cystic Fibrosis lung communities, we demonstrate that the focal pathogen Pseudomonas aeruginosa can socially exploit antibiotic resistance in the presence of Stenotrophomonas maltophilia bacterium that can hydrolyse imipenem antibiotic. In contrast, the presence of Staphylococcus aureus, another commonly co-occurring CF pathogen, provided P. aeruginosa with no protection, but instead, made P. aeruginosa more susceptible to antibiotic due to intensified competition. These findings reveal that social exploitation of pre-occurring antimicrobial resistance, and inter-specific competition, can have a large effect on the efficacy of antibiotic treatments, highlighting the importance of microbial ecology for understanding antibiotic resistance evolution.

Myxobacteria are Gram-negative bacteria, notable for their predatory and antimicrobial activities, which dictate the outcomes of their interactions with neighbouring organisms. They are abundant and widespread in nature, and can significantly affect the microbiome of an environment.

We hypothesise that there are underlying molecular mechanisms in prey specieswhich govern the prey’s susceptibility/resistance to the antimicrobial activity of myxobacteria. In this work we attempt to define the mechanisms by which Pseudomonas aeruginosa PA14 resists predation bythe model myxobacterium Myxococcus xanthus.

Pseudomonas aeruginosa is an opportunistic pathogen of humans and plants. With the rise in antibiotic resistant organisms, Pseudomonas spp. are categorised as World Health Organisation priority 1 antibiotic-resistant bacteria and are our prey of choice in this study.

In collaboration with Dr N. Tucker (Strathclyde), and using a strain of M. xanthus expressing mCherry (courtesy of E. Hoiczyk, Sheffield), we developed 96-well plate assays of predation which measured the optical density of both predator and prey and the florescence of predator at different point intervals. Predation was assayed againsta library of approximately 5700 PA14 mutants to identify strains with increased/decreased susceptibility to predation. Responses of PA14 mutants varied with time and between mutants, allowing us to create a shortlist of candidate genes involved in the prey response to predation.

We are currently performing a preliminary analysis of the data using the Integrated Genomic Viewer and Circos plots to assess the genomic organisation of the prey genes that influence susceptibility and resistance to predation.

Several eukaryotic lineages gained the ability of photosynthesis by acquiring plastids in the events of primary endosymbiosis with cyanobacteria, or secondary endosymbiosis with plastid-bearing eukaryotes. Plastids possess genomes (ptDNA) with genetic contents considerably reduced as a result of gene losses and transfers to the host’s nucleus. Still, ptDNA encodes components of various metabolic processes, including photosynthesis. Plastid genomes usually retain quadripartite structure with two rDNA-bearing inverted repeats, but the reason for its conservation, and the consequences of its decline, have not been fully understood.

As the model group to study plastid genome evolution, we chose euglenids (Euglenophyta), whose ancestor acquired the secondary plastid by endosymbiosis with a green alga. The organization of ptDNA in this lineage is rather diverse: we have shown that loss of one repeat occurred at least three times, while some species in the genus Euglena possess tandemly repeated rDNA copies. The ptDNA of euglenids is also intron-rich, but we did not confirm the previously proposed strong correlation between the prevalence of introns and quantity of maturases.

Although euglenophytes are predominantly photosynthetic, a few of them lost their photosynthetic capabilities independently. Thus far, only Euglena longa has been shown to possess vestigial plastids with reduced genome; we observed that another strain lost its plastid genome completely. Currently, we are investigating the loss and retention of metabolic functions in the plastids of other non-photosynthetic euglenophytes. This, along with investigation of ptDNA structure, will bring new insights into the evolutionary processes shaping the diversity of eukaryotic plastids.

The airways of persons with cystic fibrosis (CF) provide a nutritionally rich environment that is prone to colonisation by a diverse and dynamic community of microbes (including both bacteria and fungi). Traditionally, Pseudomonas aeruginosa (PA) was thought to be the dominant pathogen associated with CF airway infections. However, it is becoming increasingly clear that interspecies interactions play a crucial role in modulating the physiology and gene expression profile(s) of the protagonists, and also impact on the response to therapeutic intervention. However, there are currently no suitable experimental models that permit long-term successful co-cultivation of PA with other CF-associated pathogens. Simply mixing bacterial or fungal species together and hoping for the best is not a recipe for success, since PA rapidly outcompetes most other species when grown in batch culture. In this work, we rectify this by describing a “3Rs-compliant” continuous-flow in vitro co-culture model. Using our model, it is possible to maintain remarkably stable steady-state co-cultures of major CF-associated pathogens (PA, Staphylococcus aureus and Candida albicans). Our findings reveal that even numerically minor (0.1%, by cell number) species can have a profound impact on quorum sensing and virulence factor production by PA. Furthermore, we show that complete polymicrobial communities derived from CF sputum can be inoculated directly into the model, thus enabling the recapitulation of the entire microbiome associated with CF airway infections to be studied under physiologically relevant conditions.

Varroa destructor is an ectoparasitic mite associated with significant losses of honey bee colonies globally. The mite vectors a range of pathogenic viruses, most important of which is Deformed Wing Virus (DWV, (+)ssRNA). Overwintering colony losses, accounting for the death of ∼25% of all colonies each year, are associated with high levels of Varroa-DWV infestation. Effective miticide treatments are available to control Varroa. However, the absence of coordinated treatment means environmental transmission of mites continued unchecked. We aim to determine whether rational, coordinated treatment is beneficial, using features of the DWV population as an indicator of colony health. This study uses coordinated treatment of Varroa in a geographically isolated environment (Isle of Arran, Scotland). It is reported that a high level of a near-clonal virus population is associated with Varroa infestation and colony losses, whereas Varroa-free healthy colonies carry only low levels of a diverse population of DWV. The study area contains 50-85 colonies and 25 beekeepers. Sampling and virus analysis – strain diversity and viral loads – have been conducted before and after treatment. Changes in virus diversity are quantified by next generation sequencing analysis to determine population diversity. In the first two years we have observed a geographic decrease in Varroa and changes in the composition of the virus population. This study will inform our development of rational Varroa control strategies for beekeepers in temperate regions and could be used to inform policy changes regarding treatment regimes in Scotland and elsewhere for this global pathogen.

Alphaviruses are arthropod-borne positive-sense RNA viruses that have the capacity to cause large scale outbreaks of severe disease. As of now, there are no effective therapeutic strategies with which alphaviral disease may be treated. Thus, there is a need for research that defines the mechanism(s) by which the alphaviruses establish infection and cause pathogenesis. Our previous work identified non-assembly interactions between the SINV Capsid protein and the viral genomic RNA that were important for viral RNA stabilization early during infection. These efforts led us to evaluate the Protein:Protein interactions of the SINV Capsid protein using the BioID2 discovery approach to define the mechanism of action underlying the SINV Capsid-mediated genome stability.

To our surprise, these efforts indicated that the SINV CP protein interacts with the host IRAK1 protein in tissue culture models of infection. To validate the interface we utilized a Bimolecular Fluorescence Complementation approach to confirm the SINV Capsid-IRAK1 interaction. After confirming the novel Capsid Protein:Protein interaction we hypothesized that the SINV Capsid protein may interfere with IRAK1-dependent signaling during infection. To this end, we assessed the dose-responsiveness of several IRAK1-dependent signaling pathways, including TLRs 4 and 7 in the presence of the SINV Capsid protein. We found that the TLR-agonist response, was significantly decreased in the presence of the SINV Capsid protein. Collectively, these data are highly suggestive that the SINV Capsid protein interferes with TLR signaling during viral infection contributing to the evasion of the host innate immune response.

Background: Biocoatings are nanoporous polymer materials which encapsulate bacterial cells with carbohydrates as osmoprotectants. Here, we optimised biocoatings to offer a favourable environment for the metabolic activity of bacteria.

Methods: E. coli were used as a model organism and mixed with the colloidal polymer particles (i.e. synthetic latex), inorganic nanoparticles and different carbohydrates. Films were casted and dried to create a coalesced latex film and finally rehydrated to re-establish bacterial metabolism. The toxicity of the sterile latices to the bacteria was tested by using the colourimetric redox indicator resazurin. Visualisation of the bacteria inside the biocoatings was performed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM).

Results: We introduced halloysite (clay nanotubes) to create nanoporosity, which created voids in the structure that will permit gas exchange. The biocoatings were tested in liquid and rehydrated states with resazurin to find the most promising composition ensuring bacterial viability. Rehydrated biocoatings were visualised by CLSM by tracking the constitutively expressed yellow-fluorescent protein (YFP) for viable cells and the membrane exclusion dye propidium iodide for dead cells. The structure of the biocoatings appeared to be unaffected by freeze-drying compared to chemical fixation. Following this fixation, SEM allowed the observation of the organisation of the latex polymers, halloysite and bacteria.

Conclusions: The biocoatings were highly porous thanks to halloysite. E. coli survived the film formation process. Next, we will use E. coli and cyanobacteria to achieve higher efficiency for a variety of applications e.g. pollutant degradation, solar energy harvesting and carbon recycling.

The Sulfolobus Spindle-shaped Virus (SSV) system is a model for studying thermophilic archaeal virus biology. Several factors make the SSV system amenable to studying archaeal genetics and virus-host interactions in extreme environments. It has been shown that populations of Sulfolobus, the natural host, exhibit biogeographic structure. The acidic (pH<4.5) high temperature (65-88°C) habitats have low biodiversity, which diminishes prospects for host switch. SSVs and their hosts are readily cultured in liquid media and on plates. Given the wide geographic separation between various SSV-Sulfolobus habitats, the system is also amenable to studying allopatric versus sympatric virus-host interactions. We previously reported that SSVs exhibit differential infectivity on allopatric and sympatric hosts. We discovered a strikingly broad host-range for strain SSV9 (a.k.a., SSVK1). For decades, SSVs have been described as “non-lytic” dsDNA viruses that infect species of Sulfolobus and release virus particles via blebbing as a preferred strategy over host lysis (in reported laboratory infections). Here, we show, that SSVs infect more than one genus of the family Sulfolobaceae and, in allopatric hosts, SSV9 does not release virions via blebbing. Instead, SSV9 appears to lyse all susceptible allopatric hosts, while exhibiting canonical non-lytic virion release (historically reported for SSVs) on a single sympatric host. Lytic versus non-lytic virus release does not appear to be driven by multiplicity of infection. Data suggest that SSV9 is more stable than other SSVs in suspension; however, genetic substrates (e.g., CRISPR profiles) underlying non-lytic versus lytic virion release remain unresolved and are the subject of ongoing investigation.

The diversity of subtypes within Influenza A recently expanded with identification of H17N10 and H18N11 from bats. To study the tropism and zoonotic potential of these viruses, we successfully produced lentiviral pseudotypes bearing haemagglutinin H17 and neuraminidase N10. We investigated a range of cell lines from different species for their susceptibility to infection by these pseudotypes and show that a number of human haematopoietic cancer cell lines and the canine kidney MDCK II (but not MDCK I) cells are susceptible. Using microarrays and qRT-PCR we show that the dog leukocyte antigen DLA-DRA mRNA is over expressed in late passaged parental MDCK and commercial MDCK II cells, compared to early passaged parental MDCK and MDCK I cells, respectively. The human orthologue HLA-DRA encodes the alpha subunit of the MHC class II HLA-DR antigen-binding heterodimer. Small interfering RNA- or neutralizing antibody-targeting HLA-DRA, drastically reduced the susceptibility of Raji B cells to H17-PV. Conversely, over expression of HLA-DRA and its paralogue HLA-DRB1 on the surface of unsusceptible HEK293T/17 cells conferred susceptibility to H17-PV. The identification of HLA-DR as an H17N10 entry mediator will contribute to understanding the tropism of the virus and help to elucidate its zoonotic transmission. We also show that H17 pseudotypes can be efficiently neutralised by the broadly-neutralizing HA2 stalk monoclonal antibodies CR9114 and FI6. The lentiviral pseudotype system is a useful research tool, amenable for investigation of bat influenza tropism, restriction and pandemic preparedness, without safety issues of producing a replication-competent virus, to which the human population is naïve.

Cellular surface expression of CD81 (an essential co-receptor for HCV) is critical for successful HCV infection. Furthermore, CD81 cross-linking with HCV-E2 protein impedes activation signaling pathways in different lymphocytes (T-cells, B -cells and NK cells). The expression of CD81 on peripheral lymphocytes is known to be downregulated following successful dual anti-HCV therapy. On the other hands, no reports are yet available regarding its expression levels following the newly used treatment regimen in Egypt; direct-acting antivirals (DAAs): Sofosubvir & daclatsvir for three months. Thus, the aim of the current study was to evaluate the expression levels of CD81 on T and B lymphocytes in HCV-infected patients before and after successful treatment with DAAs. Cellular CD81 expression was measured on CD3+ (T lymphocytes) and CD19+ (B Lymphocytes) lymphocytes by flow cytometry from 19 patients with chronic HCV infection.

All the HCV viruses were of genotype 4. We found no correlation between CD81 expression on either CD3+ or CD19+ lymphocytes and viral load. The expression of CD81 on CD19+ lymphocytes was markedly reduced at the end of the treatment. On the contrary, CD81 was significantly increased on CD3+ lymphocytes following successful treatment.

Our data indicate that successful treatment of HCV infection is associated with a reduction in surface CD81 expression on B lymphocytes with a concomitant increase on T lymphocytes.

Herpes simplex virus 1 (HSV-1) is a prevalent neurotropic virus that persists for the host’s lifetime due to HSV-1 establishing latency in sensory neurons. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 1.5kb or 2.0kb major LAT intron and several microRNAs. These latency-associated non-coding RNAs (ncRNAs) have been reported to impact the establishment, maintenance and reactivation from latency. However, the molecular mechanisms of these ncRNAs are not fully characterised, especially in the context of human neurons.

This study investigated how the latency-associated ncRNAs affect the human neuronal transcriptome. We developed an experimental system to deliver the latency-associated ncRNAs to human neurons, differentiated from SH-SY5Y neuroblastoma cells. The cells were infected with a replication-defective HSV-1 mutant, in1382, that establishes a quiescent infection in which LAT is strongly expressed. Alternatively, we utilised lentiviruses engineered to express the first 3.1kb of LAT, without or with mutations in splice sites that prevents splicing of the major LAT intron, or five HSV-1 microRNAs, shown to be abundant in latently infected human ganglia. Following RNA-Seq of uninfected versus infected or transduced SH-SY5Y cells, we identified 178 host genes that had significant differential expression in response to in1382 quiescent infection and lentivirus delivery of LAT or the latency-associated microRNAs. A subset of these were validated by PCR. This work provides insight into possible roles of the latency-associated ncRNAs in neuronal cell biology and latency that could aid future investigations examining how HSV-1 latency affects human neurons.

Burkholderia species have environmental, industrial and medical significance, and are important opportunistic pathogens in individuals with cystic fibrosis (CF). Approximately 10% of Burkholderia genomes (6-9 Mb) are horizontally acquired material, representing a rich source of mobile genetic elements including prophages. There is limited research on Burkholderia bacteriophages, their contributions to genome evolution, virulence and antimicrobial resistance, or biotechnological and therapeutic applications. We investigated prophage carriage in Burkholderia and aimed to isolate and characterise inducible bacteriophages from B. vietnamiensis.

Burkholderia genomes were screened for prophages using PHASTER. Prophage genomes were compared using MASH and visualised with ProgressiveMauve. Phylogenomics was used to assess the distribution of prophages across B. vietnamiensis strains. Spontaneously induced phages were characterised to determine linkage between prophage regions and isolated phages, bacteriophage morphology and host range.

Prophage carriage across 456 Burkholderia strains (spanning 43 species) was high; 716 intact prophages were discovered and polylysogeny was common. In B. vietnamiensis alone, 115 prophages were identified from 81 strains, with evidence of shared prophage carriage between related and diverse strains. Three novel inducible phages were isolated from B. vietnamiensis strain G4 and their genomic origins localised putatively. One phage (vB_BvM-G4P1; family Myoviridae) had inhibitory activity against multiple strains of 5 B. cepacia complex species, including species prevalent in CF infections.

Prophages are numerous in Burkholderia genomes and contribute to strain diversity. There is huge potential for further investigation into the functional implications of prophage carriage and its impact on genome evolution, in addition to the isolation of novel bacteriophages.

Isoprene is the most abundant biogenic volatile organic compound (BVOC) on Earth, with annual global emissions almost equal to those from methane. Due to its volatile nature and high reactivity, isoprene plays a complex role in atmospheric chemistry and hence, climate. However, very little is known about its biological degradation in the environment. The vast majority of isoprene (500 Tg ·y-1) is produced by terrestrial plants and oil palm is considered one of the highest isoprene-producing trees, with estimated emissions of 175 μg·g-1 dry leaves ·h-1. Oil palm is also a heavily cultivated crop since it is the source of 30% of the vegetable oil in the world and in countries such as Malaysia represents >85% of total agricultural land. The vast expansion of a single crop that emits such high amounts of isoprene have raised serious concerns about its impact on air quality and climate change. We performed DNA Stable Isotope Probing (DNA-SIP) to study the isoprene-degrading community of oil palm trees in a Malaysian plantation and identified novel genera of isoprene-utilising bacteria in both oil palm soils and leaves. isoA amplicon sequencing data also confirmed that oil palm trees harbour a novel diversity of isoA genes, which encode the alpha subunit of the isoprene monooxygenase, a key enzyme in isoprene metabolism. In addition, metagenome assembled genomes (MAGs) were reconstructed from metagenomes from oil palm soil and leaf incubations and analysed to identify isoprene degradation gene clusters in these microorganisms. Finally, analysis of unenriched metagenomes showed that isoA-containing bacteria are more abundant in soils than in the oil palm phyllosphere.

Human cytomegalovirus (HCMV) is a pathogenic beta-herpesvirus that establishes a lifelong infection in hosts. It causes significant morbidity and mortality in the immunocompromised and is associated with a range of birth defects following congenital infection. Current therapeutic approaches that target key viral proteins are toxic and antiviral drug resistance is common. Thus, targeting host genes and cellular pathways essential for HCMV infection offers an alternative strategy for the development of antivirals. Here we show that host oxidative/nitrosative stress responses to CMV are critical for virus replication. Oxidative/nitrosative stress occurs due to accumulation of reactive oxygen/nitrogen species (ROS/RNS). Using a range of ROS/RNS scavengers, we identified that peroxynitrite, a powerful oxidant and nitrating agent, dramatically promoted virus replication in both in vitro and in vivo models of CMV infection. HCMV rapidly induced production of intracellular peroxynitrite upon infection. Inhibition of peroxynitrite within the first 24 hours alleviates efficient HCMV infection in both cell-free and cell-associated infection systems, indicating that peroxynitrite may influence pathways necessary for HCMV entry and/or replication. Furthermore, peroxynitrite inhibition also inhibited HCMV reactivation from latency. Interestingly, the neurotransmitter and naturally-occurring peroxynitrite antagonist 5-hydroxytryptamine, commonly known as serotonin, also impinged on HCMV-induced peroxynitrite production and exhibited anti-HCMV activity. Thus, overall, our study demonstrates a novel role for intracellular peroxynitrite in CMV pathogenesis and implies that peroxynitrite could be targeted as a novel approach to inhibiting CMV infection.

Shiga toxin-producing Escherichia coli O157: H7 (STEC) is a zoonotic pathogen that is globally dispersed, causing severe gastroenteritis when transmitted from ruminants to humans through direct or indirect contact with animals, their environment or contaminated food. Symptoms are varied in severity; from mild to bloody diarrhoea with more serious sequalae including hemolytic uremic syndrome (HUS) which can be fatal. Although there is compelling evidence that the Shiga toxin sub-type is a key predictor of disease severity, differences in virulence potential of strains with the same Shiga toxin profile are often observed. In this study, we employ machine learning algorithms to explore the relationship between the STEC genome with clinical outcome.

Kmer-counts of variable length (9-100 base pair) from 1148 isolates of STEC O157:H7, representing two years of routine surveillance in England, were matched to their respective clinical outcome data. A Random Forest classifier was developed and validated with the objective of inferring the clinical symptoms associated with a given STEC genome. Clinical outcomes were categorised into asymptomatic, diarrhoea, bloody diarrhoea and HUS. The model correctly classified 160 out of 190 cases of bloody diarrhoea, 81 out of 128 cases of diarrhoea and 7 out of 12 cases of HUS, with average AUC ROC score of 90%. Kmers deemed important for distinct classification were characterised and matches related to Shiga toxin 2a phage integration and excision genes and adhesion and transporter proteins were identified. This is consistent with reported virulence factors in the literature, supporting this approach of de novo pathogen characterisation.

Chlamydia abortus is the most commonly diagnosed cause of abortion in small ruminants around the world [1]. Control of chlamydial abortion is achieved in several European countries using an attenuated live 1B C. abortus vaccine strain, which can be distinguished from virulent wild-type strains by PCR-RFLP analysis [2]. Application of this method has provided molecular evidence that the 1B strain can cause abortion in ewes [3, 4]. The objective of this study was to define the distribution of lesions and bacterial load in cotyledons from ewes vaccinated with the 1B strain compared to normal wild-type infections.

A Chlamydia-free flock of 75 multiparous adult ewes were vaccinated twice, two years apart, each prior to mating, with the commercial 1B vaccine. In the second lambing season following the last vaccination, placentae (n=116) were collected and analysed by C. abortus real-time qPCR [3]. Only two of the placentae, both from the same ewe, were found to be positive. Viable organisms were isolated from these placentae and confirmed by RFLP-PCR [3] to be vaccine-type. All cotyledons from these placentae were analysed by histopathology and immunohistochemistry [5], and compared with those from wild-type infected placentae. The lesions in the vaccine-type infected placentae were indistinguishable from the wild-type infected placentae in terms of their severity, load and distribution.

These results suggest that the 1B vaccine strain of C. abortus can cause chlamydial abortion in ewes producing typical placental lesions to wild-type infected animals, and could be circulating with the potential to cause natural infection and disease.

Virus infectivity is commonly investigated with in vitro monolayer cell cultures or in vivo animal models. Ease of growth and manipulation and low cost characterise standard cell culture. Animal models allow investigation of infectivity in the context of tissue structure and environment but are costly and can be limited by species variations. Neither approach can recapitulate the human context, the tissue microenvironment and human immune components. Three-dimensional organotypic raft tissue models can provide most of the advantages of in vitro and in vivo models.

We successfully established HSV and oncolytic HSV (HSV1716) infection in 3D raft cultures of epithelial non-tumour (HaCaT) and tumour (SiHa, OVCAR3 and TOV21G) cell lines. Our 3D models allowed the evaluation and quantification of virus replication and the recovery of the virus both in culture media and tissues.

We developed a complex 3D co-culture of epithelial cells with human immune cells in order to mimic the tissue microenvironment. This innovation allowed us to study the effect of immune cells in cell killing by HSV1716 in the in vitro tissues. In HSV1716-infected co-culture tissues, immune cells were identified throughout the tissue and some migrated to the areas of infection. The immune activity was identified through increased IL-8 release. Moreover, combining infection with immune cell infiltration increased tumour cell killing in the 3D co-culture model. This new co-culture model could be further developed to identify the role of immune cells in oncolytic viroimmunotherapy and to dissect the involvement of specific single immune cell subpopulations.

Deep learning (DL) is a subset of Artificial Intelligence employing neural networks that require the use of a training set and are modelled on circuit pathways in the human brain. Whilst AI is a burgeoning field today, its roots are in the 1950s. DL algorithms use multiple layers to progressively extract higher level features from the raw input. Many different architectures of neural network exist, and for the most part are involved in applications with image recognition. Some recent uses of DL include self-driving cars; there are also creative projects using DL to create fake faces or human poses for use as models, composing music, creating novel art from different styles of art and writing fake news.

Streptomyces spp. are well-known as important producers of bioactive compounds such as antibiotics. These bioactive compounds are often encoded as secondary metabolites in the organisms by large gene clusters such as non-ribosomal peptide synthases (NRPS) and polyketide synthases (PKS). The NRPS and PKS assemble peptides using enzymatic units arranged in modules that can function in an iterative or sequential fashion independent of messenger RNA. Each NRPS or PKS is capable of assembling one type of peptide. Fusions of the two also exist.

Here we have trained deep learning neural networks to provide us with simulated “fake” secondary metabolite sequences. We examine the characteristics of these sequences and how they could be used to guide us with antibiotic discovery.

Background. BK virus (BKV) reactivation is a significant cause of BK Polyomavirus associated nephropathy (BKPyVN) resulting in acute graft rejection in 1-10% of post-renal transplant recipients. However, the association of BKV genotypes with development of BKPyVN is poorly understood. Here we aimed to determine the prevalence of BKV genotypes in post-renal transplant recipients, and its association with BKPyVN disease progression.

Method. Two methods were utilised to genotype BKV. A 800bp fragment of virus VP1 antigen region was amplified using nested (PCR) followed by sequencing. The genotypes were determined according to a previously developed algorithm based on analysing 100bp region of the VP1 gene. Furthermore, the logarithm results were validated with the constructed phylogenetic tree. The results were correlated with patient viral loads and development of BKPyVN.

Results. BK virus DNA was detected in 32 (69%) of 46 post-renal transplant recipients with BK viremia, while BKPyVN was only reported in two (4.3%)patients. 30 out of 32 samples were successfully genotyped (93.7%) with 23 (76.6%) belonging to the BKV Ib-2 subtype and seven (23.3%) belonging to the BKV Ib-1 subtype and with no cases representing genotype II, III and IV. All cases with confirmed BKPyVN matched to the BKV Ib-2 genotype. Additionally, no significance differences were observed between BKV genotypes in regards to viral loads, development of viremia, HLA mismatch, age or sex.

Conclusion. The results indicate no correlations between BKV genotypes and the development of BKPyVN. Furthermore,a high distribution of BKV genotype Ib-2 was found among BKV infected patients within this cohort.

Cyanobacterial blooms are a serious threat to public health and water quality due to the production of cyanotoxins as a result of nutrient pollution from industry, agriculture, domestic waste as well as global warming. The microcystins (MCs) are the most abundant cyanotoxins consisting of >200 analogues causing both acute and chronic toxicity, sometimes resulting in death. In Asian countries, such as Sri Lanka, reports of kidney disease are constantly increasing. Although no direct link between metal and pesticide contamination in water and kidney disease has been found, high concentration of cyanobacteria cells in drinking water wells implies that the nephrotoxic effects of cyanotoxins might play a key factor in the reports of Chronic Kidney Disease of unknown aetiology (CKDu) in Sri Lanka. Therefore, we propose a nature-based approach for water treatment which will study the hypotheses that cyanotoxins can cause CKDu. Sri Lankan bacterial isolates (Alcaligens sp., Roseateles sp., Bacillus sp., and Micrococcus sp.) known to degrade microcystins, were used to form biofilm on biochar from Sri Lankan crop residues, such as coconut shells. The immobilisation of the microbes was assessed via a high-throughput colourimetric assay, followed by monitoring the biodegradation rate of microcystins when added to the immobilised cultures. Biodegradation products were analysed and identified through molecular networking and quantified via LC-MS/MS. Ultimately, this project will provide safe water in line with UN Sustainable Development Goal 6.1 as well contributing in sustainable goals 7 (Affordable and Clean Energy), 11 (Sustainable Cities and Communities) and 12 (Responsible Production and Consumption).

Enterohaemorrhagic E. coli (EHEC) may instigate bloody diarrhoea and haemolytic uraemic syndrome (HUS) due to Shiga toxin (Stx) production. Stx has been detected within outer membrane vesicles (OMVs), which are membrane-derived nanosized proteoliposomes. During colonisation, EHEC encounters many environmental surroundings such as the presence of bile salts and carbon dioxide (CO2). Here, the influence of different intestinal cues on EHEC OMV production was studied. OMV yield was quantified by densitometric analysis of outer membrane proteins F/C and A, following OMV protein separation by SDS-PAGE. Compared to cultures in Luria broth, higher OMV yields were attained following culture in human cell growth medium and simulated colonic environmental medium, with further increases in the presence of bile salts. Interestingly, lower yields were attained in the presence of T84 cells and CO2. The interaction between OMVs and different human cells was also examined by fluorescence microscopy. Here, OMVs incubated with cells showed internalisation by semi confluent but not fully confluent T84 cell monolayers. OMVs were internalised into the lysosomes in confluent Vero and Caco-2 cells, with Stx being transported to the Golgi and then the Endoplasmic reticulum. OMVs were detected within polarised Caco-2 cells, with no impact on the transepithelial electrical resistance by 24 hours. These results suggest that the colonic environmental factors influences OMV production in vivo. Additionally, results highlight the discrepancies which arise when using different cells lines to examine the intestine. Nevertheless, coupled with Stx, OMVs may serve as tools of EHEC which are involved in HUS development.

Venezuelan Equine Encephalitis Virus (VEEV) is a positive sense RNA virus in the family Togaviridae. VEEV circulates in the Americas, causing occasional large scale epidemics. Our group has previously discovered and described the anti-VEEV compound ML336. We found that ML336 inhibits viral RNA synthesis during infection. This RNA synthesis inhibition is highly specific for VEEV, and ML336 has no effect on the closely related chikungunya virus, or on cellular RNA synthesis. We also found that this activity was maintained in a cell-free viral RNA synthesis system, supporting our hypothesis that ML336 is a direct acting antiviral compound. We recently discovered that treatment with ML336 reduces the amount of double stranded RNA present in infected cells. This reduction supports that ML336 is interfering with the synthesis of viral RNA. This was measured qualitatively with microscopy, and quantitatively with flow cytometry. We have also reported that resistant viral mutants emerge when grown in the presence of inhibitory compounds and these mutations mapped to the N terminal domains of both nsP2 and nsP4. This region of nsP2 has recently been shown to be a helical region which serves as an accessory domain to the viral helicase. However, the region of nsP4 in question currently lacks known function. Based on this genetic data we hypothesized that ML336 and related compounds interact with these two domains to interfere with the activity of the replicase complex. We are currently examining this interaction using ectopically expressed protein.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe respiratory infections in people who are immunocompromised. P. aeruginosa possesses the Type VI Secretion System (T6SS), a bacterial weapon that injects effectors into neighbouring prokaryotes and eukaryotes. The T6SS is crucial for bacterial warfare, allowing P. aeruginosa to kill its competitors, which promotes its dominance in mixed microbial environments. P. aeruginosa has three T6SSs, H1/2/3-T6SS, these are structural homologs but deliver unique effectors. Effectors are delivered via the secreted component, a Hcp tube topped with a VgrG and PAAR spike. Only the first three identified effectors are delivered by Hcp1. Since then, there has been a bias in identification of VgrG or PAAR delivered effectors, mostly as these are encoded next to the spike proteins. Some P. aeruginosa effectors not only kill bacteria but have a dual role in pathogenesis. Our aim was to identify a comprehensive set of Hcp-delivered effectors for all three systems. Using Hcp1/2/3, systematic pull-down screens were performed to identify novel interaction partners. After confirming interaction, antibacterial toxicity was evaluated, identifying new Hcp delivered T6SS effectors for Hcp2 and Hcp3, which are toxic in the bacterial cytoplasm. These new anti-bacterial effectors may kill bacteria in novel ways, which could lead to novel antibiotics. Additionally, a toxin fusion proved too large for secretion and blocked the T6SS, revealing a Hcp-delivered effector size limit. Future work will focus on fully characterising these new toxins, as well as to look into the potential eukaryotic role of other interaction partners.

Ion channels are a diverse class of transmembrane proteins that selectively allow ions across membranes, influencing a multitude of cellular processes. Modulation of these channels by viruses is emerging as an important host-pathogen interaction that regulates critical stages of the virus multiplication cycle including entry, replication and egress.

Human respiratory syncytial virus (HRSV) causes severe respiratory tract infections globally and is one of the most lethal respiratory pathogens for infants in developing countries, with frequent development of bronchiolitis. Furthermore, it is the most significant cause of hospitalisation of infants in the UK. Evidence also indicates that severe childhood HRSV infection contributes towards the increased incidence of adult asthma. No HRSV vaccine is available, and currently the only treatment is immunoprophylaxis which is prohibitively expensive and only moderately effective; thus new treatment options are required.

Utilising GFP-expressing HRSV in combination with an extensive panel of channel specific pharmacological inhibitors, we have identified an important role of cellular chloride (Cl-) channels during HRSV infection. Interestingly, pharmacological inhibition of specific Cl- channel families has ruled out involvement of the CFTR and instead highlighted a critical requirement for calcium-activated Cl- channels (CaCCs). Time of addition studies using CaCC blockers have indicated that these channels play a post-entry role during HRSV infection. Using genetic knockdown techniques we have isolated a single channel of interest and are now further investigating its role in facilitating HRSV multiplication, as well as assessing the importance of Cl- channels in replication cycles of other negative sense RNA viruses.

Recombination is a process of extensive genetic exchange that is known to contribute to virus evolution and has been frequently observed in positive-sense RNA viruses. Zika virus (ZIKV) is an emerging arbovirus of the family Flaviviridae with two distinct lineages – African and Asian. While some phylogenetic evidence suggests that recombination in the envelope-encoding region of the ZIKV genome has occurred during evolution, there has been no experimental evidence for ZIKV recombination to date. We conducted co-infections of mammalian and insect cells, using the prototype African ZIKV strain (MR766) and an Asian isolate from the 2015-16 ZIKV outbreak in Brazil (BeH819015), and used a recombinant-specific PCR assay to detect recombinant sequences from total cell RNA extracts. In brief, a 564bp fragment spanning the boundary between the structural and the non-structural genes of the viral genome was amplified using a primer pair consisting of an Asian-specific and an African-specific primer. A total of 24 individual sequences were screened. All were in-frame recombinants and they formed 10 unique junctions. Several of the detected recombinant sequences were chosen for construction of full-length infectious clones to test the viability and phenotype of the recombinant viruses. This study represents the first isolation of recombinant ZIKV sequences from co-infected cultured cells and demonstrates the capacity of ZIKV to recombine in an experimental system. Further investigation is required to better understand the evolutionary potential of this mechanism and its putative role in the emergence of ZIKV.

The European H5N8 highly pathogenic avian influenza virus (HPAIV) epizootic during 2016-2017 resulted in both wild bird and poultry deaths throughout the EU. This in vivo study investigated the potential for indirect infection of naïve birds through contaminated drinking water or feed, to assess potential disease incursion into a high biosecurity commercial floor housed setting.

Three-week-old Ross 308 chickens were exposed to H5N8 A/wigeon/Wales/52833/2016 (H5N8-2016) virus at a high (1 x 106 EID50/ml) or low (1 x 104 EID50/ml) dose, in either drinking water or feed for a 24 hour period. Chickens directly-infected with a high dose of H5N8-2016 (intra-nasal) acted as positive controls. Viral shedding, environmental contamination and clinical signs were monitored for ten days post infection (dpi).

All directly-infected birds shed virus and were humanely terminated at 3 dpi. Immunohistochemical analysis of nasal epithelium and caecal tonsil lymphoid tissue, obtained at post mortem from directly-infected chickens (2 dpi), showed the presence of influenza antigen in both tissues. Only birds exposed to high dose virus in drinking water, shed virus and showed clinical disease presentation (67% mortality). Interestingly low levels of antigen were detected in the nasal epithelium, whereas higher levels were detected in the caecal tonsil.

All surviving chickens from each group, remained uninfected and did not seroconvert. Our findings suggest virus bio availability in different substrates is variable (feed and water) and possible routes of viral contamination leading to disease ingress at poultry premises may have different outcomes including disease presentation.

Biofilms are communities of microorganisms that attach to various surfaces and are widely associated with infections. Our investigation is focussed on a current and growing concern: the formation of biofilms in washing machines. Many countries wash clothes at reduced temperatures (30°C to 40°C) rather than at higher temperatures above 60°C that would kill the bacteria. Survival of the bacteria is associated with biofouling, malodour and an increased infection risk due to the distribution of human pathogens such as Pseudomonas aeruginosa, one of the predominant bacteria found in washing machines.

Little is known about environmental microniches present in biofilms. Here, we focus on the pH variation throughout P. aeruginosa biofilms knowing that the pH can influence biofilm formation and could be an important aspect for the prevention of biofilms. We use novel pH-sensitive optical nanosensors that penetrate P. aeruginosa biofilms and emit fluorescence in response to pH variation. Using time lapse imaging, pH changes were tracked in real time at a single cell level which will ultimately facilitate monitoring of environmental changes induced as biocides penetrate biofilms. We also look at the isolation and identification of P. aeruginosa from household washing machines. Whole genome analysis was performed to identify different genomic features relevant to antimicrobial resistance (AMR) and biofilm formation. Furthermore, testing of different washing detergent formulations revealed a range of abilities to disrupt biofilm formation or kill P. aeruginosa, which will facilitate the development of more effective washing agents to limit the emergence of AMR within biofilms.

Promyelocytic leukaemia (PML) bodies are nuclear organelles implicated in post-translational modification by small ubiquitin-like modifier (SUMO) proteins and in the antiviral host cell response to infection. The 72-kDa immediate-early protein 1 (IE1) is considered the principal antagonist of PML bodies encoded by the human cytomegalovirus, one of eight human herpesviruses. Previous work has suggested that the interaction between IE1 and PML proteins, the central organisers of PML bodies, and the subsequent disruption of these organelles serve a critical role in viral replication by counteracting intrinsic antiviral immunity and the induction of interferon (IFN)-stimulated genes. However, this picture has emerged largely from studying mutant IE1 proteins known or predicted to be globally misfolded und metabolically unstable. We systematically screened for stable IE1 mutants by clustered charge-to-alanine scanning. We identified a mutant protein (IE1cc172-176) selectively defective for PML interaction. Functional comparisons between the mutant and wild-type protein revealed that IE1 can undergo modification by mixed polymeric SUMO chains and that it targets PML and Sp100, the two main constituents of PML bodies, via distinct mechanisms. Unexpectedly, IE1cc172-176 supported viral replication almost as efficiently as wild-type IE1. Moreover, lower instead of higher (as expected) levels of tumor necrosis factor alpha, IFN-beta, IFN-lambda and IFN-stimulated gene expression were observed with the mutant compared to the wild-type protein and virus. These results suggest that the disruption of PML bodies is linked to induction rather than inhibition of antiviral gene expression. Our findings challenge current views regarding the role of PML bodies in viral infection.

Air pollution is a major global health problem, with around 91% of the world’s population living in areas that exceed the WHO air pollution guidelines. This complex mix of pollutants almost always includes particulate matter (PM), and this has the greatest impact on human health. PM exposure contributes to a range of diseases such as COPD, heart disease and respiratory infections. Our recent publication was the first to document that as well as damaging the host, PM has a direct impact on bacteria that can cause respiratory infections. We showed that Black Carbon (BC) exposure results in species-specific alterations in biofilm structure in both Streptococcus pneumoniae and Staphylococcus aureus, altered biofilm protectivity against antibiotic exposure, and S. pneumoniae bacterial colonisation in vivo.

Following on from this ground-breaking work, our current data show that the bacterial response to BC occurs at the genetic level, altering the transcription of key genes involved in biofilm formation, colonisation and virulence. Bacterial adhesion to and invasion of human epithelial cells is significantly increased when S. aureus are pre-exposed to BC prior to infection compared to naïve S. aureus cells. In a murine respiratory colonisation model, both S. aureus co-infected alongside BC, and crucially S. aureus pre-exposed to BC, show increased colonisation of the nasopharynx and lungs. These data suggest that the bacteria are responding and adapting to exposure to air pollution, and this has an impact on how the bacteria infect the host.

Coccolithophores are calcifying microalgae that carry characteristic calcite platelets (coccoliths) on their surfaces. Most coccolithophore species exhibit diploid and haploid life cycle stages, each adjusted to different environmental conditions. The diploid life cycle stage of the coccolithophore C. braarudii is heavily calcifying with calcification rates that exceed the rates of photosynthesis. Haploid life-cycle stages are often weakly calcifying, generating significantly less H+ from the intracellular calcification reaction. We show how these different cellular “H+ burdens” require substantially different physiological molecular strategies to regulate intracellular pH under changing environmental conditions. Voltage-gated H+ channels (Hv) have been shown to play a role in the release of H+ in the diploid life cycle previously (Taylor et al. 2011). Combining scanning electron microscopy, electrophysiology, gene expression approaches and physiological measurements, we here show a direct link between the function of proton channels and coccolith formation of the diploid but not the haploid life-cycle stage. Our data also indicate how the different mechanisms for acid-base regulation of the diploid and haploid life-cycle stages may result in different sensitivities towards ocean acidification.

A major challenge in microbial ecology is to understand the stability of interspecies interactions when progressing from pairs of interacting species to multispecies interaction networks. A lack of direct evidence, and a conceptual framework to explore how direct and indirect effects shape cellular responses in species-rich networks has hindered progress in our understanding of these combined effects. Here we aimed to investigate whether higher-order interactions shape community dynamics and transcriptional profiles of all interacting partners in a simplified microbial community that includes a primary producer (Nannochloropsis oceanica CCAP849/10) and two heterotrophic bacteria (Marinobacter sp. FDB33 and Alteromonas sp. FDB36). By combining co-cultivation assays, quantification of absolute abundances, nutrient analysis, and simultaneous RNA-sequencing, we reveal genome-wide transcriptional responses in all binary co-cultivation partners and show that the third partner can profoundly alter binary interactions at the phenotypic and transcription level. Our study demonstrates the context-dependency of binary interactions, whereby environmental conditions and the presence of specific organisms can affect the cellular physiology of the interacting partners and ultimately the stability of the community. Furthermore, our approach provides a powerful tool for probing the molecular basis of emergent properties in more complex systems.

The Tup1-Cyc8 (Ssn6) complex is a powerful epigenetic repressor of genes in the yeast Saccharomyces cerevisiae. The highly conserved complex brings about a repressive chromatin structure at regulatory regions of its target genes or prevents the recruitment of factors needed for activation of transcription. A gap in the current understanding is whether each of the subunits contribute differently to repression. The FLO family of genes are repressed by the Tup1-Cyc8 complex, these genes encode the proteins required for flocculation, a stress response in yeast where the cells aggregate, or form flocs, to protect cells within the floc. Interestingly, each mutant strain has a distinct flocculant phenotype. The tup1Δ strain displays large, dense flocs compared to smaller, more dispersed flocs associated with the cyc8Δ strain. RT-qPCR showed that FLO1 is highly de-repressed in the tup1Δ strain whereas it is de-repressed to a significantly lower level in the cyc8Δ strain. Using the Anchor Away (AA) technique, which allows for a nuclear protein to be conditionally sequestered to the cytoplasm, I am investigating differences in the sequence of events at the FLO1 promoter when Tup1p or when Cyc8p is removed from the nucleus. Six hours after Cyc8p is removed from the nucleus transcription of FLO1 almost reaches the maximum transcription seen in the cyc8Δstrain. However, six hours after removing Tup1p the level of transcription of FLO1 is still over ten times lower than the maximum transcription in tup1Δ. This difference indicates that each of the subunits have independent functions within the complex.

Production of polyhydroxyalkanoates (PHAs) as bio-alternative to petroleum-based plastics is an important field in the biorefinery to move forward in the development of the circular economy. PHAs are bioplastics stored inside microbial cells as carbon reservoirs and can be produced from a broad range of renewable resources such as waste streams. One important waste stream is food waste that can be converted into volatile fatty acids (VFAs) by anaerobic digestion. The produced effluent from food waste is not only rich in VFAs but also, other nutrients such as nitrogen and phosphorus that can be used by the microorganisms to produce PHAs. The aim of this research is to convert VFAs produced from food waste into PHAs, in which two approaches have been studied. The first approach was to use microbial mixed cultures (MMCs) while the second used microbial pure cultures.

The MMCs were enriched in sequencing batch bioreactor cultivations, where nitrogen and carbon starvation were combined to enhance the selection phase. PHA accumulation of the selected cultures was studied in nitrogen-limited fed-batch cultivations. The second approach studied five different PHA producing bacteria: Cupriavidus necator, Burkholderia cepacea, Bacillus megaterium, Bacillus cereus and Bacillus cereus. To select the most promising bacteria, synthetic medium with the same VFAs composition as in MMCs study was used for pre-screening experiments. Both, pure and mixed culture studies, resulted in the production of PHAs containing (R)-3-hydroxybutyrate, (R)-3-hydroxyvalerate and (R)-3-hydroxyhexanoate as monomers and VFAs were consumed with a high rate by the microorganisms.

The Human Gut Microbiota (HGM) comprises two major phyla, the Bacteroidetes and Firmicutes, although important members of the Actinobacteria (Bifidobacterium) and Verrucomicrobia (Akkemansia) also make an important contribution to this ecosystem.

Accumulating datasupport the notionthat the HGM can be modulated by probiotics and prebiotics to prevent or revert common diseases of the gastrointestinal tract (GIT) such as Inflammatory Bowel Disease. Because it is believed that these GIT diseases are linked to the fact that current Western populations follow a more fat-based diet, significant efforts have been made to search for novel prebiotics/probiotics in order to restore and improve gut health.

So far, no publications have described probiotic properties of Bacteroidetes. Nonetheless, a case can be made that certain Bacteroides species present primary glycan degraders that interact in a syntrophic manner with other members of the microbiota, such as bifidobacteria, which are considered beneficial members of the microbiota.

In this study, we present the simbiotic interactions between Bacteroides and Bifidobacterium spp. acting on yeast beta-glucan (1,3/1,6 mixed linkage beta-glucan). Bacteroides cellulosilyticus and Bacteroides ovatus act as keystone organism to share beta-1,3/1,6-glucooligosaccharides with other members of the HGM, including Bifidobacterium breve UCC2003 and Bifidobacterium bifidum. We show in these Bifidobacterium spp. a specific beta-1,3-glucosidase, which degrade some of these sharing oligosaccharides. Also, we have identified the specific sugar symporter, which incorporate these oligosaccharides into the cytoplasm of B. breve UCC2003. With the help of RT-qPCR, we have quantified and monitored how these two members of the HGM are able to symbioticly use this dietary glycan.

The propensity of pathogens to evolve resistance to antibiotics used in clinical infectious disease therapeutics has been an increasing concern in recent decades. Acquisition of resistance often translates into treatment failure and puts patients at risk of serious adverse outcomes. Current laboratory testing of antibiotic susceptibility does not account for the different microenvironments that bacteria encounter within the human body, providing results that often do not translate into the clinic. Our goal is to better understand evolutionary strategies employed by Staphylococcus aureus in development of resistance in distinct environments.

We used adaptive laboratory evolution (ALE) to generate isogenic strains resistant to several antibiotics. Different media were used to mimic distinct environments and multi-omics approaches applied in the understanding of resistance mechanisms.

Evolved strains presented phenotypes similar to those observed in clinical resistant isolates. Mutational analysis indicated that resistance was specific and condition-dependent. Distinct mutations led to resistance phenotypes under a particular environmental condition, but these mutations did not necessarily translate into resistance under a different environmental condition. Furthermore, resistant strains possessed distinct transcriptional landscapes, even when the same systems were mutated, suggesting that similar evolutionary paths translate into distinct resistance mechanisms.

We identified several resistance mechanisms employed by S. aureus that were not only environment-dependent, but also environment specific. Additionally, we showed that ALE can be applied in pathogens of interest to study antibiotic resistance evolution and prediction of clinical resistance mechanisms, as supported by the significant overlap of mutations identified via ALE and those reported in clinical isolates.

Large-scale mining and analysis of bacterial datasets contribute to the comprehensive characterization of complex microbial dynamics within a microbiome and among different bacterial strains, e.g., during disease outbreaks. The study of large-scale bacterial evolutionary dynamics poses many challenges. These include data-mining steps, such as gene annotation, ortholog detection, sequence alignment, and phylogeny reconstruction. These steps require the use of multiple bioinformatics tools and ad-hoc programming scripts, making the entire process cumbersome, tedious and error-prone due to manual handling. This motivated us to develop the M1CR0B1AL1Z3R web server, a ‘one-stop shop’ for conducting microbial genomics data analyses via a simple graphical user interface (Avram, et al., Nucleic Acids Res., 2019). Some of the features implemented in M1CR0B1AL1Z3R are: (i) extracting putative open reading frames and comparative genomics analysis of gene content; (ii) extracting orthologous sets and analyzing their size distribution; (iii) analyzing gene presence-absence patterns; (iv) reconstructing a phylogenetic tree based on the extracted orthologous set; (v) inferring GC-content variation among lineages. M1CR0B1AL1Z3R facilitates the mining and analysis of dozens of bacterial genomes using advanced techniques, with the click of a button. M1CR0B1AL1Z3R is freely available at https://microbializer.tau.ac.il/ [https://microbializer.tau.ac.il/].

The secretion of extracellular enzymes by soil microbes is rate-limiting in the global recycling of biomass. Fungi and bacteria compete and collaborate for nutrients in the soil, with wide ranging ecological impacts. Within soil microbiota, the Bacteroidetes tend to be a dominant bacterial phylum, just like in human and animal intestines. The enzymology of Bacteroidetes in the dynamic and competitive soil environment is under-explored compared to their cousins from the human and ruminant gut ecosystems. We are exploring carbohydrate binding and deconstruction by Chitinophaga pinensis. This species was isolated from the leaf litter of a pine forest, and our ongoing microbiological, biochemical, and proteomic analyses show that C. pinensis has a marked metabolic preference for carbohydrates (glycans) of microbial, rather than plant, origin. The species has a repertoire of enzymes that degrade components of the fungal cell wall, and we are characterising several important enzyme activities, including some with unusual substrate specificity.

Several features of the C. pinensis “cazome” make it note-worthy. In particular, there is a significantly reduced reliance on the Polysaccharide Utilisation Loci that define glycan acquisition in most well-studied gut symbiont Bacteroidetes. Instead, C. pinensis produces some large multi-modular enzymes that convey multiple complementary carbohydrate-binding and -degrading functions, and which are often secreted via the phylum-specific Type IX Secretion System.

This presentation will highlight our latest enzyme characterisation data, discussed in the context of the environmental functions of soil bacteria, as well as the use of enzymes for industrial biotechnology.

Fermented foods are consumed by a very large population in Africa but the products have many drawbacks ranging from shelf life instability to contamination and toxicity. These foods therefore require an upgrade through improved fermentation processes. This work determined the phenotypic characteristics of the fermenting microorganisms and microbial ecological succession during fermentation of cassava and maize to determine the predominant fermenting microorganisms. Cassava roots and maize grains were fermented using the traditional method of processing them into fufu and ogi for 72 h and 48 h respectively. Samples were drawn every 12 h for analysis. Enumeration and characterization of lactic acid bacteria were carried out on MRS medium with subsequent microscopic examination, physiological, biochemical reaction tests and API 50 CH gallery. Yeast isolates were identified by their morphological characteristics. Thirteen lactic acid bacteria were isolated from the fermenting cassava and 6 from the fermenting maize. The Isolates were Gram positive and catalase negative. Lactobacillus plantarum, L. fermentum and L. pentusus predominated in both fermentations while Candida tropicalis, C. krusei and Saccharomyces cerevisae also predominated in both fermentations. Candida inconspicuo was found only in cassava fermentation. The results of this work revealed the microbial ecology of fermented cassava and maize which is a prerequisite to the understanding needed to develop a multifunctional starter culture for these fermentations for their upgrade.

Keywords: Cassava, Maize, Fermentation, lactic acid bacteria, Yeasts.

Gonorrhea is the second most commonly reported notifiable sexually transmitted disease in the world. Neisseria gonorrhoeae causes 78 million cases annually of gonorrhoea worldwide. There are no vaccines and antibiotic-resistant organisms are circulating rapidly. The estradiol-treated female mouse model is the only animal model available for studying the host response against gonococci and biological significance of host-restricted bacterial-host cell interactions observed in vitro. However, mouse models have limitations such as cost, time and ethics. Therefore, there is an urgent need for the development of new alternative in vivo models. The Galleria mellonella larval model is a simple, widely available, cost-effective, and powerful tool for studying microbial infections prior to any vertebrate animal testing. Here we report, for the first time, that G. mellonella can be used as an infection model for Neisseria spp., focusing particularly on N. gonorrhoeae. We demonstrated dose-dependent larval death and recovery of viable gonococci from the host, visualised host-pathogen interactions using histopathology and confirmed the importance of insect haemocytes as an innate immune cell during infection. The model was also used to test the efficacy of antibiotics used to treat gonorrhoea. Our results demonstrate that G. mellonella can be used as a model to study pathogenesis and virulence of gonococcal infection in addition to rapid in vivo testing of antimicrobials.

Since the last influenza pandemic in 2009, H1N1pdm has been introduced into the swine population in Europe where, in combination with swine influenza A virus (IAV) lineages, it started to generate a variety of reassortant viruses of unknown zoonotic risk for humans. To study these reassortment events, we isolated a wild swine lung cell clone (C22) susceptible to IAV infection. We established conditions for co-infection and passaging of H1N1pdm and swine avian-like H1N1. After 7 passages, we plaque-purified C22-adapted strains, characterized their genome composition by next-generation sequencing and analysed replication abilities in swine and human lung cell lines as well as in human lung tissue ex vivo.

Among C22-adapted viruses isolated from co-infection, we revealed reassortants carrying PB1/PA/NA or only PB1/PA from H1N1pdm. We also detected exclusively swine H1N1-derived strains. All isolates carried distinct mutations. As expected, adapted viruses reached higher titers compared to both parental strains in swine lung cells. Furthermore, all C22-adapted viruses were able to replicate in human lung A549 cells without any prior adaptation to the human host. Strikingly, all reassortants were able to infect and efficiently replicate in human lung tissue ex vivo, indicating that these viruses might pose a zoonotic risk.

To summarize, we successfully established an in vitro swine-like model to study reassortment and adaptation of IAVs currently circulating in swine. Our results indicate that our model might be a useful tool to prospectively evaluate the compatibility of different IAV strains to generate reassortants, which might represent a threat to the human population.

Phosphorus (P) is an essential macronutrient for all living organisms and is applied as fertilizer in agroecosystems to improve crop growth. Recycling-derived fertilizers (RDFs) have been developed for nutrient recovery from Europe’s largest waste streams as a sustainable alternative to this finite resource. The impact of four RDFs (two ashes, two struvites) on the soil microbiome in comparison with a P-free control and triple super phosphate (TSP) as mineral fertilizer was investigated in a pot trial and a subsequent microcosm trial (subset of samples). For both experiments perennial ryegrass was cultivated for 54 days. The pot trial was conducted at P fertilization rates of 20 and 60 kg P ha-1 in quadruplicates. After the pot harvest the bulk soil was stored until the microcosm trial was conducted, using the control, TSP and the two ashes at 60 kg P ha-1 in six replicates. Pot trial results showed highest P bioavailability from struvites at high P rates, also resulting in higher biomass yield on average. Furthermore, P solubilization capabilities from tri-calcium phosphate was enhanced in the RDFs treatments, while the TSP treatments were negatively affected. For the microcosm trial, most probable number (MPN) analysis showed that phytate-utilizing bacterial abundance was significantly increased in one of the ashes and had also remained higher in the RDF treatments after storage. Understanding the effects of recycling-derived fertilizer application on the soil P cycle is vital for developing a more sustainable agriculture.

BK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that infects 70-90% of the world's population and causes a lifelong, silently persistent infection. In immunocompromised individuals, BKPyV replication can result in serious pathology. Bone marrow transplant patients can develop a haemorrhagic cystitis, and in kidney transplant patients BKPyV replication can provoke a nephropathy that leads to deterioration of allograft function and eventual loss of the transplanted organ. There are currently no antiviral treatments with clinical efficacy against BKPyV associated nephropathy.

While the life cycles of non-enveloped viruses are often assumed to require cell lysis to release progeny virions, we have evidence to suggest that BKPyV exits the cell via non-lytic means using an unconventional secretory pathway. We have investigated the effects of knocking out cellular genes thought to be involved in unconventional secretory pathways that bypass the Golgi apparatus on the release of BKPyV. We observe decreased BKPyV release from cells that have undergone CRISPR-mediated knockout of Golgi Reassembly Stacking Protein (GORASP) 1 or 2. Investigation of BKPyV-induced changes to the plasma membrane of infected cells demonstrated increased cell surface expression of transmembrane proteins normally resident in the endoplasmic reticulum. This appears to be inhibited by the knockout of GORASP1 or 2, suggesting that virions and ER markers are secreted via a common pathway in infected cells. These experiments are uncovering novel virus-host interactions that, when targeted, could help prevent BKPyV-associated nephropathy and allograft loss.

The Group A carbohydrate (GAC), a bacterial surface polysaccharide, is an essential virulence factor of Streptococcus pyogenes required for growth and infection of humans.In terms of its chemical composition, this peptidoglycan-anchored polymer is mainly formed by a string of rhamnose sugars, with alternated modifications of N-acetylglucosamine and glycerolphosphate. The rhamnose polysaccharide (RhaPS) that forms the backbone chain is synthesised intracellularly by the sequential action of three rhamnosyltransferases named GacB, GacC and GacG. Importantly, deletion of any of these rhamnosyltransferases causes bacterialdeath.

In this work, we used an interdisciplinary approach to demonstrate that: 1) GacB is a novel enzyme that initiates the RhaPS biosynthesis; 2) GacC catalyses the formation of a unique stem; 3) GacG elongates the RhaPS string by adding a yet unknown number of rhamnoses. Here, we also show that homologs from different streptococcal species can substitute GacB and GacC in the RhaPS production. In particular, we demonstrate that several human pathogens from the Streptococcus genus encompassed in the Lancefield serotyping scheme, and the dental pathogen Streptococcus mutans can replace S. pyogenes’ enzymes. In contrast, the homologs from S. pneumoniae sp. D39 did not, suggesting a different structural arrangement for its surface carbohydrate. Our results highlight the importance of the group carbohydrate biosynthesis pathways in the Streptococcus genus and open the door for the future development of multi-target compounds that could inhibit these enzymes in Streptococcus pyogenes and other pathogenic streptococci of clinical and veterinary importance.

Bacterial spores are of concern in food processing due to their ubiquity and resistance. This study seeks to determine the effect of ultraviolet C (UV-C) in the inactivation of spores of Bacillus subtilis and Bacillus velezensis that can result in enzymatic spoilage in foods using PBS as the suspension medium. Purified spore samples were treated under 1 pass in a UV-C reactor using 10 mL of spore inoculum with one dose of the radiation (410 mJ/cm2) for 10secs at room temperature. Aliquots of the treated samples were plated on tryptone soy agar supplemented with 0.6% glucose and the colonies counted. Flow cytometry analysis was done using 500 μL of both treated and control samples with a cell concentration of a ≥106 CFU/ml with propidium iodide (15 μM) and SYTO 9 (500 nM) used as live/dead stains. Samples were processed for microscopy (SEM and Raman-AFM Imaging). The maximum lethality is 2.5 for B. velezensis and the minimum is 0.1 for B. subtilis. Microscopic imaging of treated spores shows significant morphological disruption of the spore structure. The Raman spectroscopy analysis reveals the B. subtilis isolates to have the highest concentrations of dipicolnic acid (Ca+2DPA) as well as other compounds belonging to other functional groups. Flow cytometric analysis of treated spores reveals sub-populations unaccounted for by plate count. UV-C shows a promising application in the inactivation of resistant spores during processing of liquid foods such as milk.

It is estimated that £5 billion are invested yearly into chronic wound management by the NHS. Whilst the demand for treatment rises every year, it has become harder to treat wounds given the burden of antimicrobial resistance. Chronic wounds can easily become harbouring grounds for polymicrobial biofilms in which species interact in specific ways.

This study assessed the interactions between two commonly co-isolated chronic wound pathogens: Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (EMRSA 15), whose biofilm relation initiates a Gram-negative shift. During this phenomenon, P. aeruginosa takes over the majority of the bacterial community, at the detriment of S. aureus. The Gram-negative shift marks the turning point from an acute to a chronic wound. The pH of a chronic wound is typically alkaline, and it was hypothesised that topical dressings with an acidic pH could disrupt the onset of the Gram negative shift, and therefore chronicity. Six different topical dressings with low pH were used in achronic wound model to assess their ability to reverse or delay the Gram-negative shift. It was found that they did not have an impact on the onset of the Gram-negative shift, despite their low pH values. However, the lower the pH of the dressings, the more frequently small colony variant (SCV) bacteria were observed in the biofilm. SCVs are known for causing persistent or chronic infections. It was therefore concluded that low pH dressings alone may not be favourable for managing chronic wound infection.

The contribution of the gut microbiota to health and disease is becoming ever more apparent in the last number of years, due to developments in DNA sequencing technology and more well-defined cultivation techniques. This has resulted in the identification of health-promoting bacteria. Until recently, prebiotics, non-digestible food substrates which are selectively utilised by beneficial bacteria, were employed with a view to increasing the growth of well-established health promoting bacteria, namely Lactobacillus and Bifidobacterium. However, other beneficial bacteria recently revealed may also be targeted to enhance their growth as they establish themselves as the next generation of health-promoting microbes. These include anaerobes such as Akkermansia muciniphila, Faecalibacterium prausnitzii and Eubacterium rectale. Identification of growth substrates/bioactives through the analysis of genome sequence data can aid in elucidating which substrates may best enhance the growth of these microbes which are often difficult to grow.

The phenotypic microbial trait analyser, Traitar, can predict 67 phenotypes based on the genome sequence inputted. Some of these traits include substrates that could potentially be utilised by the bacteria. Another tool, CarveMe, which has been created with the aim of making metabolic modelling more user-friendly, was also used with the same genomes. A select number of substrates identified in both tools have been chosen to be evaluated in vitro in order to establish the accuracy of these predictive tools as well as giving an indication as to how these beneficial microbes can be modulated through dietary components.

Zebrafish (Danio rerio) are an attractive model organism for a variety of scientific studies, including host-microbe interactions. Zebrafish contain a core (i.e., consistently detected) intestinal microbiome consisting primarily of Proteobacteria. Furthermore, this core intestinal microbiome is plastic, and can be significantly altered to due external factors. The organism is particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae. As an intestinal pathogen, V. cholerae needs to colonize the intestine of an exposed host for any type of pathogenicity to occur. It is suspected that members of the resident intestinal microbial community need to be eliminated by V. cholerae in order for colonization, and subsequently disease, to occur. While numerous studies have explored various aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms, few, if any, have examined how a V. cholerae infection alters the resident intestinal community. In this study, 16S rRNA gene sequencing was utilized to investigate how various strains of V. cholerae alter the aforementioned microbial profiles following an infection. We found that V. cholerae infection and subsequent colonization induced significant changes in the zebrafish intestinal microbiome, with specific members of the microbial community targeted. Additional salient differences to the microbial profile were observed based on the particular strain of V. cholerae utilized for challenging the zebrafish hosts. We conclude that V. cholerae causes significant modulation to the zebrafish intestinal microbiome in order for infection and subsequent disease to occur.

Aeromonas are Gram-negative facultative anaerobic rods, which inhabit various aquatic environments and are pathogens of both warm and cold-blooded animals. In humans they cause gastro-enteritis and wound infections. They are motile in liquid environments by a single polar type of flagellum. The flagellum plays an important role for the bacterial colonisation and the adhesion to the host cells. The Aeromonaspolar flagella filament is a polymer composed of two flagellins, FlaA and FlaB. The flagellins are O-linked glycosylated through the addition of the unusual bacterial sugar pseudaminic acid to serine and threonine residues within the flagellins D2/D3 domain. The addition of this sugar is essential for flagella filament assembly and bacterial motility. The flagellin’s are modified by between 6 – 8 sugar residues that occupy the potential 14 sites of attachment. Motility accessory factors (Maf proteins) are candidate enzymes for transferring glycan molecules to the flagellin (glycosyltransferases transferring sugar to flagellin) due to their genetic location and motility phenotypes associated with disruption mutants.

This study utilised site-directed mutagenesis to change the potential sites of flagellin glycosylation to assess the effect of these mutations on motility by swimming assays and flagella filament formation by electron microscopy. The analysis of different numbers of site-directed mutants suggest that some sites are more important than others and that the removal of 4 sites results in greatly reduced motility.

The study was aimed at Salmonella isolation from samples of animal food products submitted for testing from various regions of the Central part of the RF and serotyping of the recovered isolates and their testing for antibiotic resistance. A total of 2,342 tests were performed and 87 (3.7%) Salmonella isolates were recovered. Most of them (54 isolates) were recovered from poultry meat and poultry meat preparation samples submitted for testing. Besides, 25 isolates were recovered from pork and pork preparation samples, 7 isolates – from beef samples, 1 isolate – from hard cheese samples. Serotyping of 64 Salmonella isolates showed that the majority of the isolates (57.8%) belonged to О7 group. Also, Salmonella isolates belonging to О9 (21.9%), О8 (9.4%), О4,5 (6.2%) and О10 (4.7%) were detected in food products. S. Enteritidis, (23.3%), and S. infantis (18.7%), were predominant based on the number of detections. Also, the following serovars were identified: S. typhimurium, S. nigeria, S. montevideo, S. derby, S. meleagridis, S. virchov, S. oranienburg. Tests of 87 Salmonella isolates for their antibiotic resistance with disk diffusion method revealed that they were highly resistant to nalidixic acid (70.1%), tetracycline (49.4%), trimethoprim/sulfamethoxazol (40.2%). Moreover, nalidixic acid-resistance was common for all identified isolates. Seventeen isolates (19.5%) demonstrated multiple antibiotic resistance and two isolates were found to be resistant to ≥7 antibiotics. All recovered isolates were susceptible to gentamicin, amikacin, meropenem and imipenem. Obtained results indicate the necessity of Salmonella antibiotic resistance monitoring to gain understanding of Salmonellas’ antibiotic resistance emergence and trends.

Microbial colonization of a neonate’s gastrointestinal tract has significant perinatal and lifetime health consequences, with some clinical outcomes that have been linked to differences in the diversity and composition of gut microbiota. The effort to engineer intestinal ecosystem has led us to preserve the cultivable commensal microbiota. Here we investigated the association of cultivable bacterial diversity of neonates meconium from Indonesian National Referral Hospital Cipto Mangunkusumo (NRHCM) with mode of delivery and feeding patterns, as well as hyperbilirubinemia. We performed a cross-sectional study of meconium and clinical data collected from 14 Indonesian neonates born at NRHCM. Culture-dependent identification of bacterial isolates was conducted by performing simultaneous microbiological and molecular 16S rDNA PCR-Sanger sequencing methods. Phylogenetic tree and principal components analysis were employed to determine the bacterial profile and their association with clinical characteristics and outcomes. Cultivable bacterial profile indicates the predominance of Firmicutes (84,41%), with an abundant population of Staphylococcus (53.24%) with top three most significant population present are, i.e. S. hominis (12.99%), S. epidermidis (11.68%), and S. haemolyticus (10.39%). Bacterial diversity was associated with mode of delivery which showed that vaginal route populated by lower diversity of cultivable bacteria but by fewer opportunistics one than that of cesarean, with Staphylococcus hominis dominates the population, whereas with feeding patterns showed that the exclusive breast-fed was most populated by Staphylococcus, whereas non-exclusive one shared the same proportion of Staphylococcus and Bacterioides. While, non-hyperbilirubinemia group showed more abundant and diverse Staphylococcus than that of the opposite group.

Type VI secretion systems (T6SSs) are contractile nanomachines widely used by bacteria to intoxicate competitors. Salmonella Typhimurium encodes a T6SS within the Salmonella pathogenicity island 6 (SPI-6) that is used during competition against species of the gut microbiota. We characterized a new SPI-6 T6SS antibacterial effector named Tlde1 (type VI L,D-transpeptidase effector 1). Tlde1 is toxic in target-cell periplasm and its toxicity is neutralized by co-expression with immunity protein Tldi1 (type VI L,D-transpeptidase immunity 1). Time-lapse microscopy revealed that intoxicated cells display altered cell division and lose cell envelope integrity. Bioinformatics analysis showed that Tlde1 is evolutionarily related to L,D-transpeptidases. Point mutations on conserved histidine121 and cysteine131 residues eliminated toxicity. Co-incubation of purified recombinant Tlde1 and peptidoglycan tetrapeptides showed that Tlde1 displays both L,D-carboxypeptidase activity by cleaving GM-tetrapeptides between meso-diaminopimelic acid3 and D-alanine4, and L,D-transpeptidase exchange activity by replacing D-alanine4 for a non-canonical D-amino acid. Tlde1 constitutes a new family of T6SS effectors widespread in Proteobacteria. This work increases our knowledge about the bacterial effectors used in interbacterial competitions and provides molecular insight into a new mechanism of bacterial antagonism.

Aedes aegypti and Aedes albopictus are the major vectors for the transmission of more than 30 disease-causing viruses as dengue fever, dengue hemorragic fever, zika, yellow fever and chikungunya. Vector control is one of the important strategies used in order to fight these diseases in tropical and subtropical countries. However, mosquito control is facing a threat because of the emergence of resistance to synthetic insecticides. The aim of this study was to identify larvicidal and adulticidal activity of secondary metabolites in extracts produced by bacteria isolated from different sources in Colombia. A total of 105 extracts produced from the same number of bacteria were evaluated for their activity against fourth instar larvae and adults of A. aegypti and A. albopictus using standard protocols defined for the WHO (World Health Organization) and CDC (Control Disease Centre, USA). Six extracts showed relevant activity (more than 50% of mosquito larvae were killed after 48 hours), two of them showed to be actives against larvae of Aedes aegypti and four against larvae of Aedes albopictus. None of the extracts showed activity against the mosquitoes in adult stage. The bacteria producing active extracts were identified using the biolog ® identification system as Serratia marcescens ss marcescens, Escherichia hermannii, Serratia marcescens ss marcescens, Bacillus marisflavi, Bacillus atropheus/subtilis and Pseudomonas chlororaphis subsp. aurantiaca. In conclusion, bacterial extracts are a good source for the search of new strategies in the control of mosquitoes. Further studies to determine the compound responsible for the insecticidal activity are in progress.

Introduction. Urinary tract infections (UTIs) are considered prevalent among humans. While Klebsiella pneumoniae and Escherichia coli are commonly isolated from patients with UTIs, K. pneumoniae is more frequently isolated from specimens isolated from hospital-acquired infections.

Aim. The current study aimed to characterise and evaluate pathogenic K. pneumoniae isolated from patients suspected with a UTI at King Abdulaziz University Hospital.

Methodology. Twenty-four urine samples obtained from patients, between 12 November 2018 and 11 January 2019, were included in the study, and the microbial content was analysed via urine culture and VITEK 2 analyses.

Results. Of the 24 urine specimens, 23 samples (95.8%) yielded significant microbial growth (> 105 CFU/mL K. pneumoniae), and one sample (4.2%) yielded non-pathogenic microbial growth (< 105 CFU/mL K. pneumoniae). Of the specimens with cell counts of >105 CFU/mL, 21 samples (87.5%) were pure cultures showing the growth of a single pathogen, and three samples (12.5%) were mixed cultures, showing the growth of two or more pathogens; 10 (41.7%) samples were extended-spectrum beta-lactamase (ESBL) producers. VITEK 2 analysis showed that the most effective antibiotic was piperacillin, with 87.5% of the strains isolated in this study showing sensitivity to it, followed by gentamicin (83.3%) and ciprofloxacin (79.2%). Antibiotic susceptibility studies identified resistance against ampicillin (45.83%), trimethoprim (41.67%), and amoxicillin (25%) in the study isolates. Differential chromogenic culture media CHROMagar ESBL and CHROMagar KPC (K. pneumoniae carbapenemase) were used for rapid screening of ESBL and KPC producers. Among the 24 K. pneumoniae isolates, six isolates (25%) formed metallic blue colonies on CHROMagar ESBL, five (20.3%) were inhibited, and nine (37.5%) showed weak pigmentation. Three isolates (12.5%) formed pink colonies on CHROMagar ESBL. Seventeen isolates (70.8%) were KPC-positive, four (16.7%) showed weak pigmentation, and three (12.5%) formed pink colonies on CHROMagar KPC. The results from CHROMagar cultures agreed with those of VITEK 2 analyses.

Conclusion. Various microbial detection methods have been used in medical microbiology laboratories to identify and screen for microbial resistance in clinical specimens. VITEK 2 and CHROMagar are amongst the commonly used methods. Here, we report a possible discrepancy between the detection techniques and that the data obtained by different methods need not be synergistic. However, the use of a chromogenic medium offers a quick and accurate method for detection, enumeration, and presumptive identification of urinary tract pathogens.

Studies on the impact of skin microbiota on human health have been gaining more attention. The skin microbiome is considered to provide several probiotics for skin therapeutic. Beneficial bacteria mixtures (bacterial cocktail) isolated from targeted organs have shown promising modulatory activities for use in skin therapeutics. The objectives of this study were to determine and identify the microbial communities on the skin that can be potentially used as probiotics-postbiotics. Determination and identification of skin microbiota were carried out simultaneously by employing next-generation sequencing (NGS) of direct sampling, as well as by bacterial cultivation; twenty bacterial isolates with different characteristics were selected and identified by both culture-based methods and 16sRNA sequencing. We found that Actinobacteria and Firmicutes are the most abundant phylum present on the skin as presented by NGS data, which constitute to 67% and 28.59% of the whole bacterial population, consecutively. Three strains, i.e., Staphylococcus hominis (AN MK968325.1), Staphylococcus warneri (AN MK968315.1), and Micrococcus luteus (MK968318.1), were obtained from cultivable samples. They were potential to be developed further as probiotics in skin microbiome therapeutic as well as for postbiotic formulation. However, the promising formula of bacterial cocktail for skin microbiome therapeutic must be elucidated thoroughly to avoid unwanted effects by performing visual observation on agar plate and by molecular approach, q-PCR.

Directly observed treatment Short course regimen has been know to be efficacious in the chemotherapy management of tuberculosis. However, co- infection with other opportunistic bacteria could limit this success to the level of clinical suspecting the emergence of false positive multidrugs resistant TB among the patients which in many cases can lead to establishment of erroneous treatment schedule. Accordingly this facility based surveillance monitors co-infection with other bacteria in TB patients attending Dots clinic at IDH Kano, Nigeria with view to ascertaining the drug susceptibility so as to proffer better chemotherapy strategies. A total 35 non - duplicated positive AFB sputum sample collected from patients with pulmonary tuberculosis on Dots where processed according to standard bacteriological procedures including macroscopic, culture and microscopic examination using both AFB gram staining and culture methods for screening and isolation of other bacterial pathogens. These were confirmed biochemically. Overall 54.3% of the patients were Still AFB positive, Streptococcus pneumoniae and Staphylococcus species we’re predominat in up to 84.2% and 15.8% of the patients respectively. This study indicated secondary bacterial pathogenesis with pulmonary symptoms resembling TB among the patients including those that were even isolated for being refrectory to Dots an false positive conclusion of drug resistant TB. However, ofloxacin and ceftriaxone which show appreciable potencies against secondary bacterial pathogens identified where used to treat successfully, patients claimed posses drug resistant TB. It’s imperative that, success of Dots therapy could be improved by augumentin with very potent non TB antibacterial such as Streptococcus pneumoniae regimen.

Keys. TB.

Introduction. Naturally-occurring variants of Human papillomavirus (HPV) genotypes have been defined as lineages and sub-lineages, but little is known about the impact of this diversity on protein function. We have previously demonstrated that variation within the major (L1) and minor (L2) capsid proteins impact the susceptibility of HPV to serum antibodies elicited by vaccination and natural infection. Higher resolution mapping of variant residues, however, requires the availability of appropriate tools, such as type-specific monoclonal antibodies (MAbs). These empirical data will improve our understanding of the consequences of natural variation on capsid antigenicity.

Methods. We investigated the susceptibility of 37 representative pseudovirus variants of HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58 to neutralization by type-specific murine MAbs raised against the A lineage of their respective genotypes. Homology models derived from available HPV L1 crystal structures were generated to permit mapping of variant residues onto the surface-exposed L1 protein for relevant variants

Results. Type-specific lineage A-specific MAbs demonstrated differential reactivity against some, but not all, variants within its respective genotype. Some of these differences were minor (<4 fold) while some variants displayed orders of magnitude reduced sensitivity. These differences in antigenicity were mapped to a limited number of variant residues on the capsid surface.

Conclusions. These data contribute to our understanding of HPV L1 variant antigenicity and may have implications for seroprevalence or vaccine immunity studies based upon L1 antigens.

In 2011 a large outbreak of enterohemorrhagic gastroenteritis and haemolytic uremic syndrome (HUS) throughout Europe resulted in almost 4,000 infections, 845 cases of HUS and 54 fatalities. This was due to a dangerous exchange of mobile genetic elements (MGE) resulting in a hybrid strain of E. coli O104:H4. This strain carried an unusual combination of EAEC- and STEC-associated virulence factors on a plasmid and phage respectively. In vitro the virulence plasmid has exhibited unusual stability under a wide range of environmental stresses, contrasting with rapid plasmid loss in the human gut. This project will characterise plasmid encoded maintenance systems responsible for its unique stability. Current investigations focus on toxin-antitoxin (TA) systems involved in postsegregationalkilling which contribute to plasmid maintenance therefore resulting in increased virulence. By inducing expression of putative TA genes cloned onto lab-made plasmid vectors, we have analysed their function in the cell. Once characterised we will further investigate the effects of various environmental conditions able to disrupt these TA systems, ultimately resulting in plasmid loss. This atypical strain displayed heightened pathogenicity and providedun foreseen treatment challenges. We aim to further our understanding of MGE carriage in O104:H4 as a model to predict and combat future outbreaks of hybrid pathovars.

A type 6 secretion system (T6SS) was recently identified in the genome sequence data of an isolate sourced from a throat swab of a volunteer that is believed to be N. subflava. The T6SS is one of the most recently discovered bacterial secretion systems and this is the first time it has been reported in Neisseriaceae. Since this discovery, genome sequence analyses for a number of other commensal Neisseria spp. has identified that in fact, two distinct T6SS types exist across Neisseriaceae. These two types are clearly defined and are different to one another in both their core gene sequences and organisation. The two systems also differ in the number of VgrG proteins required for toxic effector protein delivery, as well as type of effector associated with them. The predicted VgrG/effector combinations identified in our isolate are not common to all members of the same species and further analysis has identified a wide range of diversity in these components between different strains of the same species. The data provides possible evidence that T6SS positive commensal Neisseria spp. can acquire new VgrG/effector combinations through competitor killing. A number of putative effectors have so far been identified within the genome of our original isolate, including hydrolases, phospholipases, and nucleases. Whilst these are predicted to be antibacterial effectors, the conditions under which the T6SS system is activated, as well as demonstration of the function of the effectors still needs to be investigated experimentally.

Maedi-Visna is a lentivirus of sheep that causes lung disease and chronic wasting. It has been designated an “Iceberg disease” by the UK sheep industry levy board with a very large burden of subclinical disease that is often not apparent until losses in an individual flock become catastrophic. Disease prevalence in the UK is thought to have doubled in the last 10 years, however farmer and veterinary awareness of the disease is poor. There is no vaccine and treatment is not cost effective, meaning that the only realistic control option is culling of affected animals.

Current testing protocols use MV gag protein ELISAs. A long lag time between infection and antibody production means that many animals are missed on flock screening and repeated rounds of testing over a period of years are necessary remove all infected animals. Preliminary testing of flocks that have attempted eradication indicates that those that do not keep testing until all animals are negative fail to eliminate the disease and that prevalence rates can even increase substantially in these flocks. The viruses extreme variability confounded attempts to develop a qPCR capable of detecting all variants, indeed deep sequencing was required to establish which strains of virus are currently present in UK sheep as there has been substantial genetic drift since the last sequencing studies (performed more than 20 years ago). More promisingly virus was detectable in nasal swabs of experimental animals at least offering a possibility for sampling methods that can be done by farmers themselves.

The microorganisms associated with severe periodontitis are the periodontal pathogens of the red complex: Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. These organisms cleave sialic acids found at the terminal end of host glycoconjugates by hydrolysing the glycosidic linkages with their expressed sialidases, thereby affecting the integrity of the host periodontium and promoting disease progression. Both P. gingivalis (SiaPG) and T. forsythia (NanH) sialidase enzymes were purified using HisTag affinity chromatography and a range of putative synthetic and plant-based inhibitors were tested for their ability to inhibit both enzymes using a MUNANA cleavage assay. Investigation of sialidase inhibitory activity of these compounds revealed that the plant derived alkaloids: Epicatechin gallate (IC50 = 21.75μM and 120.9μM) and Berberine chloride (IC50 = 106.2μM and 125.5μM) were more effective inhibitors of both SiaPg and NanH enzymes than the anti-influenza drug Zanamivir, an FDA approved viral neuraminidase inhibitor. Finally, a range of newly synthesized sialic acid analogues were effective in the micromolar to nanomolar range against both SiaPg and NanH enzymes with compound 2e3aDFNeu5Ac9N3 having an IC50 of (3.846μM and 49.40nM) respectively. The data suggests several novel inhibitors of these enzymes that might have future use as novel drugs against diseases such as periodontitis, and which we are currently testing further in host-pathogen interaction studies.

Antibiotic resistance by pathogenic microorganisms is a major threat of our times, leading to a significant rise of serious untreatable infections, especially in hospital environments. In addition, biofilms further protect the microbes against antibiotics, detergents and the attacks of our immune system. This Halloween, we launched an exciting scary story competition, named Coccus Pocus 2019. The participants were encouraged to write a short horror sci-fi story between 500 and 2,000 words, including antimicrobial resistance and microbial biofilms. The evaluation committee that was composed of eight academics and researchers from the University of Hull and other institutions, ranked the stories according to intrigue of their plot, use of language, character description and scientific soundness. The prizes (online gift vouchers) were awarded to the three winning stories, during an awards ceremony. Feedback questionnaires were completed by the participants, which showed that they all found the competition very interesting and useful, allowing them to sharpen their creative writing skills and explore key microbiology topics. The event was communicated in the social media and blogs were posted in university bulletins and on microbiology websites. It is our ambition that the competition will be held again and again around the country, aiming to increase public awareness about the important problem of antimicrobial resistance and biofilms and boost the enthusiasm of young people about the fascinating field of microbiology.

A medical device comprising of biomaterials responsive to biochemical stimuli: channel for indicating the infective states of wounds and ensuring delivery of smart antimicrobial and antibiofilm agents to promote tissue regeneration and healing.

The importance of providing diagnostic wound dressings that can inform healthcare professionals on the state of infection within wounds but also provide some of the treatment required in response to at risk or infected wounds is of key interest. The aim is to investigate an innovative proof of concept diagnostic and detection system, an intelligent hydrogel wound dressing that responds to specific biochemical stimuli in wounds (MMPs and pH) enabling the selective and triggered release of antibiofilm and antimicrobial agents (‘Detect and Treat’)to the trauma site. The dressing is made of a sterile alginate core material covered in a biocompatible dry or hydrated peptide-polymer-complex film and may include a fluorescent dye which upon release during the wound healing process indicates when a change in dressing is necessary. Efficacy studies of the hydrogel dressing were performed within a drip-flow bioreactor in which regression of Pseudomonas aeruginosa biofilm was observed. A 5-log reduction in biofilm was observed in comparison to an untreated control biofilm. The hydrogel dressing indicated a clear response when in contact with biofilms produced only by pathogenic strains of bacteria when analysed. This further confirmed the adequate release and function of the antimicrobial and antibiofilm agents within the peptide-polymer-complex formulation of the hydrogel wound dressing.

HIV and malaria are the two most prevalent and deadly diseases in the world. Malaria and HIV accounted for about 255 million cases in 2017, with malaria having 86% of this distribution and HIV having 14% of the distribution. Given the overlap of their geographic distribution and resultant rates of coinfection, interactions between the two diseases pose major public health problems. This study was aimed at investigating the epidemiology of malaria –HIV co-infection in respect to sex, age and its association with CD4+ count and viral load. 230 HIV sero-positive participants and 100 HIV sero-negative participants(control) were employed for this study. 52 (22.6%) of the HIV infected participants tested positive for malaria while only 9(9.0%) of the non-HIV participants tested positive to malaria. The prevalence of malarial infection in HIV positive individuals was higher in females (23.9%) than in males (18.5%). While in age group of 30-39 showed the highest prevalence (35.3%) of co-infection. A high prevalence of 47.7% was recorded with CD4+ below 200 cells/μl than 7.6% in participants with CD4+ greater than 200 cells/μl. A highprevalence (49.2%) was also detected in patients with viral load of above 10,000 copies/μl compared to that of those with viral load less than 10,000 copies/μl(12.6%). This study showed a high prevalence of malaria in HIV patients in Awo-Omamma,Oru East, Imo state. This should be considered a great concern to public health. Thus, more effort should be put in research to curb this health issue.

Increased frequency of global travel has facilitated the spread of antimicrobial resistant organisms like extended spectrum β-lactamase (ESBL)- producing organisms and carbapenamase-producing Enterobacteriaceae through increased exposure and transient or persistent colonisation. This study investigates the impact of travel to Laos and of faecal colonisation with ESBLs aiming to help understand the diversity of organisms and also persistency of antimicrobial resistance (AMR) during the study and on return.

Daily faecal samples were collected from 23 doctors visiting Laos over a 21-day period which allowed detection of both constant colonisation and changes to the bowel colonisation over time. Bioinformatic techniques were used to identify ESBL-producing isolates obtained from participants and their contacts. Isolates were sequenced, assembled and annotated. SNP analysis was performed and phylogenetic trees constructed using the core SNP alignment. Escherichia coli was the most prevalent species with a highly diverse array of sequence types. Citrobacter and Klebsiella were the most abundant non-E. coli species. This study confirmed that Laos is an area with high levels of AMR with 28% isolates found to have mobile colistin resistance. There was also a highly diverse and extensive spread of unexpected blaCTX-M genes. Prolonged persistence of resistance genes in the three most prevalent study species found after travellers returned is another serious cause for concern emphasising the extended risk of spread of AMR from high risk to low risk countries. Future work will allow exploration of possible AMR transmission and horizontal transfer of resistance between isolates.

The resistance-nodulation-division (RND) family of efflux pumps confer clinically relevant antibiotic resistance in Gram-negative bacteria, such as Salmonella enterica. RND pumps, including AcrB, are organized as tri-partite systems, consisting of an inner membrane RND pump, a periplasmic adaptor protein (PAP) and an outer membrane channel. Previously, inactivation of the PAPs AcrA and AcrE in S. enterica has been shown to significantly increase susceptibility to antimicrobials and reduce virulence. Therefore, PAPs are seen as attractive targets for the development of efflux pump inhibitors. However, the role of PAPs in the assembly of tri-partite pumps and the residues involved in PAP-RND pump binding is poorly understood. In this study, point mutations in the predicted RND binding residues of AcrA were generated by site-directed mutagenesis. The point mutants were characterised phenotypically through ethidium bromide efflux assays and antimicrobial susceptibility testing. Furthermore, Western blotting was used to verify that the phenotypic effect of the point mutations was not due to destabilisation of the AcrA protein. Point mutations in certain residues, such as G58, F292, R294 and G363 were found to significantly impair efflux activity and increase susceptibility to various antibiotics and dyes, suggesting an important role for these AcrA residues in RND pump binding. Western blotting confirmed that these point mutants were stable and exhibited similar expression levels to the wild-type. These residues could be important targets for the design and development of PAP inhibitors to restore the activity of existing antibiotics and reduce virulence of Salmonella.

The study was to evaluate the food safety knowledge and hygiene practices of the staff of the school feeding scheme in Soweto. A total of 42 food handlers in 13 basic schools under the School Feeding Scheme, Soweto, South Africa were recruited for the study using purposive and convenience sampling methods for the respondents and institutions respectively. A piloted self-administered questionnaire was used. All the respondents were female (100%) with majority being between the ages of 31 and 40 (40%) and had secondary education (63%). About 38 (90.5%) of the respondents indicated that food safety is important, hence, identified “promotion of good health 41 (97.6%), avoidance of bacterial infection 39 (92.9%) and prevention of food poisoning 39 (92.9)” as the major importance. Frequent hand washing 40 (95.2%); cleaning and sanitizing knives/cutting boards 40 (95.3%); checking best before date 39 (92.8); keeping kitchen surfaces clean (80.9%) and checking freshness/appearance of the food upon delivery (88.1%) were indicated as very important food safety and hygiene practices. However, they failed to agree that frozen foods, particularly meat are to be thawed using room temperature (4.8%) and also in the lower shelf in the refrigerators (26.2%) as the best practices. Spearman’s correlation coefficient revealed that no correlation exists between food safety knowledge and hygiene practices. However, there was strong positive correlations among educational level, knowledge and practices (P>0.05). Eventhough they have good knowledge and understanding of food safety issues, they still need training and workshops particularly in HACCP to cover-up the lapses.

Methods: Fungal isolates were isolated fromsoil samples using Potato Dextrose Agar (PDA). The plates were incubated at 25°C for 72 – 96 h and identification was based on molecular techniques by targeting 18S rRNA. Isolation of bioactive compounds from the extracts was carried out by GC-MS analysis. The extracted secondary metabolites was concentrated by evaporation of the solvents at room temperature. Concentrated extract was constituted by dissolving it in DMSO and stored at 4°C for antimicrobial assay.

Results: Two hundred and fifty six (256) fungal isolates were isolated and sixteen (16) of them showed promising antimicrobial potential. Phylogenetic tree showed evolutionary trend of the fungus with 99% similar to Aspergillus terreus. The broad spectrum activity of the antimicrobial substance were observed to be24.7±4.619 and 26.0±0.000 against Gram positive and negative respectively.The bioactive compounds isolated were phenols, amines, terpenes and fatty acid esters.

Conclusion: The functional and new antibiotics can be obtained from fungal family especially in this era of multi-drug resistant (MDR) bacteria. It is observed that there are functional antimicrobial substances associated with Aspergillus terreus. The extracted substances was found to be active against MDR bacteria in vitro. The bioactive compounds obtained from the GC MS analysis revealed the nature of compounds and some of these compounds have been documented in different areas of antimicrobial and related properties.

Introduction: Gram-negative bacteria are a major cause of urinary tract infections (UTIs) and particularly Klebsiella pneumoniae (K. pneumoniae), which is a causative agent of 60-70% of community-acquired infections, about 30% of nosocomial UTIs and 20% of recurrent infections.

Materials and methods: Nine urine samples were collected from patients from various clinical departments in King Abdulaziz University Hospital from 2/3/2019 to 2/4/2019. The microbial contents in the urine samples was analysed by urine culture and VITEK analyses. Here, we compared K. pneumoniae proteins profiles to find possible proteins which could shed a light on host-pathogen interactions. The colonies were suspended in a lysing buffer, which then were sonicated, and the proteins contents were separated using 1D SDS-PAGE, analyzed using liquid chromatography-mass spectrometry LC/MS. Proteins showing different expressions in samples were identified by TripleTOF 5600 mass spectrometer.

Results: All of the Klebsiella pneumoniae isolates were ESBL+ and KPC+ as shown on ChromAgar plates. There was no available data on resistance, ESBL or KPC from VITEK2. Hence, ESBL+ or KPC+ data were only obtained from ChromAgar. Additionally, proteomics analysis revealed the following, the total number of different proteins that are expressed from all of the isolates is 2958 proteins. Where in sample U-102, 328 different proteins expressed, 300 different proteins expressed from isolate U-871, 350 different proteins expressed from isolate U-713, in isolate U-755, 378 different proteins were expressed, 207 different proteins expressed from isolate U-754, 305 different proteins expressed from sample U-134, 290 different proteins expressed from isolate U-968, 600 different proteins expressed from isolate U-104, and 200 different proteins expressed from isolate U-659.

Conclusion: The different proteins between the UTI patients indicated specific host pathogen interaction, each isolate expressed different proteins than the other isolate could be reasoned by host pathogen interaction. Host pathogen interaction are influenced by numbers of factor: age, gender, immunity, underling health conditions, antibiotic treatment, acute UTI or recurrent infection all of these factor could have an impact on the type of proteins that are expressed during infection. Even though the isolates are Klebsiella pneumoniae from young females with UTI, they expressed different proteins, these proteins could explain evolutionary development of pathogens and survival in urinary niche, as pathogens need to express certain type of proteins to enable them to live and survival in urinary niche.

Absorbent hygiene wastes like nappies and incontinence pads are ubiquitous in municipal and healthcare waste streams around the world as they are convenient products used in child-care and adult incontinence management. Absorbent Hygiene Product (AHP) manufacturing is resource-intensive as the products are required to be of the highest value as they are in almost-constant contact with sensitive body parts. The potential for recovering such valuable resources such as cellulose-based fibres and super-absorbent polymers for reuse in non-food sectors like the construction and wastewater industries has been considered in this study. Appropriate decontamination via chemical methods have been examined using AHPs contaminated with human-associated bacteria.

Findings suggest that for simulated AHP wastes inoculated with 108–109 CFU g-1 of human-associated bacteria like Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes, a 1:1 ratio of 0.5% calcium hypochlorite/AHP waste is adequate to inactivate the bacteria particularly when combined with an inorganic salt for at least 60 min. Specifically, 4 to 5 log10 reductions were observed. Following such disinfection, material storage and temperatures above 25ºC minimise incidences of microbial regrowth. The disinfection protocol was not found to adversely affect the AHP quality. Overall, such findings suggest that AHP recycling is a potential alternative to current AHP waste disposal practices like incineration (with or without energy recovery) and landfilling.

Weak and unstable soils can limit the building of new infrastructure. Current soil strengthening techniques such as chemical grouting have detrimental effects on the environment from greenhouse gas production, soil pH modification and groundwater contamination. Microbial-induced calcium carbonate precipitation (MICCP) is a technique that utilises the ability of bacteria to precipitate calcium carbonate, which can be used for a variety of applications including binding adjacent soil particles and filling the pore spaces of soils to increase their mechanical properties. A commonly used bacterium is Sporosarcina pasteurii. A range of factors influences MICCP which presents challenges with process optimisation. Some studies have made use of computational models to predict biocementation at a larger scale, however aspects of models are based on assumption of conditions instead of experimental data.

An aim of this project is to investigate urease activity in S. pasteurii by comparing different growth media, growth stages, pH and temperatures. Ureolysis kinetics of S. pasteurii will be investigated at different urea and calcium chloride concentrations in liquid media. Finally, the biocementation of S. pasteurii in sand syringe setups will also be investigated to compare the effects of changing influencing factors such as growth stage and cell concentration of S. pasteurii, sand particle size, cementation media concentration, duration between cementation media applications and overall number of cementation treatments. Experimental work will be particularly focused towards gaps in the experimental data used in computational models, to help improve these models and bring MICCP biocementation closer to commercial use.

Klebsiella spp. are associated with 3 to 7% of nosocomial infections and can be responsible for a range of conditions including pneumonia, bloodstream infections, meningitis, and necrotizing enterocolitis in infants. The role of Klebsiella pneumoniae in causing disease is well-characterised but, to date, the closely related species Klebsiella oxytoca has not received the same attention, despite often encoding extended-spectrum beta-lactamases and carbapenemases in clinical settings. K. oxytoca is the causative agent of Clostridiodes difficile-negative antibiotic-associated haemorrhagic colitis, a rare condition seen in some individuals receiving antibiotics. Whole-genome sequence analyses have shown K. oxytoca to be a complex comprising at least six species (K. oxytoca, K. michiganensis, K. grimontii, K. huaxiensis, ‘K. pasteurii’, ‘K. spallanzanii’). Our study aims to better characterise the K. oxytoca complex using a polyphasic approach. Preliminary investigations into the genomes of three K. michiganensis clinical isolates revealed the presence of a plasmid-borne ccdABlocus. ccdAB is a toxin-antitoxin (TA) system known to maintain plasmids in other pathogenic enterobacteria. We aim to functionally validate this TA system by cloning and conducting toxicity assays on the CcdB toxin, and cloning and assessing the ability of CcdA to function as an antidote. We also aim to sequence and generate Illumina/Oxford Nanopore hybrid genome assemblies of a larger collection of K. oxytoca complex clinical isolates and investigate their plasmids and TA systems in the same manner.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a pathogen of humans and cattle, in which it is responsible for carriage or invasive disease and subclinical mastitis, respectively. From the 1950s to 1970s, thanks to successful mastitis control programs, the prevalence of GBS fell in the Swedish cattle population, but it re-emerged in the late 1990s. GBS was thought to consist of host-specific subpopulations but recent studies have shown that human and cattle subpopulations overlap, with different accessory genome elements providing survival advantages in each host species. We hypothesized that cattle-adapted GBS was eradicated and replaced by new GBS strains of human origin.

Our aim was to explore the differences in GBS cattle population over six decades (pre-post non-detection), with a focus on the possible role of MGE in the evolution of these strains. Historical (n = 44, 1953 to 1978) and contemporary (n = 76, 1997 to 2012) GBS isolates from bovine milk samples were sequenced and analysed for WGS-MLST. Phylogenetic network analysis revealed the presence of six major clades: two of these were detected only up to 1970, two were only detected after 2004, and two were detected in both periods. Historical isolates were all tetracycline sensitive, whereas 51% of recent isolates harboured tet(M), which is considered a marker of human adaptation. Our data support the elimination of a bovine specific clade (CC61/67) and the emergence of new clades (CC1, CC103/314) that are likely of human of origin.

Staphylococcus aureus is routinely found in sputum samples obtained from people with Cystic Fibrosis (CF). However, its role in the progression of the disease is unclear. This is important, as antibiotic clearance of S. aureus in CF yields unclear clinical results and there is debate around the utility of anti-Staphylococcal antibiotic treatment. We used an ex vivo porcine lung model (EVPL) to compare the growth and virulence of S. aureus isolates from acute CF exacerbations, with isolates from the same donors when they were stable.

There was no significant difference in mean bacterial load between donors, strains or clinical state. However, when we compared the variance in bacterial load of each pair of exacerbation/stable isolates across experimental replicates of the lung model, we found that stable samples grew more consistently in the EVPL compared to those taken from the same donor during an exacerbation.

Virulence factor assay results were mixed, with results implying greater virulence in either stable or acute samples after passage through the EVPL. We could not detect the AIP quorum sensing signal, which control expression of numerous acute virulence factors, using a reporter assay. We hypothesise that S. aureus might down-regulate Agr expression in the model, consistent with a role as a silent persister, rather than as a pathogenic agent. Further work using the EVPL model will determine how well this reflects the clinical reality in CF.

Staphylococcus aureus uses several strategies to evade the host immune reaction including expression of the genes on the immune evasion cluster (IEC), which target the innate immune response, and tarP, which alters the structure of the bacterial wall teichoic acids to avoid binding of host antibodies. IEC is carried on an ΦSa3 prophage, while the tarP gene is found located on diverse prophages. Livestock-associated methicillin-resistant S. aureus of clonal complex 398 (LA-MRSA CC398) typically lacks the IEC, but a number of Danish isolates have been found to carry the elements regardless.

In this study, whole-genome sequences of 96 isolates from humans and 45 isolates from pigs from the North Denmark Region were used to establish a maximum-likelihood phylogeny from core-genome SNPs and to investigate the prevalence of IEC and tarP. Furthermore, epidemiological and national surveillance data were used to investigate household transmission and the prevalence of IEC in LA-MRSA CC398 isolates from the general population. The study documents several independent acquisitions of IEC on distinct ΦSa3 prophages in humans and an almost 3-fold higher human-to-human transmission rate of LA-MRSA CC398 in households with strains carrying IEC than in households with strains lacking it. No such effect was found for tarP, which is widespread among LA-MRSA CC398 isolates from both humans and pigs. Moreover, IEC also seems to promote spread of LA-MRSA CC398 in the general population. Thus, LA-MRSA CC398 is capable of re-adapting to the human host by acquisition of human-specific immune evasion factors encoded on mobile genetic elements.

The evolutionarily ubiquitous multidrug and toxin efflux (MATE) proteins mediate anticancer and antibiotic resistance, while transporting toxins, ions and flavonoids in plants. MATEs of the model amoeba Dictyostelium discoideum have not been studied although sequences of its pair group with the two Homo sapiensMATEs. Ddmate1 and 2 are both transcribed, Ddmate2 more so, with peaks in vegetative and slug life-cycle stages. Ddmate1 was upregulated in response to a toxin, ethidium bromide, at the lowest concentration tested. Removing MATE function by inhibitor or mutation increased intracellular levels of various compounds, confirming these as efflux transporters. Plasma membrane localisation was revealed using a GFP-MATE1 reporter-line. MATE1 and MATE2 phenotypes indicated roles beyond detoxification: on Klebsiella lawns these mutants produced significantly smaller plaques than WT, and their axenic growth rates were also lower. The transporters’ impact on use of Dictyostelium for novel drug research was tested using flavonoids. LCMS and fluorescence-imaging revealed differential flavonoid uptake. Flavanones such as naringenin did not cross into cells, whereas flavonols localised to mitochondria and cytoplasm. Ddmate1 transcription was upregulated, however, in response to naringenin, which is known to reduce levels of kidney-disease protein PKD2 in both Dictyostelium and animal cells. Increased flavonol intracellular concentrations confirmed that efflux not import was impeded in MATE1 and MATE2, and kaempferol therefore further reduced MATE1-cells’ growth. These D. discoideum MATEs may usefully model the HsMATEs, aid understanding of flavonoids’ effects, and should be considered when using this model eukaryote to screen drugs.

The global uncontrolled rise of antimicrobial resistance (AMR) is a major societal threat and it is well documented that AMR is already negatively affecting healthcare and intensive livestock farming systems. Nonetheless, capture fisheries are still essential to the global food supply providing over 50% of the world’s aquatic organism production. To facilitate improved management, it is therefore imperative that we better understand reservoirs of AMR and how gene transmission occurs in the aquatic environment. In order to discern which AMR bacteria and genes are present in the marine environment, sediment and seawater samples were collected and Atlantic Mackerel were captured at the beginning of summer and autumn in the Solent Strait (Portsmouth, U.K). In addition, commercially available Atlantic mackerel were purchased at a local fish market. Using culture-dependent techniques we obtained more than 700 bacterial and 20 fungal isolates from skin, intestinal lining, and intestinal content. Ten different cefotaxime-resistant Pseudomonas spp. were isolated from the seawater and market fish skin samples, and one cefotaxime-resistant Rhanella sp. was isolated from wild fish digesta. Results from ongoing whole-genome shotgun metagenomics analysis will be discussed, as well as the connection between AMR bacteria and AMR gene presence within the marine coastal environment and the local fish markets, which are the last link between capture fisheries and consumers.

The opportunistic pathogen Pseudomonas aeruginosa is a major cause of nosocomial infections, and has been categorised by the World Health Organisation as a “Priority 1: Critical” target for research and development of novel antibiotics owing to its intrinsic multi-resistance and ability to acquire novel resistance mechanisms.

One strategy for discovering novel antibiotics is the identification and characterisation of metabolites with antimicrobial activity. Members of the bacterial phylum Actinobacteria are historic source of these metabolites, in particular the genus Streptomyces. However, other genera have not received this same level of interest despite sharing the capacity to biosynthesise a diverse array of metabolites. One such genus is Micromonospora, responsible for production of the broad-spectrum aminoglycoside antibiotic gentamicin (M. purpurea).

Here we present three Micromonospora species isolated from the Atacama Desert, Chile; that possess anti-pseudomonad bioactivity inducible by culture on International Streptomyces Project (ISP) Media. In addition, preliminary data indicates that this activity can be affected by the addition of P. aeruginosa conditioned media. In parallel, short-read Illumina sequencing was used to assemble draft genomes for these strains, enabling antiSMASH analysis of putative biosynthetic gene clusters. In addition to this, estimated Average Nucleotide Identity (ANI) as calculated by the autoMLST server indicates that these strains may all be novel Micromonospora species.

The results of this work serve to highlight the biosynthetic capacity of an understudied genus of bacteria, as well as the value of examining underexplored environments and habitats in the search for novel bioactive molecules.

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis worldwide. Annually it causes between 13,000 and 20,000 deaths mainly in South-East Asia. Vaccination programmes have drastically reduced the number of JEV cases however outbreaks do still occur. Recently G1 has overtaken G3 as the dominant genotype in most endemic countries and as all currently licensed vaccines are based on G3 it is necessary to evaluate the potential of emerging genotypes to break through current control measures.

This project seeks to generate a virus-like-particle (VLP) neutralisation assay in order to investigate the potential for emergent or divergent genotypes of JEV to infect vaccinated individuals. Using this assay, we will evaluate the ability of vaccinee sera to neutralise emergent genotypes as well as to introduce a variety of SNPs that have been shown to enhance virulence and investigate whether any combination of these are able to invalidate vaccine protection.

There is also concern that recombination between wild-type and live attenuated vaccine strains may occur in the vaccine setting which could result in rescue of the virulent potential of the attenuated virus. Evidence of this is being sought in silico using publicly available data and existing techniques.

These data will provide a clearer picture of the nature of JEV in endemic areas and whether the current strategy of vaccination is sufficient to prevent naturally acquired infection in the long term or if other strategies must now be considered.

To human health worldwide. Existing treatments are becoming inefficacious and therefore there is an urgent need for the development of treatments with alternative modes of action. The use of gallium as an antimicrobial agent has been of interest due to its unconventional mode of action involving the inhibition of iron acquisition and metabolism. The structural similarity and inability to reduce from a trivalent to divalent form under normal physiological conditions allows gallium to act as an iron mimetic and inhibit many iron-dependent biological pathways, respectively.

The antimicrobial potential of gallium maltolate (GaM), Ga(III) coordination complex of maltol, was investigated on the opportunistic pathogen Pseudomonas aeruginosa. In vitro and in vivo analyses using Galleria mellonella (greater wax moth) larvae demonstrated the potent bacteriostatic and non-toxic effect of the complex. Subsequent analysis of GaM treated P. aeruginosa via label-free quantitative proteomics provided an insight into the intrinsic mechanisms of action of GaM. Increased expression of iron-storage protein Bacterioferritin B, the HemO component of iron-sulfur clusters and several stress response proteins (Chaperone Proteins ClpB, HtpG and DnaJ) indicate cell stress in response to inhibited iron uptake. Decreased expression of LasA Protease and LasB Elastase quorum-sensing proteins and flagellar motility proteins FlgM and FlgG further demonstrate the growth inhibitory effect of GaM. These findings provide a basis for a better understanding of the mode of action of GaM, a requirement for the improvement of synthesis and efficacy of the treatment.

Static microcosms are a well-established system used to study the adaptive radiation of Pseudomonas fluorescens SBW25 and the adaptive biofilm-forming mutants known as the Wrinkly Spreaders (WS). We have developed this system to investigate selection within multi-species communities using a soil-wash inoculum dominated by biofilm-competent pseudomonads. Here we present community and isolate-level analyses of one serial-transfer experiment in which replicate populations were selected for over ten transfers and 60 days. Although no significant trends in improving community biofilm characteristics or total microcosm productivity were observed, a significant shift in biofilm-formation and microcosm growth by individual isolates recovered from the initial soil-wash inoculum and final transfers indicated that these communities were subject to selection for growth in these microcosms. Surprisingly, the fitness of the archetypal WS was poor when competing against community samples, and having compared the cell densities in the low-O2 region of liquid column below the biofilm, we suggest that part of the community’s fitness advantage comes from the ability to colonise this under-utilised niche as well as to compete at the A-L interface. Samples from the community biofilms and the low-O2 region were able to re-colonize both niches and many final transfer isolates grew throughout the liquid column as well as forming A-L interface biofilms. This suggests that there is a trade-off between fast growth under highly competitive conditions at the A-L interface and slower growth with less competition in the low-O2 region, with some isolates taking a bet-hedging approach a colonizing both niches in our microcosm system.

Air pollution is the single largest environmental health risk worldwide. Particulate matter (PM) air pollution is released as a result of fossil fuel combustion and vehicle motion, breaking and tyre wear. It has been shown that exposure to PM can cause increased levels of respiratory disease, including the exacerbation of COPD, which is frequently associated with bacterial infection. Despite this, the effects of air pollution exposure on COPD associated respiratory bacteria, includingHaemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae are largely unknown. Our recent publication was the first to document that as well as damaging the host, PM has a direct impact on bacteria that can cause respiratory infections. We showed that exposure to black carbon (BC), an important component of PM, results in alterations in biofilm structure in both Streptococcus pneumoniae and Staphylococcus aureus, and increases dissemination of colonising S. pneumoniaein in vivo models.

Following on from this work, we aim to determine how BC impacts the growth, behaviour and virulence of bacteria associated with the COPD exacerbation, including non-typeable Haemophilus influenzaeand Moraxella catarrhalis. Current data show that BC exposure is decreasing the biofilm forming ability of NTHistrains 162 and 375. M. catarrhalis strain M61 biofilm formation is also decreased in the presence of BC, while its growth rate is increased. In addition, pre-exposing NTHi375 cells to BC, prior to infection of A549 cells, increases their ability to adhere to human epithelial cells. This suggests that the frequency of bacterial infection induced COPD exacerbation may be altered in patients from highly polluted areas.

The excessive use of antibiotics in agriculture is routinely described as a major contributor to bacterial resistance. Globally, antibiotics are widely used as growth supplements in livestock. This has led to concerns regarding human-use antibiotics in food and food-producing animals. Lately, organic acids (OAs) such as propionic acid (PA) and formic acid (FA) have been increasingly used as alternative antimicrobials or preservatives instead of antibiotics. These are particularly efficient at removing salmonella.

Recently, we have shown that exposure of a Crohn’s Disease associated bacterial pathotype, Adherent-invasive Escherichia coli (AIEC), to PA significantly alters its phenotype resulting in increased adhesion and invasion of epithelial cells and increased persistence through biofilm formation. AIEC are both evolutionarily and phylogenetically related to avian pathogenic Escherichia coli (APEC), however the virulence mechanisms of APEC in poultry remain unclear. The widespread use of OAs as growth supplements and antimicrobials in the poultry industry is a rising concern due to the ability of OAs to alter the bacterial pathotype. In this study, we examined the effect of FA on the phenotype of APEC. We observed that following FA-exposure, APEC showed an increased ability to adhere to and invade human intestinal epithelial cells and form better biofilms both aerobically and anaerobically. Worryingly, these isolates also showed an increased resistance to several antibiotics. These results suggest that the increasing use of alternative antimicrobial such as FA in the poultry industry may lead to APEC strains that are increasingly virulent towards human cells with a potential for increased horizontal transmission

Halogenated compounds constitute one of the largest groups of environmental pollutants and are a worldwide issue accumulating readily across even remote environments. Contamination by halogenated flame retardants such as polybrominated diphenyl ether (PBDE) is widespread, including marine and freshwater sediments, soil and human tissue. This has been hypothesised to lead to multiple health issues following exposure such as endocrine disruption and immune system alterations. Despite a recent reduction in use, PBDE containing products will continue to leach into the environment for years. Marine sponges are a natural reservoir of brominated compounds and dehalogenation activities of the associated microbiota have been previously observed. It has been postulated that mechanisms for microbial mediated degradation of halogenated compounds also exist within environmental samples commonly contaminated by PBDEs, for example widespread areas of soil sediment. In order to detect and investigate the dehalogenation capabilities of environmental microorganisms, sediment samples from recycling plants have been used to create microcosms with various PBDE congeners (PBDE 47, 99, 153, 209) added, to establish bacterial communities in the laboratory associated with potential PBDE degradation. Once dehalogenation is detected characterisation of debrominating communities from environmental samples will be performed using microbial community 16S rRNA community profiling and QRTPCR. This will provide valuable insight to bacterial communities associated with haloaromatic degradation and further useful information for potential bioremediation of PBDE contaminated soils and sediments.

The evolution of tubercle bacilli correlates closely with changes in cell envelope surface lipid composition (Donoghue et al. Diversity 2017, 9:46; Jankute et al. Scientific Reports 2017, 7:1315). Smooth, hydrophilic “Mycobacterium canettii” is the first recognisable member of the Mycobacterium tuberculosis complex, but it has reduced pathogenicity and poor aerosol transmission. In contrast, rough M. tuberculosis is very hydrophobic and readily spread in aerosols. Starting from hydrophilic surface lipids in environmental Mycobacterium kansasii, intermediate “M. canettii” adds hydrophobic lipids but retains overall cell hydrophilicity. Eliminating hydrophilic lipooligosaccharides (LOSs) and phenolic glycolipids (PGLs) from “M. canettii” leads to M. tuberculosis with a refined selection of hydrophobic lipids, namely phthiocerol dimycocerosates (PDIMs), pentaacyl trehaloses (PATs) and sulfoglycolipids (SGLs). The relative hydrophobicity of M. tuberculosis is double that of representatives of M. kansasii and “M. canettii”.

The above changes have implications both for the onset of tuberculosis and pinpointing evolutionary hosts. Tuberculosis has not been found in Homo sapiens during the Late Pleistocene, but megafauna are the most likely hosts; characteristic bone lesions have been validated by TB DNA amplification and lipid biomarkers in bison metacarpals up to 17,000 years old. Late Pleistocene enhanced TB hydrophobicity and aerosolisation may have produced megafaunal pandemics, with extinction of bison, mastodons and contemporary taxa. The oldest H. sapiens tuberculosis is from the “Fertile Crescent” back to 9-11ka BP at the start of the Holocene. Naïve humans arriving “Out of Africa” may have encountered newly virulent tubercle bacilli of megafaunal origin, recently refined through a distinct “bottleneck”.

Modernisation has thrown humanity and other forms of life on our planet into ditch of problems. Poverty, climate change, injustice, environmental degradation are few of the shared global problems. The United Nations SDGs are set as blueprint to achieve a better and more sustainable future for all. The SDGs are well structured to address the global challenges we face including poverty, inequalities, hunger, climate change, environmental degradation, peace and justice. The SDGs have been driven mainly by international donors and ‘professional’ international development organisations. The world is left with 10 years to achieve these ambitious goals and targets. Various reviews indicated that little has been achieve on overall, and the SDGs will not be reality if new strategy is not in place to bring inclusion. Microbiology, the scientific discipline of microbes, their effects and practical uses has insightful influence on our day to day living. We present how microbiology and microbiologists could increase the scorecard and accelerate these global goals. Microbiology contribution to peace, justice, gender equality, decent work and economic growth will be also highlighted among others. The pledge of Leave No One Behind will fast track progress and microbiology is better position to make this work.

Cordycepin is an anticancer metabolite produces by a zombie fungus species of Cordyceps militaris. They are capable to infect and hijack insect’s nervous neuron system. Hypoxic environment commonly must be faced by the pathogenic fungus during infection either this zombie fungus. They activate oxygen sensing mode, heme, siderophore, and sterol biosynthesis to overcome it. Underlined our previous study that liquid surfaced culture of C. militaris NBRC103752 produced a higher amount of cordycepin than submerged culture, suggesting that hypoxic conditions might induce it. However, when and how the mechanism of cordycepin production started in liquid surfaced culture is not understood, yet. In our present study, the combination of transcriptomics and gas chromatography-mass spectrometry were carried out during the production phases of cordycepin (5d, 12d, and 19d of incubation periods) and the mechanism of cordycepin production was figured out. The expression of genes in the fermentation pathway and the oxidative phosphorylation pathway were significantly upregulated and down regulated, respectively. Expression of four genes in the heme biosynthesis, including 5-aminolevulinic acid synthase (CCM_01504), delta-aminolevulinic acid dehydratase (CCM_00935), coproporphyrinogen III oxidase (CCM_07483) and cytochrome c oxidase15 (CCM_05057) were upregulated at the beginning of the exponential phase (12d). Further, the activation of Zn(2)-C6 transcription factor that regulates the iron acquisition and ergosterol biosynthesis significantly upregulated and a metabolite reporter adenosine was detected only at 12d. The results in the present study show the correlation between hypoxia and the accumulation of heme before cordycepin biosynthesis.