Abstract
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 .
Highlights
Actinobacillus pleuropneumoniae is a Gram-n egative, facultatively anaerobic, rod-shaped bacterium belonging to the family Pasteurellaceae, and is the causative agent of porcine pleuropneumonia, a disease associated with high economic burdens in the pig industry worldwide [1].Donà et al, Microbial Genomics 2022;8:000776 Impact Statementwe performed comprehensive comparative genomics of 26 full genomes focusing on core-g enome-inferred strain phylogeny and serotype-specific virulence traits, such as the lipopolysaccharide (LPS), capsular (CPS) and RTX-toxin antigenic regions, and highlighting the potential of a dynamic intra- and interspecies exchange of these factors by horizontal gene transfer
We showed that our sequencing and assembly strategy, i.e. the generation of an Oxford Nanopore Technologies (ONT)-only draft consensus sequence that is subsequently polished with high-accuracy Illumina reads, yielded the full circular chromosome sequence of the A. pleuropneumoniae serotype 1 reference strain S4074 with comparable results to a PacBio-generated sequence of the same strain [31]
An ISApl3 transposase is found immediately downstream of the type I-F CRISPR/Cas locus, which was almost identical to the one found in A. lignieresii strain NCTC10568. These observations again support the potential for interspecies exchange of different full CRISPR/Cas system clusters among Actinobacillus species. We have implemented both ONT and PacBio sequencing combined with Illumina to obtain 19 complete circular genome sequences of A. pleuropneumoniae, covering all 18 different serotypes known so far, and highlighted their main genetic features and differences
Summary
Actinobacillus pleuropneumoniae is a Gram-n egative, facultatively anaerobic, rod-shaped bacterium belonging to the family Pasteurellaceae, and is the causative agent of porcine pleuropneumonia, a disease associated with high economic burdens in the pig industry worldwide [1].we performed comprehensive comparative genomics of 26 full genomes focusing on core-g enome-inferred strain phylogeny and serotype-specific virulence traits, such as the lipopolysaccharide (LPS), capsular (CPS) and RTX-toxin antigenic regions, and highlighting the potential of a dynamic intra- and interspecies exchange of these factors by horizontal gene transfer. We used seven genomes already available on NCBI and provided the complete circular genomes of reference strains and two field isolates of Actinobacillus pleuropneumoniae, representing of the serotypes described so far. We used either long-read-only assemblies with Oxford Nanopore Technologies (ONT) followed by polishing of the scaffolds with Illumina reads, or a hybrid assembly approach combining both ONT and Illumina reads. We show that both methods yield complete circular genomes with comparable assembly qualities, representing good backup options if the method of choice fails. The in-d epth genome analysis presented here may serve as a basis for the development of diagnostic tests and vaccines, as well as for the establishment of whole-g enome-based surveillance and epidemiological studies
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