bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for oscillating circadian clock genes in the copepodCalanus finmarchicusduring the summer solstice in the high Arctic
Authors: Hüppe Lukas , Payton Laura , Last Kim , Wilcockson David , Ershova Elizaveta , Meyer Bettina
Published: July 2020

The circadian clock provides a mechanism for anticipating environmental cycles and is synchronized by temporal cues such as daily light/dark cycle or photoperiod. However, the Arctic environment is characterized by several months of Midnight Sun when the sun is continuously above the horizon and where sea ice further attenuates photoperiod. To test if the oscillations of circadian clock genes remain in synchrony with subtle environmental changes, we sampled the copepodCalanus finmarchicus,a key zooplankter in the north Atlantic, to determinein situdaily circadian clock gene expression near the summer solstice at a southern (74.5° N) sea ice-free and a northern (82.5° N) sea ice-covered station. Results revealed significant oscillation of genes at both stations, indicating the persistence of the clock at this time. While copepods from the southern station showed oscillations in the daily range, those from the northern station exhibited an increase in ultradian oscillations. We suggest that inC. finmarchicus, even small daily changes of solar altitude seem to be sufficient to entrain the circadian clock and propose that at very high latitudes, in under-ice ecosystems, tidal cues may be used as an additional entrainment cue.
Immune-challenged vampire bats produce fewer contact calls
Authors: Stockmaier Sebastian , Bolnick Daniel I. , Page Rachel A. , Josic Darija , Carter Gerald G.
Published: July 2020

Vocalizations are an important means to facilitate social interactions, but vocal communication may be affected by infections. While such effects have been shown for mate-attraction calls, other vocalizations that facilitate social contact have received less attention. When isolated, vampire bats produce contact calls that attract highly associated groupmates. Here, we test the effect of an immune challenge on contact calling rates of individually isolated vampire bats. Sickness behaviour did not appear to change call structure, but it decreased the number of contact calls produced. This effect could decrease contact with groupmates and augment other established mechanisms by which sickness reduces social encounters (e.g. mortality, lethargy and social withdrawal or disinterest).
Modulators of calcium signalling at fertilization
Authors: Stein Paula , Savy Virginia , Williams Audrey M. , Williams Carmen J.
Published: July 2020

Calcium (Ca
2+
) signals initiate egg activation across the animal kingdom and in at least some plants. These signals are crucial for the success of development and, in the case of mammals, health of the offspring. The mechanisms associated with fertilization that trigger these signals and the molecules that regulate their characteristic patterns vary widely. With few exceptions, a major contributor to fertilization-induced elevation in cytoplasmic Ca
2+
is release from endoplasmic reticulum stores through the IP3 receptor. In some cases, Ca
2+
influx from the extracellular space and/or release from alternative intracellular stores contribute to the rise in cytoplasmic Ca
2+
. Following the Ca
2+
rise, the reuptake of Ca
2+
into intracellular stores or efflux of Ca
2+
out of the egg drive the return of cytoplasmic Ca
2+
back to baseline levels. The molecular mediators of these Ca
2+
fluxes in different organisms include Ca
2+
release channels, uptake channels, exchangers and pumps. The functions of these mediators are regulated by their particular activating mechanisms but also by alterations in their expression and spatial organization. We discuss here the molecular basis for modulation of Ca
2+
signalling at fertilization, highlighting differences across several animal phyla, and we mention key areas where questions remain.
Stable body size of Alpine ungulates
Authors: Büntgen Ulf , Jenny Hannes , Galván J. Diego , Piermattei Alma , Krusic Paul J. , Bollmann Kurt
Published: July 2020

In many species, decreasing body size has been associated with increasing temperatures. Although climate-induced phenotypic shifts, and evolutionary impacts, can affect the structure and functioning of marine and terrestrial ecosystems through biological and metabolic rules, evidence for shrinking body size is often challenged by (i) relatively short intervals of observation, (ii) a limited number of individuals, and (iii) confinement to small and isolated populations. To overcome these issues and provide important multi-species, long-term information for conservation managers and scientists, we compiled and analysed 222 961 measurements of eviscerated body weight, 170 729 measurements of hind foot length and 145 980 measurements of lower jaw length, in the four most abundant Alpine ungulate species: ibex (
Capra ibex
), chamois (
Rupicapra rupicapra
), red deer (
Cervus elaphus
) and roe deer (
Capreolus capreolus
). Regardless of age, sex and phylogeny, the body mass and size of these sympatric animals, from the eastern Swiss Alps, remained stable between 1991 and 2013. Neither global warming nor local hunting influenced the fitness of the wild ungulates studied at a detectable level. However, we cannot rule out possible counteracting effects of enhanced nutritional resources associated with longer and warmer growing seasons, as well as the animals' ability to migrate along extensive elevational gradients in the highly diversified alpine landscape of this study.
Antibacterial activity of graphene oxide nanosheet against multidrug resistant superbugs isolated from infected patients
Authors: Aunkor Md. Toasin Hossain , Raihan Topu , Prodhan Shamsul H. , Metselaar H. S. C. , Malik Syeda Umme Fahmida , Azad Abul Kalam
Published: July 2020

Graphene oxide (GO) is a derivative of graphene nanosheet which is the most promising material of the decade in biomedical research. In particular, it has been known as an antimicrobial nanomaterial with good biocompatibility. In this study, we have synthesized and characterize GO and checked its antimicrobial property against different Gram-negative and Gram-positive multidrug drug resistant (MDR) hospital superbugs grown in solid agar-based nutrient plates with and without human serum through the utilization of agar well diffusion method, live/dead fluorescent staining and genotoxicity analysis. No significant changes in antibacterial activity were found in these two different conditions. We also compare the bactericidal capability of GO with some commonly administered antibiotics and in all cases the degree of inhibition is found to be higher. The data presented here are novel and show that GO is an effective bactericidal agent against different superbugs and can be used as a future antibacterial agent.
Evolution of drug-resistant and virulent small colonies in phenotypically diverse populations of the human fungal pathogen
Candida glabrata
Authors: Duxbury Sarah J. N. , Bates Steven , Beardmore Robert E. , Gudelj Ivana
Published: July 2020

Antimicrobial resistance frequently carries a fitness cost to a pathogen, measured as a reduction in growth rate compared to the sensitive wild-type, in the absence of antibiotics. Existing empirical evidence points to the following relationship between cost of resistance and virulence. If a resistant pathogen suffers a fitness cost in terms of reduced growth rate it commonly has lower virulence compared to the sensitive wild-type. If this cost is absent so is the reduction in virulence. Here we show, using experimental evolution of drug resistance in the fungal human pathogen
Candida glabrata,
that reduced growth rate of resistant strains need not result in reduced virulence. Phenotypically heterogeneous populations were evolved in parallel containing highly resistant sub-population small colony variants (SCVs) alongside sensitive sub-populations. Despite their low growth rate in the absence of an antifungal drug, the SCVs did not suffer a marked alteration in virulence compared with the wild-type ancestral strain, or their co-isolated sensitive strains. This contrasts with classical theory that assumes growth rate to positively correlate with virulence. Our work thus highlights the complexity of the relationship between resistance, basic life-history traits and virulence.
Reproduction under light pollution: maladaptive response to spatial variation in artificial light in a glow-worm
Authors: Elgert Christina , Hopkins Juhani , Kaitala Arja , Candolin Ulrika
Published: July 2020

The amount of artificial light at night is growing worldwide, impacting the behaviour of nocturnal organisms. Yet, we know little about the consequences of these behavioural responses for individual fitness and population viability. We investigated if females of the common glow-wormLampyris noctiluca—which glow in the night to attract males—mitigate negative effects of artificial light on mate attraction by adjusting the timing and location of glowing to spatial variation in light conditions. We found females do not move away from light when exposed to a gradient of artificial light, but delay or even refrain from glowing. Further, we demonstrate that this response is maladaptive, as our field study showed that staying still when exposed to artificial light from a simulated streetlight decreases mate attraction success, while moving only a short distance from the light source can markedly improve mate attraction. These results indicate that glow-worms are unable to respond to spatial variation in artificial light, which may be a factor in their global decline. Consequently, our results support the hypothesis that animals often lack adaptive behavioural responses to anthropogenic environmental changes and underlines the importance of considering behavioural responses when investigating the effects of human activities on wildlife.
Higher temperatures lower rates of physiological and niche evolution
Authors: Qu Yan-Fu , Wiens John J.
Published: July 2020

Understanding rates and patterns of change in physiological and climatic-niche variables is of urgent importance as many species are increasingly threatened by rising global temperatures. Here, we broadly test several fundamental hypotheses about physiological and niche evolution for the first time (with appropriate phylogenetic methods), using published data from 2059 vertebrate species. Our main results show that: (i) physiological tolerances to heat evolve more slowly than those to cold, (ii) the hottest climatic-niche temperatures change more slowly than the coldest climatic-niche temperatures, and (iii) physiological tolerances to heat and cold evolve more slowly than the corresponding climatic-niche variables. Physiological tolerances are significantly and positively related to the corresponding climatic-niche variables, but species often occur in climates outside the range of these tolerances. However, mismatches between climate and physiology do not necessarily mean that the climatic-niche data are misleading. Instead, some standard physiological variables used in vertebrates (i.e. critical thermal maxima and minima) may reflect when species are active (daily, seasonally) and their local-scale microhabitats (sun versus shade), rather than their large-scale climatic distributions.
A game theory model for gut bacterial nutrient utilization strategies during human infancy
Authors: Angell Inga Leena , Rudi Knut
Published: July 2020

Despite the fact that infant gut colonization patterns have been extensively studied, we have limited knowledge about the underlying ecological processes. This particularly relates to the ecological choice of nutrient utilization strategies. The aim of the current study was therefore to compare empirically determined nutrient utilization strategies with that expected from a combinatorial game theory model. Observational analyses for 100 mother–child pairs suggested mother–child transmission of specialists with the potential to use few nutrients. Generalists, on the other hand, with the potential to use many nutrients, peaked at three months of age for the children. The level of generalists was gradually replaced with specialists up to 12 months of age. Game theory simulation revealed a competitive advantage of generalists in an expanding population, while more specialized bacteria were favoured with the maturation of the population. This suggests that the observed increase in generalists in the three-month-old children could be due to an immature, expanding gut microbiota population while the increase of specialists at 12 months could be due to population maturation. The simulated and empirical data also correspond with respect to an increased α diversity and a decreased β diversity with the number of simulations and age, respectively. Taken together, game theory simulation of nutrient utilization strategies can therefore provide novel insight into the maturation of the human gut microbiota during infancy.
Testing the greater male variability phenomenon: male mountain chickadees exhibit larger variation in reversal learning performance compared with females
Authors: Branch Carrie L. , Sonnenberg Benjamin R. , Pitera Angela M. , Benedict Lauren M. , Kozlovsky Dovid Y. , Bridge Eli S. , Pravosudov Vladimir V.
Published: July 2020

The greater male variability phenomenon predicts that males exhibit larger ranges of variation in cognitive performance compared with females; however, support for this pattern has come exclusively from studies of humans and lacks mechanistic explanation. Furthermore, the vast majority of the literature assessing sex differences in cognition is based on studies of humans and a few other mammals. In order to elucidate the underpinnings of cognitive variation and the potential for fitness consequences, we must investigate sex differences in cognition in non-mammalian systems as well. Here, we assess the performance of male and female food-caching birds on a spatial learning and memory task and a reversal spatial task to address whether there are sex differences in mean cognitive performance or in the range of variation in performance. For both tasks, male and female mean performance was similar across four years of testing; however, males did exhibit a wider range of variation in performance on the reversal spatial task compared with females. The implications for mate choice and sexual selection of cognitive abilities are discussed and future directions are suggested to aid in the understanding of sex-related cognitive variation.
Sperm velocity in a promiscuous bird across experimental media of different viscosities
Authors: Schmoll Tim , Rudolfsen Geir , Schielzeth Holger , Kleven Oddmund
Published: July 2020

In species with internal fertilization, the female genital tract appears challenging to sperm, possibly resulting from selection on for example ovarian fluid to control sperm behaviour and, ultimately, fertilization. Few studies, however, have examined the effects of swimming media viscosities on sperm performance. We quantified effects of media viscosities on sperm velocity in promiscuous willow warblersPhylloscopus trochilus. We used both a reaction norm and a character-state approach to model phenotypic plasticity of sperm behaviour across three experimental media of different viscosities. Compared with a standard medium (Dulbecco's Modified Eagle Medium, DMEM), media enriched with 1% or 2% w/v methyl cellulose decreased sperm velocity by up to about 50%. Spermatozoa from experimental ejaculates of different males responded similarly to different viscosities, and a lack of covariance between elevations and slopes of individual velocity-by-viscosity reaction norms indicated that spermatozoa from high- and low-velocity ejaculates were slowed down by a similar degree when confronted with high-viscosity environments. Positive cross-environment (1% versus 2% cellulose) covariances of sperm velocity under the character-state approach suggested that sperm performance represents a transitive trait, with rank order of individual ejaculates maintained when expressed against different environmental backgrounds. Importantly, however, a lack of significant covariances in sperm velocity involving a cellulose concentration of 0% indicated that pure DMEM represented a qualitatively different environment, questioning the validity of this widely used standard medium for assaying sperm performance. Enriching sperm environments along ecologically relevant gradients prior to assessing sperm performance will strengthen explanatory power ofin vitrostudies of sperm behaviour.
How to build a virulence factor: the role of three novel enzymes in the biosynthesis of the Group A Carbohydrate
Authors: Zorzoli Azul , Meyer Benjamin , Wagstaff Benjamin , Dorfmueller Helge
Published: July 2020

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

In this work, we used an interdisciplinary approach to demonstrate that:

1) GacB is a novel enzyme that initiates the RhaPS biosynthesis;

2) GacC catalyses the formation of a unique stem;

3) GacG elongates the RhaPS string by adding a yet unknown number of rhamnoses.

Here, we also show that homologs from different streptococcal species can substitute GacB and GacC in the RhaPS production. In particular, we demonstrate that several human pathogens from the Streptococcus genus encompassed in the Lancefield serotyping scheme, and the dental pathogen Streptococcus mutans can replace S. pyogenes’ enzymes. In contrast, the homologs from S. pneumoniae sp. D39 did not, suggesting a different structural arrangement for its surface carbohydrate.

Our results highlight the importance of the group carbohydrate biosynthesis pathways in the Streptococcus genus and open the door for the future development of multi-target compounds that could inhibit these enzymes in Streptococcus pyogenes and other pathogenic streptococci of clinical and veterinary importance.
Deep splicing plasticity of the human adenovirus type 5 transcriptome as a driver of virus evolution
Authors: Donovan-Banfield I’ah , Turnell Andrew , Hiscox Julian , Leppard Keith , Matthews David
Published: July 2020

Viral genomes are characterised by having high gene density and complex transcription strategies. One of the most complex is adenovirus which has a double stranded DNA genome and is the archetypal viral system in which splicing was first discovered. Understanding the transcriptional landscape using conventional mRNA cloning or more recent Illumina-based deep sequencing methods offers insight but also has limitations, including the potential for reverse transcription or PCR amplification artefacts and bias. Here we used direct RNA long read length sequencing on an Oxford Nanopore MinION device to gain a quantitative system-wide overview of transcription and splicing as it dynamically changes during a human adenovirus type 5 infection. This global overview revealed an extensive and hitherto unappreciated complexity of alternative splicing and secondary initiating codon usage. Allied to this, analysis of viral polyadenylation patterns over time showed that most viral transcripts tended to shorter polyadenylation lengths as the infection progressed. Moreover, development and use of an ORF-centric bioinformatics pipeline for analysis of sequenced mRNA, provided both a quantitative and deeper qualitative understanding of the genetic potential of this virus. The data strikingly illustrated that across the viral genome adenovirus made multiple distinctly spliced transcripts that coded for the same ORF. Indeed, as many as 11,000 different splicing patterns were recorded across the viral genome over the three time points analysed. This constitutive low level use of alternative splicing patterns and secondary ORFs potentially enables the virus to maximise its coding potential over evolutionary timescales.
Predominance of Streptococcus pneumoniae among TB patients complicates Dots chemotherapy at infectious diseases hospital Kano, Northwest Nigeria
Authors: Shu’aibumangari Abdurrahaman , Bello Mujitapha
Published: July 2020

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

Keys. TB.
Incidence of other bacterial pathogens among the patients suspected with pulmonary tuberculosis attending Hasiya Bayero Paediatric hospital Kano, Nigeria
Authors: Bello Mujitapha , Muhammad Dauda Mukhtar
Published: July 2020

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

Keywords: Mycobacterium tuberculosis, Co-infection, Bacterial pathogens, pulmonary tuberculosis.
Selfless viruses - the biology and regulation of gene transfer agents
Authors: Fogg Paul , Sherlock David , Xuan Leong Jia
Published: July 2020

Gene transfer agents (GTAs) are genetic elements that have the potential to carry out high frequency horizontal gene transfer. GTAs are similar to small bacteriophages in many ways but, instead of prioritizing the spread of their own genes, they package and disseminate the entire genome of their bacterial host. Indiscriminate transfer of bacterial genes could clearly have a major impact on bacterial evolution, fitness and antimicrobial resistance. Over the past decade, numerous pleiotropic systems have been shown to influence GTA production (e.g. nutrient stress, quorum sensing, SOS response etc.) but all act indirectly and so the mechanism of activation has remained elusive. I will present recent work that identified the missing link that couples GTA production to host regulatory pathways. I will also offer insights into the mechanism of random DNA packaging, the evolutionary role of GTAs in the environment and their potential ecological niche.
An investigation into extended spectrum beta-lactamase colonisation in travellers to Laos
Authors: Snaith Ann , Dunn Steven , Kantele Anu , Kuenzli Esther , Corander Jukka , McNally Alan
Published: July 2020

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

Daily faecal samples were collected from 23 doctors visiting Laos over a 21-day period which allowed detection of both constant colonisation and changes to the bowel colonisation over time. Bioinformatic techniques were used to identify ESBL-producing isolates obtained from participants and their contacts. Isolates were sequenced, assembled and annotated. SNP analysis was performed and phylogenetic trees constructed using the core SNP alignment. Escherichia coli was the most prevalent species with a highly diverse array of sequence types. Citrobacter and Klebsiella were the most abundant non-E. coli species. This study confirmed that Laos is an area with high levels of AMR with 28% isolates found to have mobile colistin resistance. There was also a highly diverse and extensive spread of unexpected blaCTX-M genes. Prolonged persistence of resistance genes in the three most prevalent study species found after travellers returned is another serious cause for concern emphasising the extended risk of spread of AMR from high risk to low risk countries. Future work will allow exploration of possible AMR transmission and horizontal transfer of resistance between isolates.
A two-lane mechanism for selective biological ammonium transport
Authors: Williamson Gordon , Dias Mirandela Gaetan , Tamburrino Giulia , Boeckstaens Melanie , Bizior Adriana , Terras Eilidh , Bage Marcus , Ives Callum , Pisliakov Andrei , Maria Marini Anna , Hoskisson Paul , Zachariae Ulrich , Javelle Arnaud
Published: July 2020

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. A particular controversy has existed around the mechanism of ammonium exchange by the ubiquitous Amt/Mep/Rh transporter family, an essential process in all kingdoms of life. Here, using a combination of SSME electrophysiology, yeast functional complementation, and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+transport in two archetypal members of the family. The pathway underpins a mechanism by which charged H+and neutral NH3 are carried separately across the membrane after NH4+deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.
Assessing acute and chronic Staphylococcus aureus growth and virulence in an ex vivo model of cystic fibrosis lung infection
Authors: Crealock-Ashurst Branagh , Harrison Freya , Sweeney Esther
Published: July 2020

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

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

Virulence factor assay results were mixed, with results implying greater virulence in either stable or acute samples after passage through the EVPL. We could not detect the AIP quorum sensing signal, which control expression of numerous acute virulence factors, using a reporter assay. We hypothesise that S. aureus might down-regulate Agr expression in the model, consistent with a role as a silent persister, rather than as a pathogenic agent. Further work using the EVPL model will determine how well this reflects the clinical reality in CF.
Dissecting meningococcal disease and carriage traits using high throughput phenotypic testing
Authors: De Ste Croix Megan , Neelam Dave , Oldfield Neil , Lucidarme Jay , Turner David , Borrow Ray , Sheppard Samuel , Martinez-Pomares Luisa , Bayliss Christopher
Published: July 2020

Despite on-going vaccination programmes, Neisseria meningitidis causes over 700 cases of invasive meningococcal disease (IMD) in the UK each year. In 2017-18, the MenW and MenY capsular groups caused 38% of all IMD cases. Current policy is to generate genome sequences of all meningococcal disease isolates. Using this resource, we aim to understand how genetic variation contributes to phenotypic differences between carriage and disease isolates.

We are adapting a variety of assays, designed to mimic carriage and disease behaviours, for high throughput phenotypic testing of multiple meningococcal isolates from carriage and cases of IMD. We have selected 335 MenW cc11 and MenY cc23 isolates and are currently testing subsets of isolates in cell culture (CaLu3), growth and biofilm assays. Phenotypic differences will be utilised as input data for Genome Wide Association Studies that aim to identify the specific genomic variants, or combinations of variants, determining observed differences. Genomic data will include whole genome sequences and repeat-mediated phase variation states.

Our preliminary data has detected variation in the ability of cc11 and cc23 isolates to disrupt monolayers of CaLu3 cells, indicating that minor genetic differences in phylogentically similar organisms may be physiologically important for both carriage and disease. We will also discuss progress in establishing successful, high-throughput assays for testing multiple isolates.
What are the implications of genetic variation for the control of Japanese encephalitis virus?
Authors: Bish John , Rose Nicola , Daly Janet
Published: July 2020

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

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

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

These data will provide a clearer picture of the nature of JEV in endemic areas and whether the current strategy of vaccination is sufficient to prevent naturally acquired infection in the long term or if other strategies must now be considered.
Comparison of human gut microbial profiles of matched faecal and biopsy samples from healthy individuals using culture dependent and independent methods
Authors: Mukhopadhya Indrani , Martin Jenny , Shaw Sophie , Lazou Ahrén Irini , Larsson Niklas , Niskanen Titti , Gratz Silvia , Scott Karen
Published: July 2020

Faecal samples have often been used to characterise the gut microbiota in health and disease. There is significant debate whether faecal bacterial communities accurately reflect the mucosa associated bacterial populations, which are considered critical in the aetiopathogenesis of several gastrointestinal diseases. We simultaneously assessed faecal and mucosal microbiota from healthy volunteers to unravel the degree of concordance between the two profiles. Paired fresh rectal biopsies and faecal samples were obtained from ten healthy volunteers and processed under stringent anaerobic conditions. Composition and diversity of the microbiota were studied using next generation sequencing targeting the 16S ribosomal nucleic acid (rRNA) gene and culturomics. Bacterial richness and diversity were comparable between mucosal and faecal samples with no significant statistical differences. The relative abundance of Oxalobacteraceae, Propionibacteriaceae, Campylobacteraceae and Corynebacteriaceae were significantly increased (Corncob analysis; FDR=0.00027, 0.000046, 0.011 and 0.025 respectively) in biopsy compared to faecal samples at the family level. Conversely, there was increased abundance from the family Ruminococcaceae and Clostridiaceae (Corncob analysis; FDR=0.025 and 0.025 respectively) in faecal samples. Principal Coordinates Analysis of a Bray Curtis distance matrix generated from sequence variant tables did not show distinct clustering of biopsy and faecal samples (PERMANOVA; p=0.991). A total of 528 bacteria were isolated from a subset of 6 volunteer samples (biopsy and faeces) out of which there were 97 unique and 39 novel species identified. Our study showed good concordance between faecal and gut mucosal microbial profile, corroborating that faecal samples can act as a convenient surrogate to study gut microbiota.
In vitro reconstitution of the polymicrobial community associated with CF airway infections
Authors: O’Brien Thomas , Welch Martin
Published: July 2020

The airways of persons with cystic fibrosis (CF) provide a nutritionally rich environment that is prone to colonisation by a diverse and dynamic community of microbes (including both bacteria and fungi). Traditionally, Pseudomonas aeruginosa (PA) was thought to be the dominant pathogen associated with CF airway infections. However, it is becoming increasingly clear that interspecies interactions play a crucial role in modulating the physiology and gene expression profile(s) of the protagonists, and also impact on the response to therapeutic intervention. However, there are currently no suitable experimental models that permit long-term successful co-cultivation of PA with other CF-associated pathogens. Simply mixing bacterial or fungal species together and hoping for the best is not a recipe for success, since PA rapidly outcompetes most other species when grown in batch culture. In this work, we rectify this by describing a “3Rs-compliant” continuous-flow in vitro co-culture model. Using our model, it is possible to maintain remarkably stable steady-state co-cultures of major CF-associated pathogens (PA, Staphylococcus aureus and Candida albicans). Our findings reveal that even numerically minor (0.1%, by cell number) species can have a profound impact on quorum sensing and virulence factor production by PA. Furthermore, we show that complete polymicrobial communities derived from CF sputum can be inoculated directly into the model, thus enabling the recapitulation of the entire microbiome associated with CF airway infections to be studied under physiologically relevant conditions.
Healthy Honey Bees – analysis of the Deformed Wing Virus population to assess rational Varroa control on a Scottish island
Authors: Woodford Luke , Evans David , Highet Fiona , Bowman Alan
Published: July 2020

Varroa destructor is an ectoparasitic mite associated with significant losses of honey bee colonies globally. The mite vectors a range of pathogenic viruses, most important of which is Deformed Wing Virus (DWV, (+)ssRNA). Overwintering colony losses, accounting for the death of ∼25% of all colonies each year, are associated with high levels of Varroa-DWV infestation. Effective miticide treatments are available to control Varroa. However, the absence of coordinated treatment means environmental transmission of mites continued unchecked. We aim to determine whether rational, coordinated treatment is beneficial, using features of the DWV population as an indicator of colony health. This study uses coordinated treatment of Varroa in a geographically isolated environment (Isle of Arran, Scotland). It is reported that a high level of a near-clonal virus population is associated with Varroa infestation and colony losses, whereas Varroa-free healthy colonies carry only low levels of a diverse population of DWV. The study area contains 50-85 colonies and 25 beekeepers. Sampling and virus analysis – strain diversity and viral loads – have been conducted before and after treatment. Changes in virus diversity are quantified by next generation sequencing analysis to determine population diversity. In the first two years we have observed a geographic decrease in Varroa and changes in the composition of the virus population. This study will inform our development of rational Varroa control strategies for beekeepers in temperate regions and could be used to inform policy changes regarding treatment regimes in Scotland and elsewhere for this global pathogen.
Biocoatings: Painting bacteria on surfaces
Authors: Krings Simone , Chen Yuxiu , Hingley-Wilson Suzie , L. Keddie Joseph
Published: July 2020

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

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

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

Conclusions: The biocoatings were highly porous thanks to halloysite. E. coli survived the film formation process. Next, we will use E. coli and cyanobacteria to achieve higher efficiency for a variety of applications e.g. pollutant degradation, solar energy harvesting and carbon recycling.
Combination of Rifampicin and Colistin against antibiotic resistant Enterobacteriaceae
Authors: Nam Giyoon , Coates Anthony , Hu Yanmin
Published: July 2020

Antimicrobial resistance is one of the greatest challenges for humanity. Patients, especially, admitted to the intensive care unit have been exposed to higher risk of healthcare associated infections mostly caused by antibiotic resistance since the imprudent use of antibiotics over the years. The discovery and development of new antibiotics are facing difficulties with the continuous evolution of drug resistance in bacteria, and the reduced investment in R&D for antibiotics from pharmaceutical companies. Novel strategies are urgently needed to control this pandemic threat of antibiotic resistance. Antibiotic combination with two or more drugs may be useful in targeting resistant gram-negative bacteria by producing synergistic effect and enhanced bactericidal activates. In this study, we determined the combination of rifampicin and colistin against Extended Spectrum Beta Lactamase (ESBL), carbapenemase producing and colistin resistant Enterobacteriaceae using chequerboard method and time kill curves. We measured the combination effects based on Fractional Inhibitory Concentration index (FICI) and the efficacy of bacterial reduction comparing to that of single antibiotic. Interestingly, we found that the combination of rifampicin and colistin showed synergistic activities against the tested bacteria, indicating FIC index ≤0.5. The time kill curve shows that the two drugs combination exhibits 99% kill whilst the single antibiotic had no activities. Thus, the combination of rifampicin and colistin demonstrated synergistic activity with reduced MIC and the increased rate of killing against both ESBL and carbapenemsase producing and colistin resistant Enterobacteriaceae.
Risk factors associated with the mortality of Acinetobacter baumannii
Authors: Alrahmany Diaa , Golchinheydari Sirous , M. Ghazi Islam
Published: July 2020

Background:
Acinetobacter baumannii (AB) was declared an antibiotic-resistant “Priority 1 pathogen” by WHO. We sought to investigate the predisposing risk factors to this pathogen.

Methods: In a retrospective study, adults who were admitted to Sohar hospital during 2016-2017 and had a positive laboratory-confirmed culture of AB were studied.We classified patients into 2 groups based on 30-day, all-cause mortality and compared the characteristics. Exploratory classification and regression tree (CART) analysis was performed to explore risk factors for mortality to include to a logistic regression model.

Results: A total of 321 patients were included, age was (Mean±SD) 57.42±20.22, male gender was 180(56.07%), mortality was 140(44%). Survivors vs deceased had; length of stay 38.25±88.74 vs 51.31±79.19 (p=0.002),multi-drug resistantisolates 134(51.34%) vs 127(48.66%) p=<0.001, critical care admission 35(38.04%) vs 57(61.96%) p=<0.001, comorbidities 114(47.50%) vs 126(52.50%) p=<0.001 and history of invasive procedures 82(59.85%) vs 55(40.15%) p=0.27. Logistic regression revealed that the odds of dying increase by a factor of 1.044 for every additional year of age, 1.844 times higher for male compared to female, 4.412 times higher for patients admitted into critical care units compared to general wards, 3.138 times higher for patients admitted with a diagnosis of infection, 2.356 times higher for patients with hospital-acquired AB infection compared to community-acquired.

Conclusion: Both modifiable and non-modifiable risk factors are associated with mortality and overall health status may contribute to infection outcome. Stabilization of comorbidities and effective antimicrobial treatment could be the mainstay of successful prevention.
A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus
Authors: D. Drumm Shauna , Owens Rebecca , Mitchell Jennifer , M. Keane Orla
Published: July 2020

In Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins.

Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes.
Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages.
These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.