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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, 2022-05-01)
    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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    Genomic variations leading to alterations in cell morphology of Campylobacter spp.
    Esson, D ; Mather, AE ; Scanlan, E ; Gupta, S ; de Vries, SPW ; Bailey, D ; Harris, SR ; McKinley, TJ ; Méric, G ; Berry, SK ; Mastroeni, P ; Sheppard, SK ; Christie, G ; Thomson, NR ; Parkhill, J ; Maskell, DJ ; Grant, AJ (Springer Science and Business Media LLC, 2016-12-02)
    Campylobacter jejuni, the most common cause of bacterial diarrhoeal disease, is normally helical. However, it can also adopt straight rod, elongated helical and coccoid forms. Studying how helical morphology is generated, and how it switches between its different forms, is an important objective for understanding this pathogen. Here, we aimed to determine the genetic factors involved in generating the helical shape of Campylobacter. A C. jejuni transposon (Tn) mutant library was screened for non-helical mutants with inconsistent results. Whole genome sequence variation and morphological trends within this Tn library, and in various C. jejuni wild type strains, were compared and correlated to detect genomic elements associated with helical and rod morphologies. All rod-shaped C. jejuni Tn mutants and all rod-shaped laboratory, clinical and environmental C. jejuni and Campylobacter coli contained genetic changes within the pgp1 or pgp2 genes, which encode peptidoglycan modifying enzymes. We therefore confirm the importance of Pgp1 and Pgp2 in the maintenance of helical shape and extended this to a wide range of C. jejuni and C. coli isolates. Genome sequence analysis revealed variation in the sequence and length of homopolymeric tracts found within these genes, providing a potential mechanism of phase variation of cell shape.
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    Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations.
    Maglennon, GA ; Cook, BS ; Deeney, AS ; Bossé, JT ; Peters, SE ; Langford, PR ; Maskell, DJ ; Tucker, AW ; Wren, BW ; Rycroft, AN ; BRaDP1T consortium, (Springer Science and Business Media LLC, 2013-12-21)
    Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.
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    Intracellular demography and the dynamics of Salmonella enterica infections.
    Brown, SP ; Cornell, SJ ; Sheppard, M ; Grant, AJ ; Maskell, DJ ; Grenfell, BT ; Mastroeni, P ; Levin, S (Public Library of Science (PLoS), 2006-10)
    An understanding of within-host dynamics of pathogen interactions with eukaryotic cells can shape the development of effective preventive measures and drug regimes. Such investigations have been hampered by the difficulty of identifying and observing directly, within live tissues, the multiple key variables that underlay infection processes. Fluorescence microscopy data on intracellular distributions of Salmonella enterica serovar Typhimurium (S. Typhimurium) show that, while the number of infected cells increases with time, the distribution of bacteria between cells is stationary (though highly skewed). Here, we report a simple model framework for the intensity of intracellular infection that links the quasi-stationary distribution of bacteria to bacterial and cellular demography. This enables us to reject the hypothesis that the skewed distribution is generated by intrinsic cellular heterogeneities, and to derive specific predictions on the within-cell dynamics of Salmonella division and host-cell lysis. For within-cell pathogens in general, we show that within-cell dynamics have implications across pathogen dynamics, evolution, and control, and we develop novel generic guidelines for the design of antibacterial combination therapies and the management of antibiotic resistance.
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    Publisher Correction: Genomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis.
    Weinert, LA ; Chaudhuri, RR ; Wang, J ; Peters, SE ; Corander, J ; Jombart, T ; Baig, A ; Howell, KJ ; Vehkala, M ; Välimäki, N ; Harris, D ; Chieu, TTB ; Van Vinh Chau, N ; Campbell, J ; Schultsz, C ; Parkhill, J ; Bentley, SD ; Langford, PR ; Rycroft, AN ; Wren, BW ; Farrar, J ; Baker, S ; Hoa, NT ; Holden, MTG ; Tucker, AW ; Maskell, DJ ; BRaDP1T Consortium, (Springer Science and Business Media LLC, 2019-11-22)
    An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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    Overexpression of antibiotic resistance genes in hospital effluents over time.
    Rowe, WPM ; Baker-Austin, C ; Verner-Jeffreys, DW ; Ryan, JJ ; Micallef, C ; Maskell, DJ ; Pearce, GP (Oxford University Press (OUP), 2017-06-01)
    Objectives: Effluents contain a diverse abundance of antibiotic resistance genes that augment the resistome of receiving aquatic environments. However, uncertainty remains regarding their temporal persistence, transcription and response to anthropogenic factors, such as antibiotic usage. We present a spatiotemporal study within a river catchment (River Cam, UK) that aims to determine the contribution of antibiotic resistance gene-containing effluents originating from sites of varying antibiotic usage to the receiving environment. Methods: Gene abundance in effluents (municipal hospital and dairy farm) was compared against background samples of the receiving aquatic environment (i.e. the catchment source) to determine the resistome contribution of effluents. We used metagenomics and metatranscriptomics to correlate DNA and RNA abundance and identified differentially regulated gene transcripts. Results: We found that mean antibiotic resistance gene and transcript abundances were correlated for both hospital ( ρ  = 0.9, two-tailed P  <0.0001) and farm ( ρ  = 0.5, two-tailed P   <0.0001) effluents and that two β-lactam resistance genes ( bla GES and bla OXA ) were overexpressed in all hospital effluent samples. High β-lactam resistance gene transcript abundance was related to hospital antibiotic usage over time and hospital effluents contained antibiotic residues. Conclusions: We conclude that effluents contribute high levels of antibiotic resistance genes to the aquatic environment; these genes are expressed at significant levels and are possibly related to the level of antibiotic usage at the effluent source.
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    Genome-wide fitness analyses of the foodborne pathogen Campylobacter jejuni in in vitro and in vivo models.
    de Vries, SP ; Gupta, S ; Baig, A ; Wright, E ; Wedley, A ; Jensen, AN ; Lora, LL ; Humphrey, S ; Skovgård, H ; Macleod, K ; Pont, E ; Wolanska, DP ; L'Heureux, J ; Mobegi, FM ; Smith, DGE ; Everest, P ; Zomer, A ; Williams, N ; Wigley, P ; Humphrey, T ; Maskell, DJ ; Grant, AJ (Springer Science and Business Media LLC, 2017-04-28)
    Campylobacter is the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is a lack of insight into the mechanisms driving C. jejuni growth and survival within hosts and the environment. Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during chicken colonisation, survival in houseflies and under nutrient-rich and -poor conditions at 4 °C and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq). A total of 331 homologous gene clusters were essential for fitness during in vitro growth in three C. jejuni strains, revealing that a large part of its genome is dedicated to growth. We report novel C. jejuni factors essential throughout its life cycle. Importantly, we identified genes that fulfil important roles across multiple conditions. Our comprehensive screens showed which flagella elements are essential for growth and which are vital to the interaction with host organisms. Future efforts should focus on how to exploit this knowledge to effectively control infections caused by C. jejuni.
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    Identification and initial characterisation of a protein involved in Campylobacter jejuni cell shape.
    Esson, D ; Gupta, S ; Bailey, D ; Wigley, P ; Wedley, A ; Mather, AE ; Méric, G ; Mastroeni, P ; Sheppard, SK ; Thomson, NR ; Parkhill, J ; Maskell, DJ ; Christie, G ; Grant, AJ (Elsevier BV, 2017-03)
    Campylobacter jejuni is the leading cause of bacterial food borne illness. While helical cell shape is considered important for C. jejuni pathogenesis, this bacterium is capable of adopting other morphologies. To better understand how helical-shaped C. jejuni maintain their shape and thus any associated colonisation, pathogenicity or other advantage, it is first important to identify the genes and proteins involved. So far, two peptidoglycan modifying enzymes Pgp1 and Pgp2 have been shown to be required for C. jejuni helical cell shape. We performed a visual screen of ∼2000 transposon mutants of C. jejuni for cell shape mutants. Whole genome sequence data of the mutants with altered cell shape, directed mutants, wild type stocks and isolated helical and rod-shaped 'wild type' C. jejuni, identified a number of different mutations in pgp1 and pgp2, which result in a change in helical to rod bacterial cell shape. We also identified an isolate with a loss of curvature. In this study, we have identified the genomic change in this isolate, and found that targeted deletion of the gene with the change resulted in bacteria with loss of curvature. Helical cell shape was restored by supplying the gene in trans. We examined the effect of loss of the gene on bacterial motility, adhesion and invasion of tissue culture cells and chicken colonisation, as well as the effect on the muropeptide profile of the peptidoglycan sacculus. Our work identifies another factor involved in helical cell shape.
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    The use of genome wide association methods to investigate pathogenicity, population structure and serovar in Haemophilus parasuis.
    Howell, KJ ; Weinert, LA ; Chaudhuri, RR ; Luan, S-L ; Peters, SE ; Corander, J ; Harris, D ; Angen, Ø ; Aragon, V ; Bensaid, A ; Williamson, SM ; Parkhill, J ; Langford, PR ; Rycroft, AN ; Wren, BW ; Holden, MTG ; Tucker, AW ; Maskell, DJ ; BRADP1T Consortium, (Springer Science and Business Media LLC, 2014-12-24)
    BACKGROUND: Haemophilus parasuis is the etiologic agent of Glässer's disease in pigs and causes devastating losses to the farming industry. Whilst some hyper-virulent isolates have been described, the relationship between genetics and disease outcome has been only partially established. In particular, there is weak correlation between serovar and disease phenotype. We sequenced the genomes of 212 isolates of H. parasuis and have used this to describe the pan-genome and to correlate this with clinical and carrier status, as well as with serotype. RESULTS: Recombination and population structure analyses identified five groups with very high rates of recombination, separated into two clades of H. parasuis with no signs of recombination between them. We used genome-wide association methods including discriminant analysis of principal components (DAPC) and generalised linear modelling (glm) to look for genetic determinants of this population partition, serovar and pathogenicity. We were able to identify genes from the accessory genome that were significantly associated with phenotypes such as potential serovar specific genes including capsule genes, and 48 putative virulence factors that were significantly different between the clinical and non-clinical isolates. We also show that the presence of many previously suggested virulence factors is not an appropriate marker of virulence. CONCLUSIONS: These genes will inform the generation of new molecular diagnostics and vaccines, and refinement of existing typing schemes and show the importance of the accessory genome of a diverse species when investigating the relationship between genotypes and phenotypes.
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    Motility defects in Campylobacter jejuni defined gene deletion mutants caused by second-site mutations.
    de Vries, SPW ; Gupta, S ; Baig, A ; L'Heureux, J ; Pont, E ; Wolanska, DP ; Maskell, DJ ; Grant, AJ (Microbiology Society, 2015-12)
    Genetic variation due to mutation and phase variation has a considerable impact on the commensal and pathogenic behaviours of Campylobacter jejuni. In this study, we provide an example of how second-site mutations can interfere with gene function analysis in C. jejuni. Deletion of the flagellin B gene (flaB) in C. jejuni M1 resulted in mutant clones with inconsistent motility phenotypes. From the flaB mutant clones picked for further analysis, two were motile, one showed intermediate motility and two displayed severely attenuated motility. To determine the molecular basis of this differential motility, a genome resequencing approach was used. Second-site mutations were identified in the severely attenuated and intermediate motility flaB mutant clones: a TA-dinucleotide deletion in fliW and an A deletion in flgD, respectively. Restoration of WT fliW, using a newly developed genetic complementation system, confirmed that the second-site fliW mutation caused the motility defect as opposed to the primary deletion of flaB. This study highlights the importance of (i) screening multiple defined gene deletion mutant clones, (ii) genetic complementation of the gene deletion and ideally (iii) screening for second-site mutations that might interfere with the pathways/mechanisms under study.