bluuebunny/crossref_metadata_2025 huggingface dataset:
Study on seroprevalence of IgG antibody of Varicella–Zoster virus in pregnant mothers, neonates and children of six of months old
Authors: Yasmin Selina , Chowdhury Mosharof Hossain
Published: April 2019

This was a cross sectional and observational study, conducted in the Department of Microbiology, Sylhet MAG Osmani Medical College, Sylhet. The study period was from July 2010 to June 2011 in objective to explore the Varicella-Zoster virus immune status (VZV-IgG) in pregnant mothers and neonates up to six months post birth. For this purpose, 60 pregnant women, 60 new born babies and 60 infants aged six months were selected. The mean age of the pregnant woman was 28.8 (SD±4.7) years. The sex of new born babies and six months aged infants was identical [29 (48.3 %) male vs 31 (51.7 %) male; P=0.715]. Seroprevalence rate of IgG antibody of Varicella-Zoster Virus in Pregnant, mothers 81.7%, in new born babies 78.3 % and in infants aged six months were 10.0 %. The seropositivity of VZV IgG level in both pregnant mothers and newborn babies were almost similar but the infant aged six months were significantly lower than that of new born babies (p This showed that a significant proportion of Bangladeshi pregnant mother is susceptible to varicella and infant aged six months is highly susceptible to varicella. Any vaccination strategy must have to take into account these epidemiological variability of the country.
Comparison of methods for detection of Candida in bronchoalveolar lavage (BAL) of cancer patients
Authors: Shoukat Samra , Zeeshan Basit
Published: April 2019

An accurate and rapid identification of Candida species in cancer patients with pulmonary symptoms can provide important information for effective treatment. Candida infections represent an increasing cause of morbidity and mortality in patients receiving immunosuppressive chemotherapy for cancer, organ transplantation or in immunocompromised. We used conventional methods such as culture, fermentation reactions, morphology and molecular methods based on the ribosomal DNA repetitive regions or the Internal Transcribed Spacer (ITS) for the identification of Candida species. Seventy bronchial specimens of Bronchoalveolar Lavage (BAL) from cancer patients at Shaukat Khanum Memorial Cancer Hospital and Research Center. Lahore, Pakistan were included in this study. Seventy cancer patients were diagnosed on the basis of histological profile. Candida detected by conventional methods using Sabouraud Dextrose Agar (SDA), potassium hydroxide (KOH) preparation, germ tube test, fermentation reactions and Gomori methanamine-silver stain (GMS). Thirty (42 %) positive isolates of Candida species were obtained by culture, twenty (28 %) isolates were germ tube test positive while thirty isolates (42 %) were positive by PCR method. In conclusion, the results of our study showed that the PCR based detection methods are significantly better and can detect Candida with more accuracy and specificity as compared to conventional methods. Our study would pave the path for optimization of protocols for detection of Candida in cancer patients.
Characterization of prophages in Salmonella Typhimurium definitive type 8
Authors: Thapa Salina , Mohammed Manal
Published: April 2019

Background

Salmonella Typhimurium DT8 has been associated with human outbreaks. Anderson phage typing scheme has been used for more than four decades for subtyping of Salmonella Typhimurium, but it has shown some limitations. Here, we characterize prophages among Salmonella Typhimurium DT8 strains to test the potential use of prophage sequence profiles for subtyping.

Method

A total of 54 isolates of Salmonella Typhimurium DT8 were selected for this study which was part of an outbreak study by Ashton et al. (2015)*. Twenty-six isolates were associated with an outbreak in the States of Jersey in 2013 and 28 isolates were non-outbreak-associated isolates. Comparative genomics using a range of bioinformatic tools was carried out including SPAdes for de novo assembly of WGS data. PHASTER was used for characterisation of prophages (http://phaster.ca/).

Result

All DT8 strains are lysogenic for three prophages (RE-2010, ST64B and Gifsy-2). Moreover, the three prophages showed identical sequence among outbreak and non-outbreak isolates. Interestingly, prophage SSU5 was detected in one non-outbreak isolate. Prophage SSU5 is closely related to cryptic plasmid pHMC2 that infects only rough strains of Salmonella Typhimurium. This study is the first to report the presence of prophage SSU5 in Salmonella Typhimurium DT8.

Conclusion

It is crucial to use accurate, reliable and highly discriminative subtyping methods for epidemiological characterisation and outbreak investigation of Salmonella Typhimurium. Prophage profiling might be unsuitable subtyping method. The emerging genetic analysis should be combined with the conventional method for epidemiological surveillance until WGS-based analysis can be improved and standardized. *https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336196/
Genome-guided screening of bacterial isolates to identify potential antibiotic producers
Authors: Awolope Opeyemi , O’Driscoll Noelle , Lamb Andrew
Published: April 2019

The urgent need for new antibiotics cannot be overemphasized. Bacterial secondary metabolites remain a relatively untapped source of new therapies. The ability to produce these bioactive compounds is however not universal to all bacterial species. Two key indicators are bacterial genome size (>3 Mb), and the presence of antibiotic-encoding biosynthetic gene cluster (BGCs) within the genomes. BGC distribution is largely determined by phylogeny. Another attribute of some antibiotic producers is the ability to withstand nutritional stress. We exploited these attributes to isolate and identify potential antibiotic producers. A minimal substrate medium was used to isolate nutritionally versatile bacterial strains from topsoil collected from the rhizosphere. The genera of isolates were identified by 16S rRNA gene sequence comparison as Pseudomonas, Hafnia and Obesumbacterium. The typical genome size of species in these genera are 6.2 Mb, 4.7 Mb and 5.0 Mb respectively. The antiSMASH database was browsed by phylogeny to determine the distribution pattern of BGCs in these genera. Pseudomonas strains have an average of 7 BGCs within their genomes that may encode antibiotics, whilst Hafnia and Obesumbacterium strains have 2 and 0 respectively. Therefore, the isolated Pseudomonas strain has the greatest potential to biosynthesis antibiotics. However, the biosynthetic potential of other isolates may be understated given the typical genome size of species in their genera, and their ecological origin. Consequently, all isolates are prime candidates for the next stage of the project which involves genome mining for cryptic or silent genes that may encode novel compounds with antibiotic properties. More isolates are also being recovered.
Unraveling the role of C. difficile S-layer in infection and disease
Authors: Vaz Filipa , Wilson Gill , Kirk Joe , Salgado Paula , Fagan Rob , Douce Gill
Published: April 2019

Clostridium difficile is a Gram-positive spore-forming bacterium. Gut colonization is associated with a wide spectrum of gastrointestinal diseases, ranging from mild to severe, acute to persistent. Bacterial survival and multiplication are essential processes dependent in the maintenance of homeostasis, which is, in turn, inherently dependent on the integrity of the cell wall. Bacteria present different mechanisms to maintain cell wall integrity, particularly, to protect it from direct contact with the external environment, such as phosphate polymers, capsule, outer membrane and S-layer. The S-layer is an evolutionary conserved macromolecule present in almost all bacterial types, however its role(s) remain(s) to be clarified. The S-layer is ubiquitous in C. difficile strains and is comprised of a conserved and a variable region that confers strain specificity and faces the external environment. In C. difficile FM2.5 strain, the absence of S-layer resulted in impaired pathogenicity. Gut colonization of FM2.5 infected mice was significantly lower than mice infected with the wild-type strain R20291. In addition, FM2.5 showed compromised toxin activity and in vitromotility. Accordingly, FM2.5 failed to cause disease in mice and trigger a strong inflammatory response, contrary to mice infected with R20291. Reversion of the S-layer gene restored motility, toxin activity and the abilities to colonize and cause disease. It appears that the S-layer plays a crucial role in various bacterial processes, hence FM2.5 loss of virulence may be due both to the absence of S-layer at the cell surface and its role in other processes that aid to bacterial virulence.
Selection of specific peptides recognised by polyclonal antibody from Salmonella enteritidis infected chicken using next generation phage display technology
Authors: Naqid Ibrahim , Maddison Ben , Owen Jonathan , Gough Kevin
Published: April 2019

Salmonella Enteritidis is an important cause of human salmonellosis and food-poisoning associated with consumption of contaminated chicken eggs and poultry products. Objective of the study was to develop a serological diagnostic testfor the recognition of different Salmonella serovars in chickens. Here, phage peptide libraries were screened against 9 IgY samples from chickens infected with S. Enteritidis and 9 IgY samples with S. Hadar and the individual peptide binders were then identified using NGS. Twenty-nine peptides were identifiedin silico assessmentas being enriched specifically against IgY from multiple chickens infected with S. Enteritidis compared to those infected with S. Hadar. Twenty Nine peptides identifiedin silico assessmentwere thentested by both training and test cohorts of chicken IgY samples in ELISAs. The training set of samples was made upof IgY from 9 chickens infected with S. Enteritidis and 9 infected with S. Hadar. Seventeen peptides were selected as the most recognized specific peptides against S. Enteritidis infection and were then used against IgY samples from10 birds infected with S. Enteritids and20 birds with S. Typhimurium as a test cohort. Overall, for both training and test cohorts the peptide ELISA assay sensitivity and specificity were 90 % for detecting infections. The most discriminatory peptides by ELISA test were AEGEFEPQSARPS and AEGEFFVNRALINQ. The data demonstrated that the NGPD method could identify peptides that represented serovar-specific epitopes/mimotopes, these peptide have potentially important applications for the development of peptide based immuno-diagnostic assays for the recognition of Salmonella Enteritidis in chickens.
Causative agents of early childhood caries: challenges and solutions in culturing and sequencing
Authors: Erickson Daryn , Whealy Ryann , Cocking Jill , Hepp Crystal , Fofanov Viacheslav
Published: April 2019

Dental caries, also known as cavities or dental decay, effects children at a rate five times higher than asthma in the United States. This disease is highly preventable but causes extreme pain and costs millions of dollars to treat every year. The presence of Streptococcus mutansand Streptococcus sobrinusplays a major role in the development and progression of tooth decay; therefore, it is important to establish colonization of these bacteria to assess the risk of developing the disease. Streptococcus bacteria are difficult to grow, extract, and sequence due to strict necessary growth conditions. In this study, we evaluated the performance of different storage and culturing protocols and developed strategies for reducing interference by unwanted bacterial and fungal genomes when sequencing extracted samples. We compared storage conditions of samples at various temperatures, and with and without glycerol. We decided the best storage method was at −80 °C in a specialized solution known as Aimes. When sequencing cultures, we encountered various unwanted bacterial and fungal genomes. To reduce this, we modified our culturing methods by including growth in anaerobic conditions and using serial isolation streaking. These modifications have limited the growth of aerobic specimens and increased culture purity before extraction. With this study, we will be able to better understand the oral microbiome and aim to identify virulence factors in S. mutans and S. sobrinus that contribute to the high rates of dental caries in children.
Species-specific restriction of Bluetongue virus replication correlates to host resilience
Authors: Hardy Alexandra , Stewart Meredith , Shaw Andrew , Varela Mariana , Wilson Sam , Randall Richard , Palmarini Massimo
Published: April 2019

Bluetongue is a vector-borne disease of ruminants caused by bluetongue virus (BTV). BTV can infect essentially all domestic and wild ruminants but the clinical outcome of infection differs substantially between host species. Clinical disease induced by BTV, including haemorrhagic fever in severe cases, is normally evident only in sheep. Conversely, cattle are more resilient to BTV infection, as they develop high levels of viremia and can be reservoirs of infection, but rarely show clinical signs. Here, we concentrated on BTV-host cell interactions using primary cells as an experimental system. First, we determined that BTV reaches higher titres in ovine cells, compared to bovine cells although it induces comparable levels of antiviral cytokines in both cell types. Importantly, these differences are abolished by inhibiting the Jak/Stat pathway. In addition, pre-treatment with interferon (IFN) severely hampers BTV replication in bovine, but not in ovine, primary cells. These data suggest that bovine, unlike ovine, IFN-stimulated genes (ISGs) are effective in controlling BTV replication. Using a high-throughput flow cytometry approach, we screened an expression library of over 300 bovine ISGs to identify genes with antiviral properties against BTV. We have identified ∼10 bovine ISGs that negatively impact BTV replication (by at least 50%). Currently, we are assessing the sheep orthologues to the bovine ISGs of interest in order to investigate host-species differences. Our study provides novel insights on how bovine cells restrict BTV replication and could provide an intellectual framework to understand the host determinants involved in disease severity.
Understanding the population dynamics of organisms exposed to the predatory activity of myxobacteria
Authors: Sydney Natashia , Whitworth David
Published: April 2019

Predatory myxobacteria have an antimicrobial nature that dominates their interactions with neighbouring organisms. They are abundantly found in soil, water, dung of herbivores, and have the potential to significantly affect the microbiome of an environment. In this project, we hypothesize that potential prey organisms evolve in response to the selective pressure exerted by predatory microbes. Using a variety of nutrient media, we isolated bacteria from soil samples to test their susceptibility and resistance to the laboratory strain Myxococcus xanthus DK1622 and its predatory secreted outer membrane vesicles (OMVs). Soil (with and without heat treatment) was spread onto plates which had been pre-inoculated with myxobacteria, OMVs, or no pre-treatment. Plates with myxobacteria lawns or OMVs exhibited reduced diversity of isolates compared to control plates. The yield and diversity of isolates obtained also depended on the media used. Heat pre-treated soil gave rise to distinct morphologies and fewer slime producers. Co-existence and competition were exhibited by soil isolates, which were identified using 16S rRNA gene sequencing and phylogenetic analysis. Purified isolates were also characterised for their resistance and/or susceptibility to predatory attack by a variety of myxobacteria. The bacterial isolates obtained varied when exposed to seven different myxobacteria predators. Our data suggest that the addition of myxobacteria to isolation plates biases isolation towards relatively predation-resistant prey organisms. Our next goal is to isolate myxobacterial predators from the same soil samples (on different prey isolates) and test predator-prey interactions quantitative using pure strains. The genetic basis of differential predatory activity and prey susceptibility can then be investigated.
Mapping 4D pH evolution in Streptococcus mutans biofilms using fluorescent ratiometric pH-sensitive nanosensors
Authors: Perkins Mark , Blunk Birte , Walsh Dean , Chauhan Veeren , Aylott Jonathan , Hardie Kim
Published: April 2019

Streptococcus mutans is a pre-dominant bacterial species found in oral biofilms and participates in the production of dental caries via the generation of organic acids. The production of these acids results from the fermentation of carbohydrates present in a sugar-laden diet. As the acidity of an oral biofilm decreases, the demineralisation of the enamel of a tooth increases; leading to the formation of dental caries. To detect and measure the pH change occurring following a sugar challenge, ratiometric, fluorescent, pH-sensitive nanosensors were incorporated into oral biofilms. Confocal laser scanning microscopy revealed that the addition of glucose (1 % w/v) to an S. mutans biofilm resulted in a gradual reduction in the fluorescence intensity ratio during a 30 min period. This reduction in the fluorescence intensity ratio indicated a reduction in pH of the biofilm over time as the glucose was being fermented, resulting in the production and secretion of acids into the extracellular matrix of the biofilm. Additionally, a reduction in pH was detected – using widefield microscopy – in starved, planktonic S. mutans when treated with glucose. Over the course of 30 min, the pH of the medium was reduced from pH 5.3 to pH 3.3 as the glucose was fermented by the bacteria. These findings will help us map pH changes in oral biofilms as we examine potential methods of preventing the acidification of oral biofilms and the eventual demineralisation of the enamel; leading to the reduction in dental caries and an improvement in the standard of living of those effected.
Genome-led discovery of novel microbial natural products
Authors: Russell Alicia , Lacret Rodney , Truman Andrew
Published: April 2019

Microorganisms have been increasingly exploited for their remarkable ability to produce diverse natural products, which display bioactivities ranging from antimicrobial and anticancer to signalling and developmental. Genome sequencing has revealed that bacteria harbour many more biosynthetic gene clusters (BGCs) for natural products than are currently characterised. Whilst several genome mining tools have been developed to aid discovery, some classes of BGCs remain difficult to identify. One such class is ribosomally-synthesised and post-translationally modified peptides (RiPPs). RiPP BGCs are small with few regions of homology, and the short precursor peptides are rarely annotated in genomes. After developing a targeted genome mining approach for RiPPs, we identified a novel family of BGCs spanning over 200 actinobacterial genomes. The presence of diverse biosynthetic enzymes and sequence variation of the precursor peptides suggest that these BGCs may produce several structurally diverse molecules. We have successfully TAR cloned one such BGC from Streptomycesand expressed it heterologously, and metabolomic analysis led to identification of the pathway product. Gene deletion experiments have also confirmed the involvement of individual biosynthetic enzymes. The compound has been purified and the structure elucidated by NMR. Further work will focus on exploring RiPP BGCs from other species and investigating the biological role of these molecules. The discovery of such a diverse and highly conserved group of BGCs highlights that we are still scratching the surface of the huge biosynthetic capacity of microorganisms, and the use of more sophisticated genome mining tools could help unveil many more important molecules in the future.
Dimerisation of the influenza virus RNA polymerase during viral genome replication
Authors: Walker Alexander , Fan Haitian , Carrique Loic , Keown Jeremy , Bauer David , Grimes Jonathan , Fodor Ervin
Published: April 2019

Influenza virus encodes a heterotrimeric RNA-dependent RNA polymerase (RdRP), composed of subunits PB1, PB2 and PA, that carries out both transcription and replication of the viral RNA genome segments in the context of ribonucleoproteins. Replication of negative-sense viral RNA (vRNA) is a two-step process, progressing via a positive-sense complementary RNA (cRNA) intermediate. The mechanism of viral genome replication is mostly unknown, though there are multiple reports indicating RdRP dimerisation may be central for the process. Purified RdRPs from human and avian influenza A viruses both form dimers of heterotrimers in solution. Using a combination of X-ray crystallography, SAXS and cryo-EM, we identify the interface involved in RdRP dimerization, which is primarily located on the PA C-terminal domain. We use bimolecular fluorescence complementation (BiFC) to show that influenza RdRP forms dimers in mammalian cells through the interface identified in solution. Using a combination of cell-based and in vitro assays, we show that influenza RdRP dimerisation via the PA-C terminal domain is necessary for copying cRNA back into vRNA during viral genome replication. In addition, we show that a nanobody (a small-domain antibody) that interferes with dimerisation attenuates influenza A virus growth in cell culture. These data provide insight into the mechanism of influenza viral genome replication, and identify a potential novel drug target against influenza A virus.
The O6 antigen of Escherichia coli strain CFT073 is a target for Myoviridae
Authors: Moynihan Sinead , Smith Stephen
Published: April 2019

Extraintestinal pathogenic
Escherichia coli
strain CFT073 is a prototypic urosepsis isolate. CFT073 is of serotype O6:K2:H1 and thus has an O-antigen. The wzy gene encodes the O-antigen polymerase Wzy, which catalyses the polymerisation of O subunits into a long chain polysaccharide. A CFT073 Δwzy mutant was constructed using the λ Red recombination system. The lack of O-antigen was confirmed by LPS purification and staining with Pro-Q Emerald 300. CFT073 Δwzy and wild-type CFT073 were tested for their bacteriophage sensitivity. ΦEB49, a member of the Myoviridae, can mediate generalised transduction in strain CFT073. CFT073 Δwzy and wild-type CFT073 mixed with the same concentrations of ΦEB49 phage were compared. Confluent growth was evident on all CFT073 Δwzy plates, whilst lysis was apparent on all CFT073 plates. The inability of ΦEB49 to lyse CFT073 Δwzy suggested that this phage binds to O antigen, hence the decreased O antigen of CFT073 Δwzy, compared to wild-type CFT073, prevented phage lysis from occurring. These data indicate that CFT073 Δwzy is not a susceptible host for ΦEB49.
Analysis of hepatitis C virus genotype 3 resistance to direct acting antivirals
Authors: Rodrigues Fernandes Campos Guilherme , Paulo Vilela Rodrigues João , Bittar Cintia , Forte Aguiar Bruna , Gama Florêncio Chacha Silvana , Fernandes Souza Fernanda , Régis Leira Pereira Leonardo , de Lourdes Candolo Martinelli Ana , Harris Mark , Rahal Paula
Published: April 2019

Treatment of hepatitis C virus (HCV) with direct acting antivirals (DAAs), results in a sustained virological response (SVR) that varies according to the viral genotype. Genotype 3 is the second most prevalent in Brazil and presented the lowest response among all genotypes, this was associated with the occurrence of resistance substitutions in the viral genome. The aim of this study was to analyze genotype 3 viruses circulating in Brazilian patients who do not respond to treatment with Daclatasvir to investigate the presence of novel substitutions within NS5A, and to investigate the role of these substitutions in resistance to different NS5A inhibitors in vitro. A total of 60 patients have been analyzed, including 4 relapsers. Samples collected before (all patients) and after treatment (relapsing patients) were analysed by RNA extraction, cDNA synthesis, amplification by Nested-PCR and direct Sanger sequencing. Many previously undefined substitutions were indeed observed. These included S98G which, although detected in 15 % of pre-treatment samples, substitution appeared in 75 % of post-treatment samples from the non-responding patients and only in 10.7 % of responding individuals. For post-treatment samples in relapsing patients, this substitution had a prevalence of 50 %. Due to the high prevalence, this substitution showed potential to be associated with therapy failure. The phenotypes of this substitution and others identified in the study are being evaluated in the context of a genotype 3a subgenomic replicon and infectious virus to determine their effect on DAA resistance and/or potential fitness cost.
Cyclic di-nucleotides – what is their role in biofilm formation and pathogenicity of Fusobacterium nucleatum?
Authors: Muchova Maria , Slade Daniel J. , Cameron Gary A. , Milward Michael R. , Cooper Paul R. , Kuehne Sarah A.
Published: April 2019

Introduction

Cyclic di-nucleotides (CDNs) act as important second messengers in bacteria, regulating multiple cellular functions, including biofilm formation. Fusobacterium nucleatum is a key player in disease-associated biofilms in periodontitis (gum disease). Previous studies revealed the importance of CDNs in the virulence of other dental pathogens such as Porphyromonas gingivalis, but their function in F. nucleatum virulence remains elusive. Here, we aim to elucidate their importance in the pathogenicity of F. nucleatum.

Methods

Using bioinformatics, we identified a putative dual adenylyl/guanylyl cyclase in the genome of F. nucleatum ATCC 23726, the only currently genetically tractable strain. Consequently this target gene was deleted from the chromosome. Wild-type and mutant strains were grown in single- and multi-species biofilms, the amount of biomass quantified by crystal-violet assay and the biofilm topography analysed using scanning electron microscopy. Additionally, intracellular CDNs were quantified using LC-MS/MS.

Results

Differences in biofilm formation comparing wild-type, mutant and further F. nucleatum subspecies will be presented. Furthermore the level of CDN production of those strains will be shown.

Conclusions

Disease-associated biofilms in periodontitis affect over 50 % of the adult UK population. The disease can be debilitating, potentially leading to tooth loss and the bacteria involved have also been associated with systemic diseases such as cardiovascular disease, arthritis or certain types of cancer. Understanding the involvement of CDNs in the pathogenicity of F. nucleatum and its interaction with various periodontal pathogens might provide new insights into prevention and treatment of periodontitis and other conditions.
The communities that colonise the cold
Authors: Hay Melanie , Edwards Arwyn , Mitchell Andrew
Published: April 2019

The Polar Regions have excellent potential for bioprospecting because the microorganisms that live there have adapted to extreme environmental conditions; and these adaptations can be harnessed in a variety of biotechnological applications. In this study, we describe the community structure of a range of different cryospheric habitats to identify environments appropriate for specific bioprospecting activities. Samples were collected from Midtre Lovénbreen glacier and surrounds in Spitzbergen, Svalbard in late June to early July 2017. Many cryospheric environment types were sampled including air, snow, slush, meltwater, cryoconite, proglacial water, soil and seawater. DNA was extracted and prepared for 16S amplicon sequencing. Amplicon sequence variants (ASVs) were assigned using DADA2 and contaminants were removed using the Decontam package in R. We searched the scientific literature for biotechnological applications of abundant community members in each environment. Each environment type displayed a unique community structure, with some physically linked environment types, like glacial snow, slush and meltwater showing continuity through space, and synchronised changes over time. The snow, slush and meltwater habitats were low in biomass and dominated by Gammaproteobacteria, with Cyanobacteria increasing in abundance as melt progressed. Cryoconite was dominated by Cyanobacteria, while sea water was dominated by Bacteriodetes, Gammaproteobacteria and Cyanobacteria. Soil was by far the most biodiverse habitat, with many phyla represented, and a large number of Actinobacteria suitable for antimicrobial discovery. Many of the current EPS, cold-active enzymes, fatty acids, antioxidants and antifreeze proteins are sourced from Proteobacteria and Cyanobacteria, the most abundant phyla in this study.
Improved molecular typing of toxigenic Clostridium difficile strains affecting animal and human health
Authors: Nowak-Roddy Madelyn , McKenna James , Fairley Derek
Published: April 2019

Clostridium difficile is a Gram-positive, spore forming bacterium, which remains a formidable pathogen as the etiological agent of C. difficile infection (CDI). Substantial effort goes into diagnosis of CDI and characterisation of circulating toxigenic C. difficile strains for epidemiology and infection prevention and control. Currently, molecular typing of C. difficile requires 9 days following diagnosis through PCR ribotyping and multilocus variable number tandem repeat (VNTR) analysis. There is a need for more rapid typing methods to investigate possible linkage between CDI cases in healthcare settings. This study developed a one-step, closed tube real-time PCR and high resolution melt (HRM) assay targeting the intergenic spacer region (ISR) and several VNTR loci, with results generated in 2.5 h. The discriminatory power of the PCR-HRM assay was investigated by typing previously characterised toxigenic clinical and animal C. difficile isolates (n=90). Through comparison of HRM profiles targeting the ISR of isolates belonging to 17 PCR ribotypes, 13 HRM genotypes were recognised with 11 PCR ribotypes resolved from each other. Using correlation between HRM data and known VNTR repeat numbers at the B7, C6 and G8 loci, VNTR repeat numbers for isolates could be predicted within an average absolute difference of 1.8 at the B7 locus, 2.1 at the C6 locus, and 2.5 at the G8 locus. These results suggest that a PCR-HRM assay with a multilocus panel targeting ISR and selected VNTR loci could form part of an improved molecular typing scheme for toxigenic C. difficile strains that is faster than currently available methods.
The Cathelicidin antimicrobial peptide (LL-37) stimulates the growth and pathogenicity of the pulmonary lung pathogen Aspergillus fumigatus
Authors: Sheehan Gerard , Bergsson Gudmundur , McElvaney Noel G. , Reeves Emer P. , Kavanagh Kevin
Published: April 2019

The pulmonary mucus of Cystic Fibrosis (CF) patients displays elevated levels of the Cathelicidin antimicrobial peptide LL-37. The interactions between the pulmonary antimicrobial peptide arsenal and Aspergillus fumigatus, a common pathogen of CF patients, have been neglected in the literature. Exposure of A. fumigatus to LL-37 and its derived fragment RK-31 (1.95 µg ml−1) for 24 h, has a stimulatory effect on growth (199.94±6.17 %, P<0.05) and (218.20±4.63 %, P<0.05) respectively, whereas scrambled (SCR)-LL-37 did not. Exposure of mycelium to 5 µg ml−1 LL-37 for 48 h increased hyphal wet weight (4.37±0.23 g, P<0.001) compared to the SCR-LL-37 treatments. The levels of Immunosuppressive metabolite gliotoxin was significantly increased at 24 h from LL-37 exposed hyphae (169.1±6.36 ng mg−1 hyphae, P<0.05) compared to the SCR-LL-37 treatments. The whole cell proteomic response A. fumigatus to LL-37 revealed an increase in proteins associated with growth (eIF-5A), tissue degradation (aspartic endopeptidase) and allergic reactions (Asp F13). By 48 h there was an increase in proteins indicative of cellular stress, growth and virulence. A. fumigatus conidia incubated in LL-37 and RK-31 displayed increased pathogenicity and fungal burden in the Galleria mellonella larvae model of aspergillosis. These results find that endogenous LL-37 paradoxically stimulates A. fumigatus growth and this may result in increased fungal growth and secretion of toxins in the lungs of CF patients.
Proteome-wide analysis of CD8+ T cell responses to EBV lytic infection
Authors: Forrest Calum , Hislop Andrew , Rickinson Alan , Zuo Jianmin
Published: April 2019

Epstein-Barr virus (EBV) is one of 9 Human herpesviruses that can develop lifelong persistence. These viruses provide an antigenically complex challenge that induce strong CD8+T cell immunity during primary infection and continue to shape this immunity through recurrent lytic reactivation. Here is described the first lytic proteome-wide analysis of CD8+T cell responses to EBV and the first to compare primary vs memory CD8+T cell responses to any human herpesvirus. Primary CD8+T cells were mitogenically expanded directly from the blood of infectious mononucleosis (IM) patients. Comparatively, memory CD8+T cells required pre-enrichment using autologous dendritic cells loaded with a lytically-infected EBV cell lysate and FACS selection based upon the activation marker 4-1BB. Enriched cells were then expanded in vitro as for IM cells. Preparations from 7 IM patients and 7 healthy carriers were screened against each of the 70 EBV lytic cycle proteins in combination with the donors’ HLA-I alleles. Multiple reactivities were identified across the full lytic cycle with 146 responses identified amongst the 7 IM patients and 96 amongst the 7 healthy carriers. However the distribution of responses varied between the 2 cohorts with primary responses targeting IE and a small group of E proteins whereas memory responses targeted all phases but with some prominent responses against L proteins. This infers that responses in primary infection therefore appear to be shaped by presentation on the infected cell surface prior to the activity of viral evasins. However long-term carriage appears to re-shape the virus-specific response.
Identification of novel antimicrobial-producing bacteria from an ancient water source by Oxford Nanopore Whole Genome Sequencing and Natural Product Chemistry
Authors: Walker Tim , Stapleton Paul , Gibbons Simon
Published: April 2019

Microbially-derived antimicrobial compounds are a rich source of clinical antibiotic leads. However, discovery rates have declined over the past 40 years due, in part, to high rediscovery rates of known compounds from traditional soil-based screening approaches. In this study, an ancient hot-spring water source was tested for the presence of antimicrobial-producing bacteria using culture techniques which led to isolation of two organisms capable of inhibiting the growth of multiple bacterial species. Oxford Nanopore whole genome sequencing was used to identify these two isolates as being in one of two genera; Streptomyces and Paenibacillus. Bioinformatic analysis revealed both isolates to have multiple novel secondary metabolite gene clusters. Investigations of the Streptomyces sp. by natural product chemistry techniques showed the organism to produce multiple antimicrobial compounds, these were effective methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. This study underlines the value of investigating non-traditional habitats in the search for novel antibiotic-producing organisms.
Understanding metabolic processes shaping adaptation of E. coli to the gut
Authors: Pedro Miguel , Batista-Barroso João , Pinto Catarina , Dias Joana , Gordo Isabel , Xavier Karina
Published: April 2019

Many microbes colonise the gut establishing interactions with their host and their nutritional environment. Studying genetics and metabolism brought about the drive and potential to engineer communities to promote health and improve industrial processes. However, structuring artificial communities in predictable ways is underdeveloped. We studied Escherichia coli’s genetic targets and physiological mechanisms during gut colonisation and adaptation and how metabolic environment/microbiota complexity shape these processes. We introduced a tractable E. coli K-12 in mice Germ-free or with polymicrobial communities. Whole Genome Sequencing identified potential adaptive targets. Here, we established phenotypic assays as well characterising effects of key mutations and metabolomics was performed with 1H-NMR of intestinal contents. Genes for sugar alcohol metabolism (gat) was the only target common to both mouse models, evidencing specificity. Facing complex microbiota E. coli targeted use of sugar alcohols (srlR, kdgR) and anaerobic respiration (dcuB, focA) [1] whereas alone, we observed instead mutations pointing to increased ability for amino acid use (lrp, dtpB, alaA). Mutations selected correlated dinamically with metabolomics: our results fit the model whereby other microbiota members scavenge oxygen and breakdown complex sugars, limiting E. coli to anaerobically respire simple by-product carbon sources. In the opposing scenario (functional absence) improved amino acid use are favoured colonisation factors. Through experimental evolution we gained insight on shaping E. coli’s metabolic traits through genetic engineering to colonise specific host environments. This work also highlights the versatility of E. coli as potential biotic sensor. [1] Barroso-Batista, J. et al. The first steps of adaptation of Escherichia coli to the gut are dominated by soft sweeps, 2014.
Fungal transformation of cobalt-bearing minerals and metal bioprocessing applications
Authors: Ferrier John , Gadd Geoffrey
Published: April 2019

Geoactive fungi such as Aspergillus niger play a significant role in bioweathering processes and element cycling. These organisms are able to secrete a range of organic acids, such as oxalic acid, into their microenvironment. This enables them to mediate mineral dissolution, leading to metal solubilization and precipitation in the form of secondary biominerals. In this investigation, such biotransformation processes were explored as a means of cobalt bioprocessing, an E-tech element identified as being of key strategic importance, in addition to other mineralogically related metals. A range of Co-bearing mineral phases were investigated, including a Co-bearing lithiophorite [(Al,Li)MnO2(OH)2] and erythrite (Co3(AsO4)2·8H2O), in addition to seafloor ferromanganese nodules. Bioleaching and bioprecipitation studies were carried out to investigate the ability of A. nigerto leach cobalt and related metals from Co-bearing minerals, and to precipitate them in biomineral form as a means of cobalt biorecovery. The objective of the work is to investigate the natural biotransformation of cobalt-bearing minerals, to investigate the factors that influence cobalt bioprocessing and to optimise the maximal yield of cobalt biominerals.
Elucidating the contribution of the Pseudomonas aeruginosa CPX system in biofilm formation
Authors: Paiva Ana , Alexander Morgan , Williams Paul
Published: April 2019

An investigation of Pseudomonas aeruginosa attachment to a diverse range of polymers in a high-throughput microarray format resulted in the discovery of novel biofilm-resistant and biofilm-stimulating materials. These findings raised questions about the nature of the surface interactions involved and in particular the sensory mechanisms use by P. aeruginosa to distinguish between different surface chemistries. To further investigate this, Tn5 mutants of the P. aeruginosa PAO1 Washington sub-line were tested for biofilm formation on the polymer microarrays. This revealed that a Tn5::cpxR mutant had a significant difference in biofilm formation when compared with PAO1-W. In Escherichia coli cpxR forms an operon with cpxA and cpxP. Together they form the CPX two-component signalling system controlling biofilm formation in response to cell envelope stress. To further characterise the P. aeruginosa cpxsystem, ΔcpxR,ΔcpxP and ΔcpxA deletion mutants were constructed. No significant differences were observed in their growth or in the swimming, swarming or twitch motility phenotypes normally required for surface colonization. Unlike E. coli the PAO1-W ΔcpxAmutant was able to invade lung epithelial cells and did not display increased sensitivity to antibiotics. However, the ΔcpxRmutant showed increased biofilm formation on glass and eDNA secretion in both biofilm and liquid modes of growth. This work highlights the relationship between biofilm formation and the CPX system in P. aeruginosa. However, further assays need to be conducted in order to understand the sensory mechanism(s) involved in surface sensing via the CPX system.
Identification and quantification of antibiotic resistant bacteria & Genes in an aquaculture facility which uses a novel bioactive filtering system
Authors: O’Flaherty Aidan , Walsh Fiona
Published: April 2019

The Advanced Biotechnology for Intensive-Freshwater Aquaculture Wastewater Reuse (ABAWARE) project, which is part of the European Commission’s Water Joint Programming Initiative 2016 Joint Call, aims to increase the efficiency and resilience of water use in aquaculture and minimise its negative impact on the environment and human health. This research, which forms one part of the total ABAWARE project, aimed to ascertain the impact of using microbiota and certain plant species, in conjuncture with a more traditional Recirculated Aquaculture System (RAS), as a filtering system had on the arb and G abundance in various samples taken from an aquaculture facility. Sediment and water samples were taken from the inflow, the main fish basin, after the bioactive ponds before filtration, and after filtration. The resistance genes present in these samples were detected using the Wafergen smartchip real-time qPCR system. This system allows for the simultaneous quantification of 348 distinct Antibiotic Resistance Genes for each sample. The samples also underwent microbiome analysis via 16S rRNA metagenomic sequencing. Mothur was used to analyse the sequencing data. This data informs us of the changes in the microbial population changes that are enacted by the various stages within the aquaculture facility.
A retrospective regional audit of Cytomegalovirus (CMV) laboratory diagnostics in Crohn’s/Colitis patients in Northern Ireland – ‘towards a diagnostic algorithm’
Authors: Feeney Susan , Turner Graham , Murphy Seamus , Tham Tony , Jacob George , Morrison Graham , Watson Peter , Kelly Paul , Patterson Lynsey , Coyle Peter V
Published: April 2019

Introduction

Concurrent cytomegalovirus (CMV) in inflammatory bowel disease (IBD) related colitis, (Ulcerative Colitis (UC) or Crohn’s Disease (CD) is an important yet complex scenario associated with high rates of colectomy and other morbidity. This regional audit aimed to identify baseline standards for a virological diagnostic-based approach for the identification of those at risk of CMV reactivation (flare) in the setting of IBD.

Methods

Retrospective, cross-sectional study over a three year time-period, January 2010–March 2013, involving all five Northern Ireland HSC Trusts. Sample cohort n=277 IBD patients of which n=106 further grouped as SRC ‘severe acute colitis and/or steroid refractory colitis’. Seven audit standards were assessed including specimen(s) submission for CMV diagnostic analysis, antiviral therapy, ascertainment of colectomy rate, and result communication protocol.

Findings

Audit primarily found a need to better define test requesting protocols (optimal sample required for optimal serology and molecular diagnostics), and a need to work towards timeliness in both test requesting and result reporting. This audit also identified the high risk patient group (SRC) when test positive for CMV (PCR and/or histopathology) three times more likely to undergo colectomy (OR 3.16, 95 % CI (0.74, 13.21; P=0.06).

Conclusions

Diagnostic-based patient identification is optimal to identify those most at risk of CMV reactivation in the IBD setting. The serological IgG profile is key in risk assessment of potential reactivation, together with quantitative CMV PCR in colonic tissue, with/without supportive histopathology, as the most sensitive and timely means of identify those high risk patients.
Identification of host proteins that interact with non-structural proteins-1α and -1β of PRRSV-1
Authors: Riccio Sofia , Jackson Ben , Hiscox Julian , Graham Simon , Seago Julian
Published: April 2019

Porcine reproductive and respiratory syndrome viruses (PRRSV) are responsible for the most important infectious disease affecting the global pig industry, causing respiratory disease in piglets and reproductive failure in sows. Both species of PRRSV (PRRSV-1 and −2) are rapidly evolving and existing vaccines are failing to control the PRRS panzootic. PRRSV produces 16 non-structural proteins (NSPs) that are involved in viral replication and/or modulating the host immune response. Previous studies have shown that PRRSV NSP1α and NSP1β modulate host cell responses; however, the underlying molecular mechanisms remain to be fully elucidated. PRRSV-1 strains predominate in Europe but have been studied far less than PRRSV-2, therefore, this project aims to identify and characterise novel PRRSV-1 NSP1-host protein interactions. NSP1α and NSP1β from a representative PRRSV-1 subtype 1 strain (215-06) were screened using the yeast-2-hybrid (y-2-h) system and a cDNA library generated from porcine alveolar macrophages – the primary target cell of PRRSV-1. The screens identified 62 and 127 putative binding partners for NSP1α and NSP1β, respectively. Potential binding partners involved in IFN signalling, the NF-κB pathway, ubiquitination and nuclear transport have been selected for confirmation and characterisation. Identifying and characterising these novel interactions will increase our understanding of how PRRSV-1 NSP1α/β modulates the host cellular immune response, which could subsequently be exploited to rationally attenuate PRRSV-1 as a basis for improved vaccines.
Penicillium spp. strains as a possible weapon to fight microbial infections
Authors: Broomfield Abigail , Filipa Simões Marta
Published: April 2019

Bacteria are becoming increasingly resistant to antibiotics, leading to untreatable infections and constituting a major public health hazard (Lewis, 2013). This problem is further increased by the reduction in the number of effective antibiotics to tackle resistant strains (Penesyan et al. 2015), so the search for new compounds is seen as a vital priority. Our study consisted in the analysis of four different environmental strains of Penicillium spp., with screening for their extracellular metabolites for potential antimicrobial activity. Several methods were combined and tested for metabolite production and extraction. The strains were grown on: solid media (I-potato dextrose agar and II-malt extract agar), broth (malt extract broth with III-0 and IV-5% NaCl), and V-tapwater; extractions were performed using three different solvents: A-methanol, B-butanol and C-ethyl acetate. All extracts were tested on three different model organisms: Micrococcus luteus, Escherichia coli and Mycobacterium smegmatis. These are models for Gram-positive and Gram-negative bacteria, and for Tuberculosis. The extracts were compared and analysed in order to determine minimal inhibitory concentrations for each of the microorganisms tested. This research presents preliminary results on the development of potential new chemical compounds to help us circumvent the problem of drug resistance. Lewis, K., 2013. Platforms for antibiotic discovery. Nature reviews Drug discovery, 12(5), pp.371–387. Penesyan, A., Gillings, M. and Paulsen, I.T., 2015. Antibiotic discovery: combatting bacterial resistance in cells and in biofilm communities. Molecules, 20(4), pp.5286–5298.