bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of essential residues for polyprenol phosphate mannose synthase and protein O-mannosyl transferase activities required for protein glycosylation in Streptomyces
Authors: Holman Nathaniel , Banana-Dube Pamela , Wilkinson Tony , Tosin Manuela , Smith Maggie
Published: April 2019

Actinobacteria have a protein O-glycosylation system that resembles eukaryotic protein O-mannosylation. Both M. tuberculosis and S. coelicolor have growth retarded phenotypes when protein-O-mannosyl transferase (Pmt), which transfers mannose from polyprenol phosphate mannose to a target protein, is absent. Moreover, S. coelicolor pmt- mutants are resistant to φC31 phage infection and have increased susceptibility to vancomycin and multiple b-lactams. S. coelicolor strains that lack polyprenol phosphate mannose synthase (Ppm1), which transfers mannose from GDP-mannose to polyprenol phosphate, are even more susceptible to antibiotics and a ppm1- mutant in M. tuberculosis is lethal. Pmt and Ppm1 are therefore possible new targets for the isolation of antimicrobials to be used against M. tuberculosis. Our aim is to further understand the structure and function of these enzymes. Sequence alignments and structural bioinformatics were used for S. coelicolor Ppm1 and Pmt to identify site-directed mutagenesis targets. Mutant alleles were introduced into ppm1- (DT3017) or pmt- (DT2008) S. coelicolor strains using conjugative integrative plasmids and scored for their ability to complement phage sensitivity and antibiotic hyper-susceptible phenotypes. Twenty-two highly conserved Pmt residues were each changed to alanine and six mutants failed to complement DT2008, indicating essentiality. Modelling these six residues indicated that five are positioned close to the predicted catalytic DE motif. For Ppm1, ten mutant alleles were tested and eight were essential for DT3017 complementation, with four residues positioned close to the predicted catalytic DXD motif. Whilst some of the mutations were predicted to impair catalytic activity, others may have affected localisation or substrate binding.
Application of Next Generation Phage Display technology to study cross reactivity in closely-related Flavivirus species
Authors: Varghese Anitha , Daly Janet , Gough Kevin , Rocchi Mara
Published: April 2019

Flaviviruses are a large family of viruses, some members of which cause human and veterinary disease and pose a potential risk of death. The transmission route is typically through the bite of arthropods such as ticks or mosquitoes and. They are a major cause of emerging and re-emerging viral infections. One of the major issues faced when carrying out serology diagnostics is the risk of cross reactive antibodies among closely related species. Next Generation Phage Display is a molecular biology technique combines phage display with next generation sequencing. A phage library is created by inserting peptide sequences into a phagemid vector. Each phage in the resultant library displays a particular peptide on its external surface and the resultant phagemid is packaged within the phage particle. The main advantage of phage display is linking the phenotype (peptide binding properties) with genotype (the peptide gene within the phagemid) Serum antibodies from flavivirus infected species are immobilised on a solid support and incubated with the phage library. Following washing steps to remove non-specific binding, the phage are rescued and propagated in bacteria. This process is called biopanning and is repeated up to 4 times. The phage genomes are sequenced using Ion Torrent sequencing and through analysing the sequences, the antigenic peptide regions can be identified. The most antigenically potent sites can then be used to create a diagnostic assay such an ELISA.
Investigating evolution of the paediatric cystic fibrosis lung microbiota using induced sputum sampling and culture-independent techniques
Authors: Weiser Rebecca , Oakley Juliette , Jones Cerith , Mahenthiralingam Eshwar , Forton Julian
Published: April 2019

Background

Children with cystic fibrosis are frequently non-productive of sputum even during exacerbation. Current routine sampling methods are either invasive (Bronchoalveolar lavage; BAL) or insensitive (cough swab) which makes pathogen surveillance challenging. We investigated induced sputum (IS) as a promising complementary sampling technique, looking at both its comparison to BAL and the evolution of the lung microbiota across children aged 0.5–18 years.

Methods

BAL and IS samples were collected as part of the CF-SpIT study (UKCRN14615; ISRCTNR12473810). DNA was extracted from samples and Illumina NextSeq sequencing of the 16S rRNA gene V4 region was performed. Bioinformatics data processing was carried out using Mothur. Microsoft Excel and R statistical software were used for downstream analyses.

Results

Comparison of the microbiota of 30 BAL-IS matched patient samples indicated that at a presence/absence level, IS captured >80 % of the pathogens observed in BAL samples. These findings validated the expansion of the study to 136 un-matched IS sputum samples, and we found that as patient age increased, bacterial diversity decreased, and changes in the abundance of key genera occurred over time. Both Neisseria and Haemophilus decreased with age, whilst Pseudomonas and a sub-group of Prevotella increased.

Conclusions

Here we uniquely demonstrate using culture-independent techniques that IS captures the majority of the bacterial diversity observed in BAL samples. The age associated decline in lung microbiota diversity, previously documented using BAL samples, can also be captured using IS samples. This suggests that surveillance of microbiota evolution may be possible using this method.
Old drugs learn new tricks – repurposing phenothiazines to uncover effective antimicrobial
Authors: Ferreira Daniela Alves , Martins Marta
Published: April 2019

Thioridazine (TZ) is an antipsychotic drug that acts against antibiotic resistant bacteria. The main aim of this study was to uncover the mechanism of action of TZ using Salmonella enterica serovar Thypimurium as a model bacterium. The antibacterial activity of TZ was initially determined based on its minimum inhibitory concentration (MIC). Membrane permeability assays were performed and fluorescence measured using the Ethidium Bromide accumulation assay. Salmonella was exposed to TZ and its effects on membrane potential and cell wall assessed by flow cytometry and Transmission Electron Microscopy, respectively. Effects on the bacterial proteome were assessed through 2D gel electrophoresis. Infection assasys were performed in THP-1 ad RAW 264.7 cells treated and non-treated with TZ. The MIC of TZ against Salmonella was 200 mg l−1. Our in vitro data demonstrates that TZ mechanism(s) of action involves primarily Salmonella’s membrane by affecting its permeability and potential after 15 min of exposure to TZ. At half of the MIC, and only after 15 min, TZ disrupts the bacterial membrane leading to leakage of the cellular contents and lysis of Salmonella. Proteomic profiling revealed 75 upregulated and 62 downreuglated proteins. Infected macrophages treated with sub-MIC of TZ, showed a reduction on intracellular c.f.u./mL. This may be indicative of TZ’s ability to enhance the killing activity of infected macrophages. The results obtained suggest that TZ may act in vitro by targeting the bacterial cell-envelope. Due to its effect on infected macrophages, TZ may be considered a useful adjuvant to current therapeutics.
Identification of novel factors associated with severe respiratory syncytial virus disease in infants
Authors: Parke Hong Guo , Groves Helen , Broadbent Lindsay , Touzelet Olivier , Douglas Isobel , Campos Guillermo Lopez , Canning Paul , Lyons Jeremy D. , Coyle Peter , Heaney Liam , Shields Michael D. , Power Ultan F.
Published: April 2019

Background

Almost all infants are infected with RSV by 2 years. 1–3 % of RSV-infected infants are hospitalised with severe disease. Reasons for susceptibility to severe disease remain obscure. We aimed to identify factors that might explain such susceptibility.

Methods

We generated well-differentiated primary nasal epithelial cell (WD-PNEC) cultures from infants with histories of severe or mild RSV disease. Following infection with RSV BT2a (clinical isolate), virus growth kinetics, cytopathogenesis, chemokines, and IL-29/IFNλ1 responses and differential gene expression were determined. In an exciting development, 1 differentially expressed gene, ptn, encodes pleiotrophin (PTN), which interacts with nucleolin (NCL), an RSV entry co-factor. The PTN antiviral activity was determined in BEAS-2B cells and WD-PBECs (where B=bronchial).

Results

Viral growth kinetics, cell tropism, IP-10/CXCL10, TRAIL and RANTES/CCL5 responses did not differ significantly between cohorts. However, apical cell sloughing and IL-29/IFNλ1 were diminished in severe WD-PNECs following infection, while expression of isg15, ifi6, irf9, duox2 and tap1 was also reduced. Interestingly, diminished ptn expression was evident in severe WD-PNECs, irrespective of RSV infection. Furthermore, pre-treatment with PTN blocked RSV infection in BEAS-2B cells and WD-PBECs, while neutralisation of PTN with either antibodies or siRNAs resulted in increased RSV replication.

Conclusions

Diminished apical cell sloughing and expression/secretion of IL-29/IFNλ1 and specific interferon stimulated genes in WD-PNECs were associated with severe RSV. Importantly, PTN was identified as a novel endogenously expressed RSV antiviral protein in human airway epithelium. Lower expression of PTN in paediatric airway epithelium may explain, in part, increased susceptibility to severe disease.
The impact of a horizontally acquired virulence plasmid on Bacillus cereus G9241, the causative agent of an anthrax-like illness
Authors: Taylor-Joyce Grace , Brooker Tom , Waterfield Nick
Published: April 2019

Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis, are Gram-positive, spore- forming bacteria and principle members of the Bacillus cereus sensu lato complex. The species are highly similar at a chromosomal level, but are phenotypically diverse due to the presence of different plasmids. The anthrax pathogen, B. anthracis contains two virulence plasmids, pXO1 and pXO2. The pXO1 plasmid carries the anthrax toxin genes which are involved in intracellular survival and suppression of immune cell function while pXO2 carries capsule genes which are required for the pathogen to evade phagocytosis. Both plasmids are required to allow B. anthracis to act as a highly virulent mammalian pathogen. As well as encoding toxins, the pXO1 plasmid encodes atxA, a transcriptional regulator that is able to control gene expression from both the plasmid and chromosome. It is proposed that AtxA is incompatible with the chromosomally encoded PlcR, a global transcriptional regulator which controls expression of secreted haemolytic and cytolytic toxins. This has led to the genetic inactivation of plcRin all B. anthracis isolates and driven the evolution of high mammalian virulence. Interestingly, there are several B. cereus isolates that possess a pXO1-like plasmid, called pBCXO1, which are capable of inducing an anthrax like illness. Importantly, genome sequencing of one such strain, B. cereus G9241, revealed intact copies of both atxA and plcRgenes. This project aims to understand how G9241 has evolved to accommodate both regulators. We have used a pBCXO1-cured strain of G9241 to study the influence of AtxA and pBCXO1 on the biology of G9241 and how it interacts with human macrophage.
Perceptions towards antimicrobial use and resistance in the UK pork supply chain
Authors: Bradford Hollie
Published: April 2019

Antimicrobial Resistance (AMR) occurs when micro-organisms develop the ability to counteract antimicrobial drugs through previous exposure. The past decade has seen an increased prevalence of AMR bacteria due to widespread antimicrobial use (AMU). A substantial share of antimicrobial consumption is attributed to animal production, particularly within the pig industry as it is recognised to be a high user of antimicrobials. Considerable research has focused on the scientific mechanisms of AMR; however, limited literature exists regarding the perceptions of food producers towards AMR. Four databases; Web of Science, PubMed, ScienceDirect and Google were used to search keywords; ‘UK,’ ‘pork,’ ‘supply’ and ‘chain’ to identify relevant papers. Each paper was inspected to ensure that a pork supply chain was illustrated. Interviews were conducted respectively with professionals working in the pork sector to verify the chain and uncover perceptions towards AMU and AMR. Results verified the accurate mapping of the pork chain, enabling professionals to highlight areas of AMU. Stakeholders perceived antimicrobials as useful for the treatment of diseases however, opinions varied regarding the transfer of AMR to humans and the effects this may have on health. To combat the problem of AMR and high use of antimicrobials within the pig industry, it is necessary to identify the key stages of AMU and to uncover stakeholder perceptions along the pork chain. Results will be verified using surveys and an intervention will be designed to enhance knowledge and understanding of AMR to influence a change in behaviour and thus, positively impact farmers on-farm practices.
Influenza A Virus interactions with complement factor H
Authors: Rabeeah Iman , Kishore Uday , Nal-Rogier Beatrice , Shelton Holly
Published: April 2019

Influenza A virus (IAV) is a major causative agent of respiratory tract infection in humans. Human IAVs cause seasonal epidemics, whilst avian IAV strains sporadically cross the host range barrier leading to zoonotic infections. The complement system is a component of the innate immune system that acts to remove pathogens including IAVs. Many bacterial pathogens and several viral pathogens have been reported to actively target components of the complement activation pathway as a defensive strategy. The aim of this project is to understand if there is an interaction between IAV and co-factor H, a critical inhibitor of the complement system and what effect any interaction may have on influenza replication. Using purified human co-factor H protein and purified human (H1N1 and H3N2) and avian (H9N2 and H5N3) IAVs we have demonstrated using ELISA assays that an interaction does occur between co-factor H and all the IAV strains tested. Far western blot analysis suggests that the interaction is with the viral Hemagglutinin (HA), which is responsible for attachment and entry of IAV to cells. Interestingly the interaction has divergent effects on the replication of the IAV strains; enhancing human H1N1 replication and restricting H3N2 replication in A549 and THP-1 cells whilst having no effect on the replication of avian H5N3 or H9N2 viruses. Understanding IAV interaction with the complement pathway could enhance our ability to produce effective vaccines.
Lineage-specific evolution in Listeria monocytogenes detected by analysis of a panel of Swiss isolates from food and human origin
Authors: Zamudio Roxana , Ralph Joseph D , Haigh Richard D , De Ste Croix Megan , Tasara Taurai , Zurfluh Katrin , Kwun Min Jung , Croucher Nicolas J , Bentley Stephen D , Stephan Roger , Oggioni Marco R
Published: April 2019

Listeria monocytogenes is the causative agent of Listeriosis, a foodborne infection and characterised by outbreaks with high mortality. In the past years, outbreak analysis has shifted from PFGE to core genome MLST (cgMLST) and a difference of ≤10 cgMLST alleles was identified as the most appropriate cut off. The cgMLST scheme contributes significantly to the analysis of outbreaks but is less suitable to provide insight in the overall population structure. In the present work, we characterize the differences in the evolution of lineage I and II and the impact they have on the identification of outbreak isolates starting from the genome sequences of a panel of 166 Swiss L. monocytogenes clinical and food isolates. We included in the analysis recently published genomes from Germany (414 isolates) and Holland (128 isolates). Using data of pairwise cgMLST and SNP differences of these 708 isolates, we can clearly identify the genetic diversity associated to outbreaks (≤10 differences), sublineages (≤150 differences) and lineages. The sublineages identified by SNP and cgMLST match the clustering of the PopPUNK software. When broken down for lineages, data show clearly that lineage II has a lower mutation rate within sublineages but a higher diversity between sublineages. Admixture analysis confirms that the increased diversity between sublineages in lineage II is due to horizontal gene transfer and recombination. While our data show that genome evolution is lineage-specific in L. monocytogenes, the cut off for the identification of outbreaks remains identical between both lineages.
Cellular cholesterol abundance regulates potassium accumulation within endosomes and is an important determinant in Bunyavirus entry
Authors: Charlton Frank , Hover Samantha , Fuller Jack , Hewson Roger , Fontana Juan , Barr John , Mankouri Jamel
Published: April 2019

The Bunyavirales order of segmented negative sense RNA viruses includes over 500 isolates that infect insects, animals, and plants, and are often associated with severe and fatal disease in humans. To multiply and cause disease, bunyaviruses must transport their genomes from outside the cell into the cytosol, achieved by transit through the endocytic network. We have previously shown that the model bunyaviruses Bunyamwera virus (BUNV) and Hazara virus (HAZV) exploit the changing potassium concentration ([K+]) of maturing endosomes to release their genomes at the appropriate endosomal location. K+ was identified as a biochemical cue to activate the viral fusion machinery, promoting fusion between viral and cellular membranes, consequently permitting genome release. In this study, we further define the biochemical prerequisites for BUNV and HAZV entry and their K+ dependence. We report four major findings: (1) BUNV and HAZV require cellular cholesterol during virus infection; (2) cholesterol is required during BUNV endosomal escape; (3) cholesterol depletion from host cells impairs their ability to accumulate K+ in maturing endosomes, revealing new insights into endosomal K+ homeostasis; (4) ‘priming’ BUNV virions with K+ prior to infection alleviates BUNV cholesterol requirement, revealing the mechanism of cholesterol dependence. Taken together, we provide a new model in which cholesterol abundance influences K+ endosomal homeostasis and consequently the efficiency of bunyavirus infection. The ability to inhibit bunyaviruses with existing cholesterol lowering drugs offers new options for future anti-bunyavirus interventions for pathogenic family members.
Investigation and genome characterisation of tatenale hantavirus in wild rodent populations in the United Kingdom
Authors: Chappell Joseph G , Tsoleridis Theocharis , McClure C. Patrick , Bennett Malcolm , Tarlinton Rachael E , Ball Jonathan K
Published: April 2019

Hantaviruses are a diverse group of single-stranded, negative sense RNA viruses, belonging to the Bunyaviridae family. They are primarily rodent-borne and transmitted into humans through the inhalation of aerosolised excreta of infected animals. Each hantavirus is typically associated with a single reservoir species, resulting in a persistent, yet asymptomatic infection. In humans, however, in addition to asymptomatic infection two disease syndromes are associated with hantavirus infection, hantavirus haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Tatenale Hantavirus (TATV) was first isolated from field voles in NW England in 2013 and has since been detected at other sites in Northern England. However, only two small fragments of the L and S segments have been sequenced, precluding full phylogenetic characterisation of the virus. Field voles were sampled at two sites in Leicestershire (N=104) and Cheshire (N=12), and their lungs subjected to a degenerate pan-hantavirus RT-PCR assay, targeting a section of the polymerase gene. Eleven Cheshire and one Leicestershire sample were TATV positive, and were 94 % homologous across the amplicon. Illumina Hi-Seq sequencing revealed the complete coding regions of the Leicestershire isolate. Analysis of the sequence showed that TATV is most closely related to Khabarovsk virus, with a homology of 91%/88%/89 % (AA) across the L/M/S Segments, respectively. ICTV species demarcation requires an AA difference of >7 % across the S and M, TATV meets the requirement of a novel species. Retrieval of full coding sequence will allow further investigation into TATV, and its potential to infect humans through pseudotyping of the glycoproteins.
High-throughput sequencing of patients with symptoms of unknown etiology
Authors: Chappell Joseph G , Tsoleridis Theocharis , McClure C. Patrick , Tarlinton Rachael , Clark Gemma , Irving William L , Ball Jonathan K
Published: April 2019

Routine clinical diagnosis of patients with a suspected viral infection involves screening with multiple assays, often limited to only a single genus or species. This approach, however, may fail to detect novel species, atypically presenting viruses (i.e a ‘respiratory’ virus causing neurological symptoms) and viruses that are imported from other countries; often due to clinicians focusing on the ‘likeliest’ candidates. High-throughput sequencing (HTS) allows for the identification viruses present within a sample, by sequencing all viral genomes present. Without the inherent bias of limiting screening targeted assays, these unusual viruses are more likely to be detected. Samples taken from patients with an illness of unknown etiology, were grouped into 5 pools; 2 Respiratory, 2 CSF and an EDTA blood pool. The CSF Pools each contained 200 samples, the Respiratory pools 100 and the EDTA blood pool 80. HTS libraries were created from each of these pools and an additional CSF sample from a single patient with encephalitis and were then sequenced using an Illumina HiSeq platform. Human Pegivirus was detected in both CSF Pools, the EDTA pool and a single respiratory pool. Picobirnavirus was detected in a respiratory pool. RT-PCR was used to screen individual samples compromising these pools. BK Polyomavirus and Mastadenovirus C were detected in the CSF of a patient who had presented with encephalitis. Coinfection of these viruses typically cause neurological symptoms only in immune-compromised patients, so this exemplifies the advantage of using HTS for the detection of atypically presenting viruses.
The Savage Dawn Peptide: an antibiotic woven from 12th century Welsh poetry
Authors: Thomas Benjamin , Gani Jurnorain , Creevey Chris , Hilpert Kai
Published: April 2019

Bacterial antibiotic resistance is widely regarded to be one of the most pressing threats facing humanity. Finding new antibiotics is a vital research area and can now be supported by a vast reservoir of readily available ‘omic data on the back of the explosion of low cost sequencing technologies. Antimicrobial Peptides (AMPs): endogenous peptides that provide a fast and effective means of defence against pathogens as part of the innate immune response. The detection of AMPs in metagenomic data is a tantalising low-hanging fruit for computational biologists. Large reservoirs of existing sequences exist and are well annotated and understood. Post-computational wet-lab work is relatively cheap with spot synthesis of peptides cheaply available from a wide array of third party companies. A well organised screening program can screen 100 s of prospects a day against model bacterial organisms to test for activity and is one of the few areas of biological science that can scale to meet the data output from computational prediction toolkits. AMPLY, an in-house tool designed at Aberystwyth University, supported by Life Science Wales and working in collaboration with Tika Diagnostics at St. George’s Hospital (London) and Queen’s University (Belfast) is part of a next wave of computational drug discovery platforms and is already uncovering a treasure trove of novel AMPs in diverse microbial environments. However, AMPLY’s abilities extend beyond the analysis of ‘omic data alone and its predictive modelling has extracted a therapeutically viable novel AMP built from a string of ancient Welsh poetry.
Pangenomic analysis of Staphylococcus pseudintermedius to better understand antimicrobial resistance profiles
Authors: MacFadyen Alison , Paterson Gavin
Published: April 2019

Although Staphylococcus pseudintermedius are commensal bacteria of dogs, they are also opportunistic pathogens, being the primary cause of canine skin and ear infections. Recently, more attention has been given to this species, due to the emergence of antimicrobial resistance. Further to this a methicillin resistant S. pseuintermedius was isolated from a human and was subsequently found to be multidrug resistant. To better understand the frequency of carriage of antimicrobial resistance, as well as the presence of multidrug resistance, we examined the pangenome of over 200 S. pseudintermedius isolates from across the globe. Focussing on methicillin resistance we were able to identify staphylococcal cassette chromosome mec (SCCmec) types unique to specific Bayesian Analysis of Population Structure (BAPS) groups, including groups which carried mec resistance genes independent of a known SCCmec element. In addition, we identified an SCCmec element, within isolates from North America, that shares 99 % nucleotide identity with a recently described non-typeable SCCmec element carried by Staphylococcus aureus isolated from a human in the Netherlands. Beyond methicillin resistance we found over 50 % of those strains analysed were putatively multidrug resistant (resistant to 3 or more antimicrobial drug classes). This highlights the diverse resistance determinants present in animal staphylococci and the importance of monitoring S. pseudintermedius.
D-Amino acids do no inhibit biofilm formation in Staphylococcus sp.
Authors: Szank Tomasz , Kealey Carmel , Brady Damien
Published: April 2019

D-amino acids are responsible for cell wall re-modelling in Staphylococcus and are capable of inhibition and mature biofilm disassembly. Staphylococcus aureus and Staphylococcus epidermidis are recognised as recurrent nosocomial pathogens and a common cause of biofilm-associated infections. The combination of amino acids used in the study consisted of d- and l- isomers of tyrosine, methionine, tryptophan and phenylalanine. A semiquantitative microplate crystal violet assay was used to assess the effect of amino acids on biofilm development. Biofilm viability staining using fluorescent microscopy was performed to assess the effect of the amino acid mixtures on biofilm development on submerged surfaces. None of the amino acids when tested individually or as a mixture could reduce biofilm formation. However, at the highest concentration tested 25 mmol 1−1 equimolar D-amino acid mixture of tryptophan, phenylalanine, tyrosine and methionine caused a considerable biofilm inhibition in three Staphylococcus strains. Microscopy analysis showed that initial surface attachment remained unaffected at 25 mmol 1−1 mixture of d-amino acids but bacteria did not proceed to form mature biofilms. This suggests inhibition of protein synthesis or a lack of polysaccharide extracellular adhesin formation as no aggregates were observed. The reported bioactivity of D-amino acid on biofilm development and disassembly has been conflictual. It has been established that D-amino acids are incorporated in the bacterial cell wall suggesting they play a role in the complexity of biofilm lifecycle. However, our study indicates that they play no direct role in the inhibition of biofilm formation in Staphylococcus.
Antibiotic resistance transfer in Enterococcus faecalis via pheromone-induced conjugation
Authors: McCarron Mary , Dooley James , Banat Ibrahim , Arnscheidt Joerg , Snelling William
Published: April 2019

It is acknowledged that the environment facilitates antibiotic resistance development and spread but not much research has been undertaken to understand the mechanisms involved. This study focuses on antibiotic resistance transfer by enterococci, normal inhabitants of the mammalian gut and important healthcare associated pathogens, under a variety of environmental conditions. These are novel environmental strains, isolated from a farm in Monaghan, Ireland. The primary aim of this project is to quantify factors controlling the horizontal gene transfer (HGT) of antibiotic resistance genes via pheromone-induced conjugation. The main objectives include; identifying enterococcal isolates capable of transferring antibiotic resistance genes under laboratory conditions; using whole genome sequencing (WGS) to characterise genetic events involved in conjugal transfer; developing markers of transfer efficiency; and developing environmentally relevant models to measure enterococcal HGT. The transfer of vancomycin resistance genes from a donor to a recipient cell have been demonstrated and transfer efficiencies range from 1.09×10−1 to 9.74×10−5. This data shows that some donors are better at donating vancomycin resistance genes than other donors and some recipients are better than other recipients at receiving them. Preliminary data also shows the transfer of trimethoprim, tetracycline and erythromycin and we can see that within a localised environment Enterococcus faecalis can spread resistance genes very differently. In conclusion this project hopes to further our knowledge of the mechanism of pheromone-induced conjugation in environmental Enterococci and to show that environmental enterococcal strains can become multi-drug resistant just as easily as clinical isolates.
Modelling biofilms on infected chronic wounds
Authors: Cheng Yanyan , De Bank Paul , Bolhuis Albert
Published: April 2019

Chronic wounds, for instance venous, pressure, arterial and diabetic ulcers, are a major health problem throughout the world. Compared with normal wounds, those that take more than four weeks to heal are defined as chronic. Interestingly, the numbers of patients suffering from chronic wounds and the cost for treatment have been increasing during the past two decades. There is increasing evidence that suggests that bacteria infect those chronic wounds and there exist as a biofilm, which affects the wound healing and success of wound treatment. To study biofilms in infected wounds, both in vitro and in vivo biofilm models have been developed. In this project, the colony biofilm assay was used to determine antibiotics effect on removing biofilm. The results of this study so far indicated that mature Staphylococcus aureus biofilms were resistant to vancomycin treatment, which works effectively on killing planktonic cells. However, other antibiotics used topically for healing infected chronic wounds, for example, gentamicin, tetracycline, fusidic acid and mupirocin, were more effective at killing mature biofilms in the colony biofilm model. These sets of experiments were also done with pig skin based on colony biofilm assay. This project aims to build up a new ex vivo dynamic model using pig skin and a 3D print flow chamber to mimic chronic wounds. Hence, the results gathered from the colony biofilm assay will be compared with those obtained for the newly developed model. This model can then be used to study the drug delivery and topical treatment of chronic wounds.
Intrageneric competition of staphylococci reveals discrete evolutionary outcomes
Authors: Alrajeh Hind , Horsburgh M. J. , Ghabban Hanaa
Published: April 2019

We previously identified the diversity of antimicrobial activity in nasal microbiomes that correlates with presence and absence of Staphylococcus aureus. The major competitor of skin surfaces is Staphylococcus epidermidis that can express diverse antimicrobial activities. Ten-fold less frequently found in skin and nasal microbiomes, Staphylococcus hominis is relatively unstudied. The aims of the research were to develop insights into the factors of S. hominis that contribute to the dynamics of competition with these major skin colonising staphylococci. One prominent inhibitory strain, S. hominis was selected to culture with both S. aureus SH1000 and S. epidermidis with aim to use genome sequencing to reveal loci contributing to competition. S. aureus evolved during the competition of S. epidermidis and inhibitor-producing S. hominis to reveal two discrete phenotypes. Non-pigmented clone expressed less alpha-haemolysin and had a SNP in agr Cencoding the receptor of the Agr quorum-sensing system. More Pigmented clone had a SNP in sig Bencoding the accessory sigma factor required for expression of the staphyloxanthin. Distinctively, competition of S. aureus SH1000 with S. epidermidis revealed evolution of S. aureus that corresponded with a SNP in the lytSgene of the LytSR two-component system controlling murein hydrolase activity and autolysis. Pacbio genome sequencing followed by use of AntiSMASH revealed the S. epidermidis inhibitory strain, which enabled persistence of S. hominis, encoded the lantibiotic gallidermin biosynthesis operon on a 39 kb plasmid. Gallidermin decreased S. aureus survival to competitor S. epidermidis, and an increased ability of S. hominis to maintain its population size during evolution experiments, which supported the dynamic relationship of the three staphylococci. Future studies will unravel explanations for the contributions the identified loci make to multi-species competition.
Swine influenza A viruses with zoonotic potential – PCR HA/NA typing, and differential detection of pandemic09 reassortants in GB and European pigs
Authors: Brookes Sharon , Reid Scott , Lewis Nicola , Chepkwony Sharon , Russell Christine , Cooper Jayne , Coward Vivien , Everett Helen , Coney Elizabeth , Byrne Alexander , Parys Anna , Vandoorn Elien , Byrne Dominic , Williamson Susanna , Van Reeth Kristien , Brown Ian
Published: April 2019

Swine influenza A virus (swIAV) causes respiratory disease and productivity loss in pigs. Swine ‘flu viruses have been known to be both zoonotic and reverse zoonotic and they contain genes of swine, avian(av) and human(hu) origin. Surveillance of swIAV subtypes is important as genotypes/phenotypes are fluid and impact with respect to epidemiology, vaccination, pig welfare, veterinary and public health. Three sub-types (H1avN1, H1N1pdm09, H1huN2) are currently found in pigs from Great Britain (GB), plus H3huN2 in Europe and their reassortants. Screening of candidate samples is carried out by RRT-PCR assays – generic detection of swIAV (M gene) followed by a specific RRT-PCR for H1N1pdm09 (HA gene), a suite of RRT-PCR assays for sub-typing (HA and NA genes) and a (differential) RRT-PCR to specifically identify reassortant swIAVs that incorporate the pandemic 2009 internal gene cassette (NP gene). Subtyping assays, conventional and/or molecular, are carried out on virus isolation-positive and –negative (RNA only) samples from clinical material (respiratory tissue and/or nasal swabs). Since 2009, the number of swIAV has expanded with the H1N1pdm09 isolates reassorting with the traditional subtypes. Many European variants arose (>25) of which some have become established – in GB including H1huN2/pdm (since 2010), and H1avN1/pdm (since 2012), and in Belgium the traditional isolates were detected plus H1pdmN1/pdm and H3huN2/pdm reassortants. PCR subtyping (2012 onwards ∼130 from GB and ∼40 from BE/NL), wholegenome sequencing and bioinformatics analysis of these isolates facilitate further diagnostic improvements and assessment of zoonotic pandemic potential (in silico and in vivo).
The effects of APOBEC3 proteins on Hepatitis B virus replication
Authors: Elbusifi Ibrahim , McGarvey Michael
Published: April 2019

Hepatitis B virus causes chronic liver infection in 257 million people worldwide. Current treatments against HBV can control, but not cure HBV infection. Therefore, new treatments for chronic HBV infection need to be developed. The APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3) proteins are cellular restriction factors, which have been shown to restrict viral replication for a number of viruses (e.g. HIV) and for retrotransposons. The aim of this study is to elucidate the role of APOBEC3 proteins in inhibiting Hepatitis B virus replication. Quantitative polymerase chain reaction (qPCR) was used to evaluate the impact of APOBEC3 family members on the Hepatitis B virus replication in HepG2.2.15 cells. The highest inhibition of intracellular capsid associated HBV DNA, extracellular virion associated HBV DNA was induced by APOBEC3DE, APOBEC3F, and APOBEC3G as compared to the other APOBEC3 proteins. However, APOBEC3DE showed no inhibition of HBV total RNA, whereas the highest inhibition of HBV total RNA was induced by APOBEC3F and APOBEC3G. The sub-cellular localisation of APOBEC3 proteins was determined by immunofluorescence using confocal microscopy. It was found that APOBEC3DE, APOBEC3F, and APOBEC3G localise to the cytoplasm, suggesting a crucial role of these proteins in HBV replication in the cytoplasm. Nevertheless, APOBEC3A and APOBEC3B localise to the nucleus. The expression and co-localisation of APOBEC3 proteins and viral and host proteins (Uracil DNA Glycosylase UNG and ATP-dependent RNA helicase DDX3) in HepG2.2.15 and HEK293T cells are being investigated in order to determine the interaction between these proteins.
Viral adaptation to alternating hosts and associated allelic variants
Authors: Ayansola Oyeronke , Dickins Benjamin , McVicker Gareth , Loughlin Michael
Published: April 2019

The long-term reproductive success of a lineage depends on its ability to tolerate a wide range of environmental conditions and for a virus a substantial part of its environment is the host that it infects. Viruses may overcome limitations of a given host environment by switching to other available hosts. Experiments addressing host switching may pave the way for an improved understanding of the emergence of new viral diseases. Here, a model bacteriophage, φX174, its canonical laboratory bacterial host strain, Escherichia coli, and a novel host, Salmonella Typhimurium were employed. Bacteriophage φX174 adaptation was studied at population level in a bioreactor. We analyzed phenotypes and genotypes arising during continuous evolution of φX174 on alternating hosts for four consecutive periods of 10 days each. The fitness and adsorption of each viral population were measured using qPCR in liquid culture. Deep sequencing analysis of isolates was carried out to determine the genetic basis of pleiotropic costs and to characterize allelic variations occurring during growth. Some alleles specific to one host were lost or reduced in frequency in the alternative host while other alleles (not present in the ancestor) were shared between hosts. The fitness effects of specific alleles were examined in isolation through targeted mutagenesis. This work contributes to a better understanding of some of the general constraints, costs and benefits influencing the evolution of parasite populations as they adapt to the complexities of a novel host environment- a key consideration during the emergence of infectious diseases.
Application of remote real-time monitoring of biological airborne particles to characterise different hospital environments
Authors: Fennelly Mehael , O’Connor David , Murphy Noel , Casey Colman , Plant Barry , O’Sullivan Niamh , Hellebust Stig , Sodeau John , Prentice Mike
Published: April 2019

Conventional microbial sampling of air in hospitals is usually carried out using settle plates or impaction air samples. This provides little information about intermittent contamination events and is unhelpful for source attribution. Direct continuous bioaerosol sampling is an established technology used to characterise ambient external air. Portable instruments such as the Wideband Integrated Bioaerosol Sensor (WIBS) combine laser particle size and shape detection with signals of biological origin (fluorescence from amino acids and NAD(P)H) characteristic of viable bioaerosols. Monitoring is continuous for weeks at a time and data collected remotely over the internet. We present evidence of the utility of WIBS analysis in characterising air in hospitals in three different environments: operating theatres (plenum ventilated and ultraclean), a respiratory ward, and a specialist cystic fibrosis outpatients. The airborne particle profile was quantitatively and qualitatively different in each environment. Plumes of biologically-relevant airborne particles were detected and source investigation of failing conventional counts in an operating theatre aided by the continuous record. Nebulised drugs contributed a detectable effect on airborne particles which lasted for several hours on the ward despite air changes. A significant effect of plasma air treatment on airborne particles in the ward was detected by WIBS and not conventional cultures. Continuous monitoring may in future allow objective standard setting for airborne particles in different hospital environments and facilitate rapid detection of airborne infection risks.
Production of Influenza C HEF pseudotyped lentiviral particles
Authors: Tatawi Y
Published: April 2019

While influenza A and B virus contain the two glycoproteins Hemagglutinin (HA) and Neuraminidase (NA) inserted into the viral membrane, influenza C virus possesses only one spike designated Hemagglutinin-Esterase-Fusion (HEF) protein which combines the functions of both HA and NA. Like HA, it recognizes and binds to a receptor on the cell surface to initiate virus entry. However, the receptor is N-acetyl-9-O-acetylneuraminic acid. HEF is the receptor-destroying enzyme, which is the function of the neuraminidase (NA) in influenza A and B virus. Although Influenza C virus infections are generally mild and self-limited, it comprises around 13 % of influenza positive cases in hospitalized children. It is also a cause of community acquired pneumonia of children. As influenza research has been more focused on Influenza A and B, it is of interest to pseudotype influenza C HEF. The full sequence of Inflenza C HEF (C/Tokyo/4/2014) was obtained and cloned into pi.18 expression plasmid. The second generation packaging construct pCMVΔR8.91, and the self-inactivating lentiviral vector pCSFLW expressing firefly luciferase, and pCSGW expressing GFP were used for lentiviral vector particle production. The original HEF sequence was mutated by site directed mutagenesis to inhibit esterase activity to prevent receptor destruction prior to pseudotype viral entry. Pseudotyped particles bearing the HEF glycoprotein were successfully produced and verified by fluorescence and Luciferase titration assay with titres of 5×107 RLU/ml. Currently, optimization is undergoing to achieve higher titres as well determining the specific protease/s utilized by HEF for its activation.
No clinical benefit of empirical antimicrobial therapyfor pediatric diarrhea in a high-usage, high-resistance setting
Authors: Thanh Tuyen Ha , Baker Stephen
Published: April 2019

Pediatric diarrheal disease presents a major public health burden in low- to middle-income countries. The clinical benefits of empirical antimicrobial treatment for diarrhea are unclear in settings that lack reliable diagnostics and have high antimicrobial resistance (AMR). In this study, conducted a prospective multicenter cross-sectional study of pediatric patients hospitalized with diarrhea containingblood and/or mucus in Ho Chi Minh City, Vietnam. Clinical parameters, including disease outcome and treatment, were measured. Shigella, nontyphoidal Salmonella (NTS) and Campylobacter were isolated from fecal samples, and antimicrobial susceptibility profiles were determined. Statistical analyses, comprising log-rank tests and accelerated failure time models, were performedto assess the effect of antimicrobials on disease outcome. Among 3166 recruited participants (median age 10 months; interquartile range, 6.5–16.7 months), one-third (1096 of 3166) had bloody diarrhea, and 25 % (793 of 3166) were culture positive for Shigella, NTS, or Campylobacter. More than 85 % of patients (2697 of 3166) were treated with antimicrobials; fluoroquinolones were the most commonly administered antimicrobials. AMR was highly prevalent among the isolated bacteria, including resistance against fluoroquinolones and third-generation cephalosporins. Antimicrobial treatment and multidrug resistance status of the infecting pathogens were found to have no significant effect on outcome. Antimicrobial treatment was significantly associated with an increase in the duration of hospitalization with particular groups of diarrheal diseases. Our results imply a lack of clinical benefit for treating diarrhea with antimicrobials in a setting using high antimicrobials; adequately powered randomized controlled trials are required to assess the role of antimicrobials for diarrhea.
First report of New Clonal groups ST706 and ST1088 from MDR Klebsiella pneumoniae Mexican strains
Authors: Córdova-Espinoza María Guadalupe , Hernández Arana Eder Daniel , Giono-Cerezo Silvia , Sierra Atanacio Erika Gabriela , Tapia Eduardo Carrillo , Carrillo Vázquez Laura Isabel , González-Vázquez Rosa
Published: April 2019

Multidrug-resistant Klebsiella pneumoniae Mexican strains were characterized for the identification of endemic and pandemic clonal groups. The aims of this study were to know population structure and to identify endemic clonal groups inside K. pneumoniae Mexican strains isolated from clinical sources. We studied 93 isolated strains from three third level hospitals and one family clinic from Mexico City. Identification of the strains was done by conventional microbiological methods and an automated system (Vitek2®). The multidrug-resistant phenotype was confirmed following CLSI recommendations, and the strains were classified as MDR, XDR and PDR. Molecular characterization was done by Multilocus Sequence Typing scheme (rpoB, gapA, mdh, pgi, phoE, infB, y tonB). All strains were isolated from hospitalized patients, the most frequent sources were urine and blood cultures. Population structure of K. pneumoniae was clonal, 30 ST were identified, six of them are commonly found. The Clonal complex ST25, ST36, S5392, ST405 and ST551 were isolated from clinical sources, ST1088 was isolated from surfaces of hospital environment.
Enhancers facilitate the birth of de novo genes and their integration into regulatory networks
Authors: Majic Paco , Payne Joshua L.
Published: April 2019

Regulatory networks control the spatiotemporal gene expression patterns that give rise to and define the individual cell types of multicellular organisms. In eumetazoa, distal regulatory elements called enhancers play a key role in determining the structure of such networks, particularly the wiring diagram of “who regulates whom.” Mutations that affect enhancer activity can therefore rewire regulatory networks, potentially causing changes in gene expression that are adaptive. Here, we use whole-tissue and single-cell transcriptomic and chromatin accessibility data from mouse to show that enhancers play an additional role in the evolution of regulatory networks: They facilitate network growth by creating transcriptionally active regions of open chromatin that are conducive to de novo gene evolution. Specifically, our comparative transcriptomic analysis with three other mammalian species shows that young, mouse-specific intergenic open reading frames are preferentially located near enhancers, whereas older open reading frames are not. Mouse-specific intergenic open reading frames that are proximal to enhancers are more highly and stably transcribed than those that are not proximal to enhancers or promoters, and they are transcribed in a limited diversity of cellular contexts. Furthermore, we report several instances of mouse-specific intergenic open reading frames that are proximal to promoters that show evidence of being repurposed enhancers. We also show that open reading frames gradually acquire specific interactions with enhancers over macro-evolutionary timescales, helping integrate new genes into existing regulatory networks. Taken together, our results highlight a dual role of enhancers in expanding and rewiring gene regulatory networks.