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    Mobility of antimicrobial resistance across serovars and disease presentations in non-typhoidal Salmonella from animals and humans in Vietnam
    Bloomfield, S ; Vu, TD ; Ha, TT ; Campbell, J ; Thomson, NR ; Parkhill, J ; Hoang, LP ; Tran, THC ; Maskell, DJ ; Perron, GG ; Nguyen, MN ; Lu, LV ; Adriaenssens, EM ; Baker, S ; Mather, AE (MICROBIOLOGY SOC, 2022-05)
    Non-typhoidal Salmonella (NTS) is a major cause of bacterial enterocolitis globally but also causes invasive bloodstream infections. Antimicrobial resistance (AMR) hampers the treatment of these infections and understanding how AMR spreads between NTS may help in developing effective strategies. We investigated NTS isolates associated with invasive disease, diarrhoeal disease and asymptomatic carriage in animals and humans from Vietnam. Isolates included multiple serovars and both common and rare phenotypic AMR profiles; long- and short-read sequencing was used to investigate the genetic mechanisms and genomic backgrounds associated with phenotypic AMR profiles. We demonstrate concordance between most AMR genotypes and phenotypes but identified large genotypic diversity in clinically relevant phenotypes and the high mobility potential of AMR genes (ARGs) in this setting. We found that 84 % of ARGs identified were located on plasmids, most commonly those containing IncHI1A_1 and IncHI1B(R27)_1_R27 replicons (33%), and those containing IncHI2_1 and IncHI2A_1 replicons (31%). The vast majority (95%) of ARGS were found within 10 kbp of IS6/IS26 elements, which provide plasmids with a mechanism to exchange ARGs between plasmids and other parts of the genome. Whole genome sequencing with targeted long-read sequencing applied in a One Health context identified a comparatively limited number of insertion sequences and plasmid replicons associated with AMR. Therefore, in the context of NTS from Vietnam and likely for other settings as well, the mechanisms by which ARGs move contribute to a more successful AMR profile than the specific ARGs, facilitating the adaptation of bacteria to different environments or selection pressures.
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    Draft Genome Sequences of the Type Strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319), Two NAD-Dependent Bacterial Species Found in the Respiratory Tract of Pigs.
    Bossé, JT ; Li, Y ; Fernandez Crespo, R ; Angen, Ø ; Holden, MTG ; Weinert, LA ; Maskell, DJ ; Tucker, AW ; Wren, BW ; Rycroft, AN ; Langford, PR ; BRaDP1T consortium, ; Gill, SR (American Society for Microbiology, 2020-01-02)
    We report here the draft genome sequences of the type strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319). These NAD-dependent bacterial species are frequently found in the upper respiratory tract of pigs and are occasionally associated with lung pathology.
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    Complete genome for Actinobacillus pleuropneumoniae serovar 8 reference strain 405: comparative analysis with draft genomes for different laboratory stock cultures indicates little genetic variation.
    Bossé, JT ; Li, Y ; Cohen, LM ; Stegger, M ; Angen, Ø ; Lacouture, S ; Gottschalk, M ; Lei, L ; Koene, M ; Kuhnert, P ; Bandara, AB ; Inzana, TJ ; Holden, MTG ; Harris, D ; Oshota, O ; Maskell, DJ ; Tucker, AW ; Wren, BW ; Rycroft, AN ; Langford, PR ; On Behalf Of The BRaDP T Consortium, (Microbiology Society, 2021-11)
    We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain.
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    Identification of genes required for the fitness of Streptococcus equi subsp. equi in whole equine blood and hydrogen peroxide
    Charbonneau, ARL ; Taylor, E ; Mitchell, CJ ; Robinson, C ; Cain, AK ; Leigh, JA ; Maskell, DJ ; Waller, AS (MICROBIOLOGY SOC, 2020-04)
    The availability of next-generation sequencing techniques provides an unprecedented opportunity for the assignment of gene function. Streptococcus equi subspecies equi is the causative agent of strangles in horses, one of the most prevalent and important diseases of equids worldwide. However, the live attenuated vaccines that are utilized to control this disease cause adverse reactions in some animals. Here, we employ transposon-directed insertion-site sequencing (TraDIS) to identify genes that are required for the fitness of S. equi in whole equine blood or in the presence of H2O2 to model selective pressures exerted by the equine immune response during infection. We report the fitness values of 1503 and 1471 genes, representing 94.5 and 92.5 % of non-essential genes in S. equi, following incubation in whole blood and in the presence of H2O2, respectively. Of these genes, 36 and 15 were identified as being important to the fitness of S. equi in whole blood or H2O2, respectively, with 14 genes being important in both conditions. Allelic replacement mutants were generated to validate the fitness results. Our data identify genes that are important for S. equi to resist aspects of the immune response in vitro, which can be exploited for the development of safer live attenuated vaccines to prevent strangles.
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    HAM-ART: An optimised culture-free Hi-C metagenomics pipeline for tracking antimicrobial resistance genes in complex microbial communities.
    Kalmar, L ; Gupta, S ; Kean, IRL ; Ba, X ; Hadjirin, N ; Lay, EM ; de Vries, SPW ; Bateman, M ; Bartlet, H ; Hernandez-Garcia, J ; Tucker, AW ; Restif, O ; Stevens, MP ; Wood, JLN ; Maskell, DJ ; Grant, AJ ; Holmes, MA ; Didelot, X (Public Library of Science (PLoS), 2022-03)
    Shotgun metagenomics is a powerful tool to identify antimicrobial resistance (AMR) genes in microbiomes but has the limitation that extrachromosomal DNA, such as plasmids, cannot be linked with the host bacterial chromosome. Here we present a comprehensive laboratory and bioinformatics pipeline HAM-ART (Hi-C Assisted Metagenomics for Antimicrobial Resistance Tracking) optimised for the generation of metagenome-assembled genomes including both chromosomal and extrachromosomal AMR genes. We demonstrate the performance of the pipeline in a study comparing 100 pig faecal microbiomes from low- and high-antimicrobial use pig farms (organic and conventional farms). We found significant differences in the distribution of AMR genes between low- and high-antimicrobial use farms including a plasmid-borne lincosamide resistance gene exclusive to high-antimicrobial use farms in three species of Lactobacilli. The bioinformatics pipeline code is available at https://github.com/lkalmar/HAM-ART.
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    Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS).
    Bossé, JT ; Li, Y ; Leanse, LG ; Zhou, L ; Chaudhuri, RR ; Peters, SE ; Wang, J ; Maglennon, GA ; Holden, MTG ; Maskell, DJ ; Tucker, AW ; Wren, BW ; Rycroft, AN ; Langford, PR ; BRaDP1T consortium, (Springer Science and Business Media LLC, 2021)
    Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.
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    Large-scale genomic analysis of antimicrobial resistance in the zoonotic pathogen Streptococcus suis
    Hadjirin, NF ; Miller, EL ; Murray, GGR ; Yen, PLK ; Phuc, HD ; Wileman, TM ; Hernandez-Garcia, J ; Williamson, SM ; Parkhill, J ; Maskell, DJ ; Zhou, R ; Fittipaldi, N ; Gottschalk, M ; Tucker, AWD ; Ngo, TH ; Welch, JJ ; Weinert, LA (BMC, 2021-09-07)
    BACKGROUND: Antimicrobial resistance (AMR) is among the gravest threats to human health and food security worldwide. The use of antimicrobials in livestock production can lead to emergence of AMR, which can have direct effects on humans through spread of zoonotic disease. Pigs pose a particular risk as they are a source of zoonotic diseases and receive more antimicrobials than most other livestock. Here we use a large-scale genomic approach to characterise AMR in Streptococcus suis, a commensal found in most pigs, but which can also cause serious disease in both pigs and humans. RESULTS: We obtained replicated measures of Minimum Inhibitory Concentration (MIC) for 16 antibiotics, across a panel of 678 isolates, from the major pig-producing regions of the world. For several drugs, there was no natural separation into 'resistant' and 'susceptible', highlighting the need to treat MIC as a quantitative trait. We found differences in MICs between countries, consistent with their patterns of antimicrobial usage. AMR levels were high even for drugs not used to treat S. suis, with many multidrug-resistant isolates. Similar levels of resistance were found in pigs and humans from regions associated with zoonotic transmission. We next used whole genome sequences for each isolate to identify 43 candidate resistance determinants, 22 of which were novel in S. suis. The presence of these determinants explained most of the variation in MIC. But there were also interesting complications, including epistatic interactions, where known resistance alleles had no effect in some genetic backgrounds. Beta-lactam resistance involved many core genome variants of small effect, appearing in a characteristic order. CONCLUSIONS: We present a large dataset allowing the analysis of the multiple contributing factors to AMR in S. suis. The high levels of AMR in S. suis that we observe are reflected by antibiotic usage patterns but our results confirm the potential for genomic data to aid in the fight against AMR.
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    Evaluation of the recombinant proteins RlpB and VacJ as a vaccine for protection against Glaesserella parasuis in pigs.
    Hau, SJ ; Luan, S-L ; Loving, CL ; Nicholson, TL ; Wang, J ; Peters, SE ; Seilly, D ; Weinert, LA ; Langford, PR ; Rycroft, AN ; Wren, BW ; Maskell, DJ ; Tucker, AW ; Brockmeier, SL ; BRaDP1T Consortium, (Springer Science and Business Media LLC, 2020-05-27)
    BACKGROUND: Glaesserella parasuis, the causative agent of Glӓsser's disease, is widespread in swine globally resulting in significant economic losses to the swine industry. Prevention of Glӓsser's disease in pigs has been plagued with an inability to design broadly protective vaccines, as many bacterin based platforms generate serovar or strain specific immunity. Subunit vaccines are of interest to provide protective immunity to multiple strains of G. parasuis. Selected proteins for subunit vaccination should be widespread, highly conserved, and surface exposed. RESULTS: Two candidate proteins for subunit vaccination (RlpB and VacJ) against G. parasuis were identified using random mutagenesis and an in vitro organ culture system. Pigs were vaccinated with recombinant RlpB and VacJ, outer membrane proteins with important contributions to cellular function and viability. Though high antibody titers to the recombinant proteins and increased interferon-γ producing cells were found in subunit vaccinated animals, the pigs were not protected from developing systemic disease. CONCLUSIONS: It appears there may be insufficient RlpB and VacJ exposed on the bacterial surface for antibody to bind, preventing high RlpB and VacJ specific antibody titers from protecting animals from G. parasuis. Additionally, this work confirms the importance of utilizing the natural host species when assessing the efficacy of vaccine candidates.
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    Research and education in public sector practice: a systems approach to understanding policy impact
    Althaus, C ; Carson, L ; Sullivan, H ; van Wanrooy, B (TAYLOR & FRANCIS LTD, 2021-07-03)
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    Can Robots Understand Welfare? Exploring Machine Bureaucracies in Welfare-to-Work
    Considine, M ; Mcgann, M ; Ball, S ; Nguyen, P (CAMBRIDGE UNIV PRESS, 2022-07)
    Abstract The exercise of administrative discretion by street-level workers plays a key role in shaping citizens’ access to welfare and employment services. Governance reforms of social services delivery, such as performance-based contracting, have often been driven by attempts to discipline this discretion. In several countries, these forms of market governance are now being eclipsed by new modes of digital governance that seek to reshape the delivery of services using algorithms and machine learning. Australia, a pioneer of marketisation, is one example, proposing to deploy digitalisation to fully automate most of its employment services rather than as a supplement to face-to-face case management. We examine the potential and limits of this project to replace human-to-human with ‘machine bureaucracies’. To what extent are welfare and employment services amenable to digitalisation? What trade-offs are involved? In addressing these questions, we consider the purported benefits of machine bureaucracies in achieving higher levels of efficiency, accountability, and consistency in policy delivery. While recognising the potential benefits of machine bureaucracies for both governments and jobseekers, we argue that trade-offs will be faced between enhancing the efficiency and consistency of services and ensuring that services remain accessible and responsive to highly personalised circumstances.