Comparative genomics of 26 complete circular genomes of 18 different serotypes of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is a Gram-negative, rod-shaped bacterium of the family Pasteurellaceae causing pig pleuropneumonia associated with great economic losses worldwide. Nineteen serotypes with distinctive lipopolysaccharide (LPS) and capsular (CPS) compositions have been described so far, yet complete circular genomes are publicly available only for the reference strains of serotypes 1, 4 and 5b, and for field strains of serotypes 1, 3, 7 and 8. We aimed to complete this picture by sequencing the reference strains of 17 different serotypes with the MinION sequencer (Oxford Nanopore Technologies, ONT) and on an Illumina HiSeq (Illumina) platform. We also included two field isolates of serotypes 2 and 3 that were PacBio- and MinION-sequenced, respectively. Genome assemblies were performed following two different strategies, i.e. PacBio- or ONT-only de novo assemblies polished with Illumina reads or a hybrid assembly by directly combining ONT and Illumina reads. Both methods proved successful in obtaining accurate circular genomes with comparable qualities. blast-based genome comparisons and core-genome phylogeny based on core genes, SNP typing and multi-locus sequence typing (cgMLST) of the 26 circular genomes indicated well-conserved genomes across the 18 different serotypes, differing mainly in phage insertions, and CPS, LPS and RTX-toxin clusters, which, consistently, encode serotype-specific antigens. We also identified small antibiotic resistance plasmids, and complete subtype I-F and subtype II-C CRISPR-Cas systems. Of note, highly similar clusters encoding all those serotype-specific traits were also found in other pathogenic and commensal Actinobacillus species. Taken together with the presence of transposable elements surrounding these loci, we speculate a dynamic intra- and interspecies exchange of such virulence-related factors by horizontal gene transfer. In conclusion, our comprehensive genomics analysis provides useful information for diagnostic test and vaccine development, but also for whole-genome-based epidemiological studies, as well as for the surveillance of the evolution of antibiotic resistance and virulence genes in A. pleuropneumoniae .

[1]  P. Langford,et al.  Proposal of Actinobacillus pleuropneumoniae serovar 19, and reformulation of previous multiplex PCRs for capsule-specific typing of all known serovars. , 2021, Veterinary microbiology.

[2]  K. Waldmann,et al.  Sero- and apx-typing of German Actinobacillus pleuropneumoniae field isolates from 2010 to 2019 reveals a predominance of serovar 2 with regular apx-profile , 2021, Veterinary Research.

[3]  Gregory J Tsongalis,et al.  Third-Generation Sequencing in the Clinical Laboratory: Exploring the Advantages and Challenges of Nanopore Sequencing , 2019, Journal of Clinical Microbiology.

[4]  Adina R. Bujold,et al.  Investigation of putative invasion determinants of Actinobacillus species using comparative genomics. , 2019, Genomics.

[5]  V. Perreten,et al.  Comparative Genomics of the First and Complete Genome of “Actinobacillus porcitonsillarum” Supports the Novel Species Hypothesis , 2018, International journal of genomics.

[6]  Claude Thermes,et al.  The Third Revolution in Sequencing Technology. , 2018, Trends in genetics : TIG.

[7]  D. Maskell,et al.  Comparative sequence analysis of the capsular polysaccharide loci of Actinobacillus pleuropneumoniae serovars 1–18, and development of two multiplex PCRs for comprehensive capsule typing , 2018, Veterinary microbiology.

[8]  W. Jachymek,et al.  Antigens of Actinobacillus pleuropneumoniae and their use in the design of vaccines, especially glycoconjugates , 2018, Postępy Higieny i Medycyny Doświadczalnej.

[9]  P. Langford,et al.  Update on Actinobacillus pleuropneumoniae—knowledge, gaps and challenges , 2018, Transboundary and emerging diseases.

[10]  A. Loera-Muro,et al.  New trends in innovative vaccine development against Actinobacillus pleuropneumoniae. , 2018, Veterinary microbiology.

[11]  D. Maskell,et al.  Proposal of serovars 17 and 18 of Actinobacillus pleuropneumoniae based on serological and genotypic analysis , 2018, Veterinary microbiology.

[12]  E Trees,et al.  Next-generation sequencing technologies and their application to the study and control of bacterial infections. , 2017, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[13]  A. Ramette,et al.  Heterogeneous Genetic Location of mcr-1 in Colistin-Resistant Escherichia coli Isolates from Humans and Retail Chicken Meat in Switzerland: Emergence of mcr-1-Carrying IncK2 Plasmids , 2017, Antimicrobial Agents and Chemotherapy.

[14]  Ryan R. Wick,et al.  Completing bacterial genome assemblies with multiplex MinION sequencing , 2017, bioRxiv.

[15]  James Hadfield,et al.  Phandango: an interactive viewer for bacterial population genomics , 2017, bioRxiv.

[16]  Roy R. Chaudhuri,et al.  Whole Genome Sequencing for Surveillance of Antimicrobial Resistance in Actinobacillus pleuropneumoniae , 2017, Front. Microbiol..

[17]  David W. Russell,et al.  Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol. , 2017, CSH protocols.

[18]  P. Langford,et al.  A Unique Capsule Locus in the Newly Designated Actinobacillus pleuropneumoniae Serovar 16 and Development of a Diagnostic PCR Assay , 2017, Journal of Clinical Microbiology.

[19]  Ryan R. Wick,et al.  Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads , 2016, bioRxiv.

[20]  Dmitry Antipov,et al.  plasmidSPAdes: Assembling Plasmids from Whole Genome Sequencing Data , 2016, bioRxiv.

[21]  D. Maskell,et al.  Complete Genome Sequence of MIDG2331, a Genetically Tractable Serovar 8 Clinical Isolate of Actinobacillus pleuropneumoniae , 2016, Genome Announcements.

[22]  M. Gottschalk.  The challenge of detecting herds sub-clinically infected with Actinobacillus pleuropneumoniae. , 2015, Veterinary journal.

[23]  Andrew J. Page,et al.  Roary: rapid large-scale prokaryote pan genome analysis , 2015, bioRxiv.

[24]  D. Maskell,et al.  Multiplex PCR Assay for Unequivocal Differentiation of Actinobacillus pleuropneumoniae Serovars 1 to 3, 5 to 8, 10, and 12 , 2014, Journal of Clinical Microbiology.

[25]  D. Leach,et al.  Bacterial Genome Instability , 2014, Microbiology and Molecular Reviews.

[26]  Yongqiang Yang,et al.  Advances in nanopore sequencing technology. , 2013, Journal of nanoscience and nanotechnology.

[27]  Ole Lund,et al.  Genotyping using whole-genome sequencing is a realistic alternative to surveillance based on phenotypic antimicrobial susceptibility testing. , 2013, The Journal of antimicrobial chemotherapy.

[28]  R. Bashir,et al.  Nanopore sensors for nucleic acid analysis. , 2011, Nature nanotechnology.

[29]  M. Monteiro,et al.  The lipopolysaccharide core of Actinobacillus suis and its relationship to those of Actinobacillus pleuropneumoniae. , 2011, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[30]  B. Korczak,et al.  A new variant of Actinobacillus pleuropneumoniae serotype 3 lacking the entire apxII operon , 2011, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

[31]  C. Friis,et al.  Comparative profiling of the transcriptional response to iron restriction in six serotypes of Actinobacillus pleuropneumoniae with different virulence potential , 2010, BMC Genomics.

[32]  P. Langford,et al.  Prevalence of Actinobacillus pleuropneumoniae serovars in England and Wales , 2010, Veterinary Record.

[33]  Yongxiang Zhang,et al.  Pan-genome sequence analysis using Panseq: an online tool for the rapid analysis of core and accessory genomic regions , 2010, BMC Bioinformatics.

[34]  Huanchun Chen,et al.  Comparative Genomic Characterization of Actinobacillus pleuropneumoniae , 2010, Journal of bacteriology.

[35]  L. Marraffini,et al.  CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea , 2010, Nature Reviews Genetics.

[36]  Erik J. Sontheimer,et al.  Self vs. non-self discrimination during CRISPR RNA-directed immunity , 2009, Nature.

[37]  M. Jacques,et al.  Actinobacillus pleuropneumoniae vaccines: from bacterins to new insights into vaccination strategies , 2008, Animal Health Research Reviews.

[38]  V. Perreten,et al.  Small multidrug resistance plasmids in Actinobacillus porcitonsillarum. , 2008, Plasmid.

[39]  Wei Li,et al.  Genome Biology of Actinobacillus pleuropneumoniae JL03, an Isolate of Serotype 3 Prevalent in China , 2008, PloS one.

[40]  Simon J. Foote,et al.  The Complete Genome Sequence of Actinobacillus pleuropneumoniae L20 (Serotype 5b) , 2007, Journal of bacteriology.

[41]  M. Jacques,et al.  Outer membrane proteome of Actinobacillus pleuropneumoniae: LC‐MS/MS analyses validate in silico predictions , 2007, Proteomics.

[42]  B. Korczak,et al.  Distribution of RTX toxin genes in strains of [Actinobacillus] rossii and [Pasteurella] mairii. , 2006, Veterinary microbiology.

[43]  P. Kuhnert,et al.  Characterization of the type I secretion system of the RTX toxin ApxII in "Actinobacillus porcitonsillarum". , 2005, Veterinary microbiology.

[44]  E. Vinogradov,et al.  Structural characterization of the antigenic capsular polysaccharide and lipopolysaccharide O-chain produced by Actinobacillus pleuropneumoniae serotype 15. , 2005, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[45]  M. Jacques.  Surface polysaccharides and iron-uptake systems of Actinobacillus pleuropneumoniae. , 2004, Canadian journal of veterinary research = Revue canadienne de recherche veterinaire.

[46]  H. van den Bosch,et al.  Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 15. , 2002, Veterinary microbiology.

[47]  M. Jacques,et al.  Actinobacillus pleuropneumoniae surface polysaccharides: their role in diagnosis and immunogenicity , 2000, Animal Health Research Reviews.

[48]  P. Kuhnert,et al.  Apx toxins in Pasteurellaceae species from animals. , 2000, Veterinary microbiology.

[49]  V. Fussing.  Genomic relationships of Actinobacillus pleuropnewmoniae serotype 2 strains evaluated by ribotyping, sequence analysis of ribosomal intergenic regions, and pulsed‐field gel electrophoresis , 1998, Letters in applied microbiology.

[50]  J. Nielsen,et al.  Comparison of virulence of different Actinobacillus pleuropneumoniae serotypes and biotypes using an aerosol infection model. , 1996, Veterinary microbiology.

[51]  J. Nicolet,et al.  Development of an efficient PCR method for toxin typing of Actinobacillus pleuropneumoniae strains. , 1995, Molecular and cellular probes.

[52]  J. Frey.  Virulence in Actinobacillus pleuropneumoniae and RTX toxins. , 1995, Trends in microbiology.

[53]  F. Haesebrouck,et al.  Actinobacillus pleuropneumoniae RTX-toxins: uniform designation of haemolysins, cytolysins, pleurotoxin and their genes. , 1993, Journal of general microbiology.

[54]  F. Haesebrouck,et al.  Comparative virulence of NAD-dependent and NAD-independent Actinobacillus pleuropneumoniae strains. , 1992, Zentralblatt fur Veterinarmedizin. Reihe B. Journal of veterinary medicine. Series B.

[55]  D. Williams,et al.  Experimental reproduction of acute lesions of porcine pleuropneumonia with a haemolysin-deficient mutant of Actinobacillus pleuropneumoniae , 1991, Veterinary Record.

[56]  Eugene V Koonin,et al.  Annotation and Classification of CRISPR-Cas Systems. , 2015, Methods in molecular biology.