24 results on '"Li, Yanwen"'
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2. Application of the MISTEACHING(S) disease susceptibility framework to Actinobacillus pleuropneumoniae to identify research gaps: an exemplar of a veterinary pathogen.
- Author
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Langford PR, Stringer OW, Li Y, and Bossé JT
- Subjects
- Animals, Disease Susceptibility veterinary, Host-Pathogen Interactions, Swine, Actinobacillus pleuropneumoniae, Microbiota, Swine Diseases microbiology
- Abstract
Historically, the MISTEACHING (microbiome, immunity, sex, temperature, environment, age, chance, history, inoculum, nutrition, genetics) framework to describe the outcome of host-pathogen interaction, has been applied to human pathogens. Here, we show, using Actinobacillus pleuropneumoniae as an exemplar, that the MISTEACHING framework can be applied to a strict veterinary pathogen, enabling the identification of major research gaps, the formulation of hypotheses whose study will lead to a greater understanding of pathogenic mechanisms, and/or improved prevention/therapeutic measures. We also suggest that the MISTEACHING framework should be extended with the inclusion of a 'strain' category, to become MISTEACHINGS. We conclude that the MISTEACHINGS framework can be applied to veterinary pathogens, whether they be bacteria, fungi, viruses, or parasites, and hope to stimulate others to use it to identify research gaps and to formulate hypotheses worthy of study with their own pathogens.
- Published
- 2021
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3. Complete genome for Actinobacillus pleuropneumoniae serovar 8 reference strain 405: comparative analysis with draft genomes for different laboratory stock cultures indicates little genetic variation.
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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, and On Behalf Of The BRaDP T Consortium
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- Animals, Genetic Variation, Genome, Bacterial, High-Throughput Nucleotide Sequencing, Serogroup, Swine, Actinobacillus Infections, Actinobacillus pleuropneumoniae classification, Actinobacillus pleuropneumoniae genetics, Swine Diseases
- Abstract
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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- 2021
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4. Proposal of Actinobacillus pleuropneumoniae serovar 19, and reformulation of previous multiplex PCRs for capsule-specific typing of all known serovars.
- Author
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Stringer OW, Bossé JT, Lacouture S, Gottschalk M, Fodor L, Angen Ø, Velazquez E, Penny P, Lei L, Langford PR, and Li Y
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- Actinobacillus pleuropneumoniae genetics, Bacterial Capsules chemistry, DNA, Bacterial genetics, Genome, Bacterial, Actinobacillus pleuropneumoniae classification, Bacterial Capsules classification, Multiplex Polymerase Chain Reaction methods, Serotyping methods
- Abstract
Two serologically and molecularly non-typeable isolates of the porcine lung pathogen Actinobacillus pleuropneumoniae have been identified from diseased swine in two different continents. Genome sequencing was carried out to identify their diagnostically relevant genotypes. Both isolates are biovar 1 and encode genes for production of ApxIV and ApxII (apxIICA structural genes, and apxIBD export genes). They both possess the same novel type II capsule locus (most similar to serovar 1, but with two capsule genes not previously found in A. pleuropneumoniae) but differ in their O-Ag loci. Strain 7213384-1 from Denmark, which we propose as the reference strain for serovar 19, has a serogroup 3/6/8/15 O-Ag locus; the Canadian isolate A08-013 has a serogroup 4/7 O-Ag locus. We have expanded the second of our two previously described A. pleuropneumoniae mPCRs to include capsule gene-specific primers for definitive detection of serovars 13-14 and 16-19., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2021
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5. Serovar-dependent differences in Hfq-regulated phenotypes in Actinobacillus pleuropneumoniae.
- Author
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Crispim JS, da Silva TF, Sanches NM, da Silva GC, Pereira MF, Rossi CC, Li Y, Terra VS, Vohra P, Wren BW, Langford PR, Bossé JT, and Bazzolli DMS
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- Actinobacillus Infections microbiology, Actinobacillus pleuropneumoniae classification, Animals, Disease Models, Animal, Gene Deletion, Gene Expression Regulation, Bacterial, Genetic Complementation Test, Host Factor 1 Protein genetics, Larva microbiology, Moths microbiology, Phenotype, Promoter Regions, Genetic, Serogroup, Swine, Actinobacillus pleuropneumoniae pathogenicity, Bacterial Adhesion, Host Factor 1 Protein metabolism, Stress, Physiological, Virulence
- Abstract
The RNA chaperone Hfq regulates diverse processes in numerous bacteria. In this study, we compared phenotypes (growth rate, adherence, response to different stress conditions and virulence in Galleria mellonella) of wild-type (WT) and isogenic hfq mutants of three serovars (1, 8 and 15) of the porcine pathogen Actinobacillus pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆hfq, which showed slightly reduced growth throughout the 24 h time course, and the complemented Ap8∆hfqC mutant had a prolonged lag phase. Differences were seen between the three serovar WT strains regarding adherence, stress response and virulence in G. mellonella, and deletion of hfq affected some, but not all of these phenotypes, depending on serovar. Complementation by expression of cloned hfq from an endogenous promoter only restored some WT phenotypes, indicating that complex regulatory networks may be involved, and that levels of Hfq may be as important as presence/absence of the protein regarding its contribution to gene regulation. Our results support that Hfq is a pleiotropic global regulator in A. pleuropneumoniae, but serovar-related differences exist. These results highlight the importance of testing multiple strains/serovars within a given species when determining contributions of global regulators, such as Hfq, to expression of complex phenotypes., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)
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- 2020
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6. Comparative sequence analysis of the capsular polysaccharide loci of Actinobacillus pleuropneumoniae serovars 1-18, and development of two multiplex PCRs for comprehensive capsule typing.
- Author
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Bossé JT, Li Y, Fernandez Crespo R, Lacouture S, Gottschalk M, Sárközi R, Fodor L, Casas Amoribieta M, Angen Ø, Nedbalcova K, Holden MTG, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, and Langford PR
- Subjects
- Actinobacillus Infections diagnosis, Actinobacillus Infections microbiology, Actinobacillus pleuropneumoniae classification, Actinobacillus pleuropneumoniae pathogenicity, Animals, Bacterial Capsules chemistry, Serogroup, Serotyping, Swine, Swine Diseases microbiology, Actinobacillus pleuropneumoniae genetics, Bacterial Capsules classification, Bacterial Capsules genetics, Multiplex Polymerase Chain Reaction methods, Polysaccharides, Bacterial genetics, Sequence Analysis, Swine Diseases diagnosis
- Abstract
Problems with serological cross-reactivity have led to development of a number of PCRs (individual and multiplex) for molecular typing of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Most of these assays were developed for detection of specific amplicons within capsule biosynthetic genes before the availability of complete sequences for the different serovars. Here we describe comparative analysis of the complete capsular loci for all 18 serovars of A. pleuropneumoniae, and development of two multiplex PCRs for comprehensive capsule typing of this important pig pathogen., (Copyright © 2018. Published by Elsevier B.V.)
- Published
- 2018
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7. Proposal of serovars 17 and 18 of Actinobacillus pleuropneumoniae based on serological and genotypic analysis.
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Bossé JT, Li Y, Sárközi R, Fodor L, Lacouture S, Gottschalk M, Casas Amoribieta M, Angen Ø, Nedbalcova K, Holden MTG, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, and Langford PR
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- Actinobacillus Infections epidemiology, Actinobacillus pleuropneumoniae immunology, Actinobacillus pleuropneumoniae isolation & purification, Animals, Bacterial Capsules genetics, Canada epidemiology, DNA Primers genetics, DNA, Bacterial genetics, Denmark epidemiology, Polymerase Chain Reaction methods, Serotyping, Swine, Swine Diseases epidemiology, Swine Diseases microbiology, Whole Genome Sequencing, Actinobacillus Infections veterinary, Actinobacillus pleuropneumoniae classification, Actinobacillus pleuropneumoniae genetics, Genotype, Serogroup
- Abstract
The aim of this study was to investigate isolates of Actinobacillus pleuropneumoniae previously designated serologically either as non-typable (NT) or as 'K2:07', which did not produce serovar-specific amplicons in PCR assays. We used whole genome sequencing to identify the capsule (CPS) loci of six previously designated biovar 1 NT and two biovar 1 'K2:O7' isolates of A. pleuropneumoniae from Denmark, as well as a recent biovar 2 NT isolate from Canada. All of the NT isolates have the same six-gene type I CPS locus, sharing common cpsABC genes with serovars 2, 3, 6, 7, 8, 9, 11 and 13. The two 'K2:O7' isolates contain a unique three-gene type II CPS locus, having a cpsA gene similar to that of serovars 1, 4, 12, 14 and 15. The previously NT isolates share the same O-antigen genes, found between erpA and rpsU, as serovars 3, 6, 8, and 15. Whereas the 'K2:O7' isolates, have the same O-antigen genes as serovar 7, which likely contributed to their previous mis-identification. All of the NT and 'K2:O7' isolates have only the genes required for production of ApxII (apxIICA structural genes, and apxIBD export genes). Rabbit polyclonal antisera raised against representative isolates with these new CPS loci demonstrated distinct reactivity compared to the 16 known serovars. The serological and genomic results indicate that the isolates constitute new serovars 17 (previously NT) and 18 (previously 'K2:O7'). Primers designed for amplification of specific serovar 17 and 18 sequences for molecular diagnostics will facilitate epidemiological tracking of these two new serovars of A. pleuropneumoniae., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2018
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8. Characterization of the Actinobacillus pleuropneumoniae SXT-related integrative and conjugative element ICEApl2 and analysis of the encoded FloR protein: hydrophobic residues in transmembrane domains contribute dynamically to florfenicol and chloramphenicol efflux.
- Author
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Li Y, Li Y, Fernandez Crespo R, Leanse LG, Langford PR, and Bossé JT
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- Actinobacillus pleuropneumoniae isolation & purification, Animals, Chloramphenicol metabolism, Conjugation, Genetic genetics, Hydrophobic and Hydrophilic Interactions, Microbial Sensitivity Tests, Pneumonia microbiology, Pneumonia veterinary, Swine, Thiamphenicol metabolism, Thiamphenicol pharmacology, Actinobacillus pleuropneumoniae drug effects, Actinobacillus pleuropneumoniae genetics, Bacterial Proteins genetics, Biological Transport genetics, Chloramphenicol pharmacology, Drug Resistance, Multiple, Bacterial genetics, Interspersed Repetitive Sequences genetics, Swine Diseases microbiology, Thiamphenicol analogs & derivatives
- Abstract
Objectives: To characterize ICEApl2, an SXT-related integrative and conjugative element (ICE) found in a clinical isolate of the porcine pathogen Actinobacillus pleuropneumoniae, and analyse the functional nature of the encoded FloR., Methods: ICEApl2 was identified in the genome of A. pleuropneumoniae MIDG3553. Functional analysis was done using conjugal transfer experiments. MIDG3553 was tested for susceptibility to the antimicrobials for which resistance genes are present in ICEApl2. Lack of florfenicol/chloramphenicol resistance conferred by the encoded FloR protein was investigated by cloning and site-directed mutagenesis experiments in Escherichia coli., Results: ICEApl2 is 92660 bp and contains 89 genes. Comparative sequence analysis indicated that ICEApl2 is a member of the SXT/R391 ICE family. Conjugation experiments showed that, although ICEApl2 is capable of excision from the chromosome, it is not self-transmissible. ICEApl2 encodes the antimicrobial resistance genes floR, strAB, sul2 and dfrA1, and MIDG3553 is resistant to streptomycin, sulfisoxazole and trimethoprim, but not florfenicol or chloramphenicol. Cloning and site-directed mutagenesis of the floR gene revealed the importance of the nature of the hydrophobic amino acid residues at positions 160 and 228 in FloR for determining resistance to florfenicol and chloramphenicol., Conclusions: Our results indicate that the nature of hydrophobic residues at positions 160 and 228 of FloR contribute dynamically to specific efflux of florfenicol and chloramphenicol, although some differences in resistance levels may depend on the bacterial host species. This is also, to our knowledge, the first description of an SXT/R391 ICE in A. pleuropneumoniae or any member of the Pasteurellaceae., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)
- Published
- 2018
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9. A Unique Capsule Locus in the Newly Designated Actinobacillus pleuropneumoniae Serovar 16 and Development of a Diagnostic PCR Assay.
- Author
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Bossé JT, Li Y, Sárközi R, Gottschalk M, Angen Ø, Nedbalcova K, Rycroft AN, Fodor L, and Langford PR
- Subjects
- Actinobacillus Infections diagnosis, Actinobacillus pleuropneumoniae genetics, Animals, DNA Primers genetics, DNA, Bacterial chemistry, DNA, Bacterial genetics, Genome, Bacterial, Molecular Diagnostic Techniques methods, Pleuropneumonia microbiology, Pleuropneumonia veterinary, Sequence Analysis, DNA, Swine, Actinobacillus Infections veterinary, Actinobacillus pleuropneumoniae classification, Bacterial Capsules genetics, Genetic Loci, Polymerase Chain Reaction methods, Serogroup, Swine Diseases diagnosis
- Abstract
Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar-designated serovar 16-of A. pleuropneumoniae ., (Copyright © 2017 Bossé et al.)
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- 2017
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10. The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering.
- Author
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Cuccui J, Terra VS, Bossé JT, Naegeli A, Abouelhadid S, Li Y, Lin CW, Vohra P, Tucker AW, Rycroft AN, Maskell DJ, Aebi M, Langford PR, and Wren BW
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- A549 Cells, Actinobacillus pleuropneumoniae genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Cell Adhesion, Cloning, Molecular, Gene Expression Regulation, Bacterial, Glycosylation, Humans, Protein Engineering, Virulence Factors metabolism, Actinobacillus pleuropneumoniae pathogenicity, Escherichia coli genetics, Operon, Virulence Factors genetics
- Abstract
Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering., (© 2017 The Authors.)
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- 2017
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11. A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae.
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Rossi CC, Bossé JT, Li Y, Witney AA, Gould KA, Langford PR, and Bazzolli DM
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- Actinobacillus pleuropneumoniae pathogenicity, Genome, Bacterial, RNA, Small Untranslated chemistry, Software, Transcriptome, Actinobacillus pleuropneumoniae genetics, Algorithms, RNA, Small Untranslated genetics, Sequence Analysis, RNA methods
- Abstract
Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies., (© 2016 Rossi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2016
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12. Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae.
- Author
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Bossé JT, Li Y, Atherton TG, Walker S, Williamson SM, Rogers J, Chaudhuri RR, Weinert LA, Holden MT, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, and Langford PR
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- Actinobacillus pleuropneumoniae genetics, Animals, Base Sequence, Chloramphenicol Resistance genetics, Molecular Sequence Data, Pasteurellaceae isolation & purification, Sequence Analysis, DNA, Swine, Thiamphenicol analogs & derivatives, Thiamphenicol pharmacology, Actinobacillus Infections microbiology, Actinobacillus pleuropneumoniae drug effects, Drug Resistance, Bacterial genetics, Pasteurellaceae genetics, Pasteurellaceae Infections microbiology, Plasmids genetics, Swine Diseases microbiology
- Abstract
The complete nucleotide sequence of a 7.7kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2015
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13. Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.
- Author
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Bossé JT, Li Y, Walker S, Atherton T, Fernandez Crespo R, Williamson SM, Rogers J, Chaudhuri RR, Weinert LA, Oshota O, Holden MT, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, and Langford PR
- Subjects
- Actinobacillus Infections microbiology, Actinobacillus pleuropneumoniae enzymology, Actinobacillus pleuropneumoniae genetics, Actinobacillus pleuropneumoniae isolation & purification, Animals, Anti-Infective Agents pharmacology, England, Genome, Bacterial, Microbial Sensitivity Tests, Molecular Sequence Data, Sequence Analysis, DNA, Sulfisoxazole pharmacology, Swine, Trimethoprim pharmacology, Actinobacillus Infections veterinary, Actinobacillus pleuropneumoniae drug effects, Plasmids, Swine Diseases microbiology, Tetrahydrofolate Dehydrogenase genetics, Trimethoprim Resistance
- Abstract
Objectives: The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England., Methods: Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids., Results: A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene., Conclusions: This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)
- Published
- 2015
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14. Galleria mellonella is an effective model to study Actinobacillus pleuropneumoniae infection.
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Pereira MF, Rossi CC, Vieira de Queiroz M, Martins GF, Isaac C, Bossé JT, Li Y, Wren BW, Terra VS, Cuccui J, Langford PR, and Bazzolli DMS
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- Actinobacillus pleuropneumoniae genetics, Actinobacillus pleuropneumoniae pathogenicity, Animals, Humans, Larva microbiology, Virulence, Actinobacillus Infections microbiology, Actinobacillus pleuropneumoniae physiology, Disease Models, Animal, Moths microbiology
- Abstract
Actinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 °C but had different LD50 values, ranging from 10(4) to 10(7) c.f.u. per larva. The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen., (© 2015 The Authors.)
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- 2015
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15. The generation of successive unmarked mutations and chromosomal insertion of heterologous genes in Actinobacillus pleuropneumoniae using natural transformation.
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Bossé JT, Soares-Bazzolli DM, Li Y, Wren BW, Tucker AW, Maskell DJ, Rycroft AN, and Langford PR
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- Chromosomes, Bacterial, Escherichia coli genetics, Mutagenesis, Insertional, Actinobacillus pleuropneumoniae genetics, Bacterial Proteins genetics, Transformation, Bacterial
- Abstract
We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains.
- Published
- 2014
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16. Multiplex PCR assay for unequivocal differentiation of Actinobacillus pleuropneumoniae serovars 1 to 3, 5 to 8, 10, and 12.
- Author
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Bossé JT, Li Y, Angen Ø, Weinert LA, Chaudhuri RR, Holden MT, Williamson SM, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, and Langford PR
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- Actinobacillus Infections microbiology, Actinobacillus pleuropneumoniae isolation & purification, Animals, DNA Primers genetics, DNA, Bacterial chemistry, DNA, Bacterial genetics, Humans, Molecular Sequence Data, Sequence Analysis, DNA, Serogroup, Actinobacillus pleuropneumoniae classification, Actinobacillus pleuropneumoniae genetics, Multiplex Polymerase Chain Reaction methods
- Abstract
An improved multiplex PCR, using redesigned primers targeting the serovar 3 capsule locus, which differentiates serovars 3, 6, and 8 Actinobacillus pleuropneumoniae isolates, is described. The new primers eliminate an aberrant serovar 3-indicative amplicon found in some serovar 6 clinical isolates. Furthermore, we have developed a new multiplex PCR for the detection of serovars 1 to 3, 5 to 8, 10, and 12 along with apxIV, thus extending the utility of this diagnostic PCR to cover a broader range of isolates., (Copyright © 2014 Bossé et al.)
- Published
- 2014
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17. PluMu—A Mu-like Bacteriophage Infecting Actinobacillus pleuropneumoniae.
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Bartsch, Lee Julia, Fernandez Crespo, Roberto, Wang, Yunfei, Skinner, Michael A., Rycroft, Andrew N., Cooley, William, Everest, David J., Li, Yanwen, Bossé, Janine T., and Langford, Paul R.
- Subjects
ACTINOBACILLUS pleuropneumoniae ,BACTERIOPHAGES ,POLYETHYLENE glycol ,TRANSMISSION electron microscopy ,MICROBIAL virulence - Abstract
Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia, an economically important lung disease in pigs. In draft genomes of two Cypriot clinical A. pleuropneumoniae isolates (MIDG3457 and MIDG3459), we previously identified single genomic regions with homology to Mu-like bacteriophage and presented preliminary evidence of active phage. Here, updated Phastest genomic analysis identified two loci in both MIDG3457 and MIDG3459 that were predicted to encode proteins with high homology to, and whose organisation was characteristic of, Mu-like phages. Phylogenetically, the closest matches were with Mannheimia Vb and Glaesserella SuMu phages. Phastest scored the loci as "complete", indicating they produced active phage. PCR amplification of the Mu-like phage c and tail genes from DNase-treated polyethylene glycol 8000 (PEG)-precipitated supernatants of MIDG3457 and MIDG3459 (grown in either Brain Heart Infusion-NAD or Grace's Insect Medium-NAD broth) indicated the presence of intact virions. The phages from MIDG3457 and MIDG3459 were named PluMu 3457-1, 3457-2, and PluMu 3459-1 and PluMu 3459-2, respectively. Transmission electron microscopy (TEM) of the PEG-precipitated supernatants of broth-grown MIDG3459 identified virions with icosahedral heads and tails, consistent with other Mu-like phages. We conclude that MIDG3459 produces an active Mu-like phage. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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18. Comparative Genome Sequence Analysis of Actinobacillus pleuropneumoniae Serovar 8 Isolates From Norway, Denmark, and the United Kingdom Indicates Distinct Phylogenetic Lineages and Differences in Distribution of Antimicrobial Resistance Genes.
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Cohen, Liza Miriam, Bossé, Janine T., Stegger, Marc, Li, Yanwen, Langford, Paul R., Kielland, Camilla, Klem, Thea Blystad, Gulliksen, Stine Margrethe, Ranheim, Birgit, Grøntvedt, Carl Andreas, and Angen, Øystein
- Subjects
ACTINOBACILLUS pleuropneumoniae ,DRUG resistance in microorganisms ,GENETIC variation ,CIRCOVIRUS diseases ,SEQUENCE analysis ,SINGLE nucleotide polymorphisms ,GENOMES - Abstract
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a disease of major impact on pig health, welfare, and productivity globally. Serovar 8 (APP) is the predominant clinical serovar in Norway and the United Kingdom (UK), and has been isolated from clinical cases in Denmark. The primary objective of this study was to characterize the genetic variability of isolates of A. pleuropneumoniae APP8 in the Norwegian population. The secondary objectives were to determine the within-host variability of APP8; to compare the APP8 bacterial populations in Norway, Denmark, and the UK, including antimicrobial resistance (AMR) gene profiles and to assess the effect of national differences in antimicrobial drug use and restricted animal movement on the occurrence of resistance. Isolates of APP8 from the UK (n =67), Denmark (n =22), and Norway (n =123) collected between 1983 and 2020 were compared using whole genome sequencing. To investigate genetic variability within individual hosts, an additional 104 APP8 isolates from the lungs of six Norwegian pigs were compared. Very low within-host variation was observed (≤ 2 single nucleotide polymorphisms). The phylogeny of 123 Norwegian APP8 isolates from 76 herds revealed some within-herd genetic variation, but substantial geographical clustering. When inferring the relatedness of the three international APP8 collections, the topology highlighted the existence of two distinct monophyletic branches characterized by the Norwegian and UK isolates, respectively. Three Danish isolates were scattered across the UK branch, whereas the remaining 19 Danish isolates clustered in two monophyletic groups nested in the Norwegian branch. Coalescence analysis, performed to estimate the divergences from a common ancestor, indicated a last common ancestor several centuries ago. The phylogenetic analyses also revealed striking differences in occurrence of AMR genes, as these were 23-times more prevalent among the UK isolates than among the Norwegian isolates. An increased understanding of the effects of population strategies is helpful in surveillance and control of infectious diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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19. Proposal of serovars 17 and 18 of Actinobacillus pleuropneumoniae based on serological and genotypic analysis
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Bossé, Janine T, Li, Yanwen, Sárközi, Rita, Fodor, László, Lacouture, Sonia, Gottschalk, Marcelo, Casas Amoribieta, Maria, Angen, Øystein, Nedbalcova, Katerina, Holden, Matthew TG, Maskell, Duncan J, Tucker, Alexander W, Wren, Brendan W, Rycroft, Andrew N, Langford, Paul R, BRaDP1T consortium, Biotechnology and Biological Sciences Research Council (BBSRC), Pfizer Limited (UK), Biotechnology and Biological Sciences Research Council, University of St Andrews. School of Medicine, University of St Andrews. Infection and Global Health Division, University of St Andrews. Infection Group, and University of St Andrews. Biomedical Sciences Research Complex
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0301 basic medicine ,Serotype ,Swine ,Biovar ,animal diseases ,Denmark ,DIVERSITY ,Polymerase Chain Reaction ,Actinobacillus Infections ,Genotype ,ASSAY ,Diagnostics ,Genetics ,Swine Diseases ,biology ,Structural gene ,Actinobacillus pleuropneumoniae ,General Medicine ,3. Good health ,PCR ,Serovar 18 ,SEROTYPE-2 ,Serovar 17 ,QR355 Virology ,Life Sciences & Biomedicine ,MULTIPLEX PCR ,0605 Microbiology ,DNA, Bacterial ,Canada ,GENES ,030106 microbiology ,Locus (genetics) ,QH426 Genetics ,Serogroup ,Microbiology ,Article ,03 medical and health sciences ,BRaDP1T consortium ,Animals ,BIOSYNTHESIS ,Veterinary Sciences ,Serotyping ,CAPSULAR POLYSACCHARIDES ,Gene ,QH426 ,Bacterial Capsules ,DNA Primers ,Whole genome sequencing ,QR355 ,Science & Technology ,General Veterinary ,IDENTIFICATION ,Whole Genome Sequencing ,0707 Veterinary Sciences ,STRAINS ,DAS ,biology.organism_classification ,bacterial infections and mycoses ,Capsule genes - Abstract
Highlights • Identification of two new serovars of Actinobacillus pleuropneumoniae. • Serological confirmation of specific reactivity with homologous antisera. • Characterization of the capsule loci of serovars 17 and 18. • Development of PCRs for molecular diagnostics., The aim of this study was to investigate isolates of Actinobacillus pleuropneumoniae previously designated serologically either as non-typable (NT) or as ‘K2:07’, which did not produce serovar-specific amplicons in PCR assays. We used whole genome sequencing to identify the capsule (CPS) loci of six previously designated biovar 1 NT and two biovar 1 ‘K2:O7’ isolates of A. pleuropneumoniae from Denmark, as well as a recent biovar 2 NT isolate from Canada. All of the NT isolates have the same six-gene type I CPS locus, sharing common cpsABC genes with serovars 2, 3, 6, 7, 8, 9, 11 and 13. The two ‘K2:O7’ isolates contain a unique three-gene type II CPS locus, having a cpsA gene similar to that of serovars 1, 4, 12, 14 and 15. The previously NT isolates share the same O-antigen genes, found between erpA and rpsU, as serovars 3, 6, 8, and 15. Whereas the ‘K2:O7’ isolates, have the same O-antigen genes as serovar 7, which likely contributed to their previous mis-identification. All of the NT and ‘K2:O7’ isolates have only the genes required for production of ApxII (apxIICA structural genes, and apxIBD export genes). Rabbit polyclonal antisera raised against representative isolates with these new CPS loci demonstrated distinct reactivity compared to the 16 known serovars. The serological and genomic results indicate that the isolates constitute new serovars 17 (previously NT) and 18 (previously ‘K2:O7’). Primers designed for amplification of specific serovar 17 and 18 sequences for molecular diagnostics will facilitate epidemiological tracking of these two new serovars of A. pleuropneumoniae.
- Published
- 2018
20. Whole Genome Sequencing for Surveillance of Antimicrobial Resistance in Actinobacillus pleuropneumoniae
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Bossé, Janine T., Li, Yanwen, Rogers, Jon, Crespo, Roberto Fernandez, Li, Yinghui, Chaudhuri, Roy R., Holden, Matthew T. G., Maskell, Duncan J., Tucker, Alexander W., Wren, Brendan W., Rycroft, Andrew N., Langford, Paul R., BRaDP1T Consortium, University of St Andrews. School of Medicine, University of St Andrews. Infection Group, University of St Andrews. Infection and Global Health Division, University of St Andrews. Biomedical Sciences Research Complex, Maskell, Duncan [0000-0002-5065-653X], Tucker, Alexander [0000-0003-0062-0843], Apollo - University of Cambridge Repository, Biotechnology and Biological Sciences Research Council (BBSRC), and Pfizer Limited (UK)
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0301 basic medicine ,PREDICTION ,QH301 Biology ,MANNHEIMIA-HAEMOLYTICA ,Respiratory tract ,SUSCEPTIBILITY ,Agar dilution ,BACTERIAL PATHOGENS ,chemistry.chemical_compound ,Ampicillin ,CONJUGATIVE ELEMENT ICE ,animal infections ,PIGS ,Tilmicosin ,Original Research ,biology ,HAEMOPHILUS-PARASUIS ,Animal infections ,Genomics ,QR Microbiology ,Antimicrobial ,Integrative conjugative elements ,PASTEURELLA-MULTOCIDA ,integrative conjugative elements ,Life Sciences & Biomedicine ,medicine.drug ,Plasmids ,Microbiology (medical) ,plasmids ,Tetracycline ,030106 microbiology ,Tylosin ,Microbiology ,03 medical and health sciences ,QH301 ,Enrofloxacin ,medicine ,genomics ,Actinobacillus pleuropneumoniae ,Science & Technology ,IDENTIFICATION ,DAS ,biology.organism_classification ,respiratory tract ,QR ,Antimicrobial resistance genes ,030104 developmental biology ,chemistry ,Pasteurellaceae ,antimicrobial resistance genes - Abstract
This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (BBSRC grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1, and BB/G018553/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis (formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium. MH was supported by the Wellcome Trust (grant number 098051). JR was funded from the former AHVLA’s Research and Development Internal Investment Fund (grant number RD0030c). The aim of this study was to evaluate the correlation between antimicrobial resistance (AMR) profiles of 96 clinical isolates of Actinobacillus pleuropneumoniae, an important porcine respiratory pathogen, and the identification of AMR genes in whole genome sequence (wgs) data. Susceptibility of the isolates to nine antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, sulfisoxazole, tetracycline, tilmicosin, trimethoprim, and tylosin) was determined by agar dilution susceptibility test. Except for the macrolides tested, elevated MICs were highly correlated to the presence of AMR genes identified in wgs data using ResFinder or BLASTn. Of the isolates tested, 57% were resistant to tetracycline [MIC ≥ 4 mg/L; 94.8% with either tet(B) or tet(H)]; 48% to sulfisoxazole (MIC ≥ 256 mg/L or DD = 6; 100% with sul2), 20% to ampicillin (MIC ≥ 4 mg/L; 100% with blaROB-1), 17% to trimethoprim (MIC ≥ 32 mg/L; 100% with dfrA14), and 6% to enrofloxacin (MIC ≥ 0.25 mg/L; 100% with GyrAS83F). Only 33% of the isolates did not have detectable AMR genes, and were sensitive by MICs for the antimicrobial agents tested. Although 23 isolates had MIC ≥ 32 mg/L for tylosin, all isolates had MIC ≥ 32 mg/L for tylosin, all isolates had MIC ≤ 16 mg/L for both erythromycin and tilmicosin, and no macrolide resistance genes or known point mutations were detected. Other than the GyrAS83F mutation, the AMR genes detected were mapped to potential plasmids. In addition to presence on plasmid(s), the tet(B) gene was also found chromosomally either as part of a 56 kb integrative conjugative element (ICEApl1) in 21, or as part of a Tn7 insertion in 15 isolates. Our results indicate that, with the exception of macrolides, wgs data can be used to accurately predict resistance of A. pleuropneumoniae to the tested antimicrobial agents and provides added value for routine surveillance. Publisher PDF
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- 2017
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21. A Unique Capsule Locus in the Newly Designated Actinobacillus pleuropneumoniae Serovar 16 and Development of a Diagnostic PCR Assay
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Bossé, Janine T., Li, Yanwen, Sárközi, Rita, Gottschalk, Marcelo, Angen, Øystein, Nedbalcova, Katerina, Rycroft, Andrew N., Fodor, László, Langford, Paul R., and Biotechnology and Biological Sciences Research Council (BBSRC)
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DNA, Bacterial ,STRUCTURAL-CHARACTERIZATION ,Swine ,Serogroup ,Polymerase Chain Reaction ,Microbiology ,SEQUENCE ,Clinical Veterinary Microbiology ,Actinobacillus Infections ,diagnostics ,Animals ,LIPOPOLYSACCHARIDE-O-CHAIN ,Bacterial Capsules ,DNA Primers ,Swine Diseases ,Pleuropneumonia ,Science & Technology ,STRAINS ,Actinobacillus pleuropneumoniae ,Sequence Analysis, DNA ,11 Medical And Health Sciences ,06 Biological Sciences ,respiratory system ,serovar 16 ,PCR ,POLYSACCHARIDE ,Molecular Diagnostic Techniques ,Genetic Loci ,07 Agricultural And Veterinary Sciences ,Life Sciences & Biomedicine ,MULTIPLEX PCR ,Genome, Bacterial - Abstract
Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar—designated serovar 16—of A. pleuropneumoniae.
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- 2017
22. Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae
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Bossé, Janine T, Li, Yanwen, Atherton, Tom G, Walker, Stephanie, Williamson, Susanna M, Rogers, Jon, Chaudhuri, Roy R, Weinert, Lucy A, Holden, Matthew TG, Maskell, Duncan J, Tucker, Alexander W, Wren, Brendan W, Rycroft, Andrew N, Langford, Paul R, BRaDP1T consortium, University of St Andrews. School of Medicine, University of St Andrews. Infection Group, and University of St Andrews. Biomedical Sciences Research Complex
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Florfenicol ,Swine ,animal diseases ,Short Communication ,Molecular Sequence Data ,NDAS ,Chloramphenicol Resistance ,Microbiology ,chemistry.chemical_compound ,Actinobacillus Infections ,Plasmid ,Drug Resistance, Bacterial ,Animals ,Actinobacillus pleuropneumoniae ,Swine Diseases ,Thiamphenicol ,Base Sequence ,General Veterinary ,biology ,Pasteurellaceae ,Nucleic acid sequence ,Sequence Analysis, DNA ,General Medicine ,biology.organism_classification ,veterinary(all) ,stomatognathic diseases ,chemistry ,Pasteurellaceae Infections ,Plasmids - Abstract
Highlights • First complete sequence of a floR plasmid from Actinobacillus pleuropneumoniae • Extended similarity to floR plasmids in other Pasteurellaceae species • Conjugal transfer between between species confirmed, The complete nucleotide sequence of a 7.7 kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally.
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- 2015
23. The generation of successive unmarked mutations and chromosomal insertion of heterologous genes in Actinobacillus pleuropneumoniae using natural transformation
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Bossé, Janine T., Soares-Bazzolli, Denise M., Li, Yanwen, Wren, Brendan W., Tucker, Alexander W., Maskell, Duncan J., Rycroft, Andrew N., and Langford, Paul R.
- Subjects
Natural transformation ,Actinobacillus pleuropneumoniae - Abstract
We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains.
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- 2014
24. Characterization of the Actinobacillus pleuropneumoniae SXT-related integrative and conjugative element ICEApl2 and analysis of the encoded FloR protein: hydrophobic residues in transmembrane domains contribute dynamically to florfenicol and chloramphenicol efflux.
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Yinghui Li, Yanwen Li, Fernandez Crespo, Roberto, Leanse, Leon G., Langford, Paul R., Bossé, Janine T., Li, Yinghui, and Li, Yanwen
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ACTINOBACILLUS pleuropneumoniae ,MEMBRANE proteins ,HYDROPHOBIC interactions ,CHLORAMPHENICOL ,SITE-specific mutagenesis ,DRUG resistance in bacteria ,TRIMETHOPRIM ,THERAPEUTICS - Abstract
Objectives: To characterize ICEApl2, an SXT-related integrative and conjugative element (ICE) found in a clinical isolate of the porcine pathogen Actinobacillus pleuropneumoniae, and analyse the functional nature of the encoded FloR.Methods: ICEApl2 was identified in the genome of A. pleuropneumoniae MIDG3553. Functional analysis was done using conjugal transfer experiments. MIDG3553 was tested for susceptibility to the antimicrobials for which resistance genes are present in ICEApl2. Lack of florfenicol/chloramphenicol resistance conferred by the encoded FloR protein was investigated by cloning and site-directed mutagenesis experiments in Escherichia coli.Results: ICEApl2 is 92660 bp and contains 89 genes. Comparative sequence analysis indicated that ICEApl2 is a member of the SXT/R391 ICE family. Conjugation experiments showed that, although ICEApl2 is capable of excision from the chromosome, it is not self-transmissible. ICEApl2 encodes the antimicrobial resistance genes floR, strAB, sul2 and dfrA1, and MIDG3553 is resistant to streptomycin, sulfisoxazole and trimethoprim, but not florfenicol or chloramphenicol. Cloning and site-directed mutagenesis of the floR gene revealed the importance of the nature of the hydrophobic amino acid residues at positions 160 and 228 in FloR for determining resistance to florfenicol and chloramphenicol.Conclusions: Our results indicate that the nature of hydrophobic residues at positions 160 and 228 of FloR contribute dynamically to specific efflux of florfenicol and chloramphenicol, although some differences in resistance levels may depend on the bacterial host species. This is also, to our knowledge, the first description of an SXT/R391 ICE in A. pleuropneumoniae or any member of the Pasteurellaceae. [ABSTRACT FROM AUTHOR]- Published
- 2018
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