Comparative Genomics of the First and Complete Genome of “Actinobacillus porcitonsillarum” Supports the Novel Species Hypothesis

Abstract

“Actinobacillus porcitonsillarum” is considered a nonpathogenic member of the Pasteurellaceae family, which phenotypically resembles the pathogen Actinobacillus pleuropneumoniae. Previous studies suggested that “A. porcitonsillarum” may represent a new species closely related to Actinobacillus minor, yet no full genome has been sequenced so far. We implemented the Oxford Nanopore and Illumina sequencing technologies to obtain the highly accurate and complete genome sequence of the “A. porcitonsillarum” strain 9953L55. After validating our de novo assembly strategy by comparing the A. pleuropneumoniae S4074T genome sequence obtained by Oxford Nanopore Technology combined with Illumina reads with a PacBio-sequenced S4074T genome from the NCBI database, we performed comparative analyses of the 9953L55 genome with the A. minor type strain NM305T, A. minor strain 202, and A. pleuropneumoniae S4074T. The 2,263,191 bp circular genome of 9953L55 consisted of 2168 and 2033 predicted genes and proteins, respectively. The lipopolysaccharide cluster resembled the genetic organization of A. pleuropneumoniae serotypes 1, 9, and 11, possibly explaining the positive reactions observed previously in serotyping tests. In contrast to NM305T, we confirmed the presence of a complete apxIICABD operon in 9953L55 and 202 accounting for their hemolytic phenotype and Christie-Atkins-Munch-Petersen (CAMP) reaction positivity. Orthologous gene cluster analysis provided insight into the differential ability of strains of the A. minor/“porcitonsillarum” complex and A. pleuropneumoniae to ferment lactose, raffinose, trehalose, and mannitol. The four strains showed distinct and shared transposable elements, CRISPR/Cas systems, and integrated prophages. Genome comparisons based on average nucleotide identity and in silico DNA-DNA hybridization confirmed the close relationship among strains belonging to the A. minor/“porcitonsillarum” complex compared to other Actinobacillus spp., but also suggested that 9953L55 and 202 belong to the same novel species closely related to A. minor, namely, “A. porcitonsillarum.” Recognition of the taxon as a separate species would improve diagnostics and control strategies of pig pleuropneumonia.

Genomic insights into methicillin-resistant Staphylococcus pseudintermedius isolates from dogs and humans of the same sequence types reveals diversity in prophages and pathogenicity islands

“…Genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. Whole-genome sequencing was performed on MinION (Oxford Nanopore Technologies (ONT), Oxford, UK) and Illumina (Illumina Inc, San Diego, US) platforms [12]. Sequencing libraries for MinION were prepared from mechanically fragmented DNA (g-TUBE, Covaris) using the ONT 1D ligation sequencing kit (SQK-LSK108) with the native barcoding expansion kit (EXP-NBD103) (Oxford Nanopore).…”

“…Advances in next generation sequencing (NGS) permit nowadays to obtain accurate complete genome sequences identifying both accessory genetic elements and single nucleotide polymorphisms (SNPs) [10]. The combination of long and short-read NGS technologies, like Oxford Nanopore Technologies (ONT, Oxford Nanopore Technologies, Oxford, UK) with Illumina 1 (Illumina, San Diego, CA), generates accurate complete genomes of bacteria, which results from the genome scaffold obtained with the long reads of ONT and corrected with the accurate sequence of Illumina [11,12]. Additionally, long read sequencing platform provides an important role for the study of structural variation (SV), including insertions, deletions, duplications, inversions, and large-scale structural rearrangements in short sequence variant [11].…”

A Novel Trimethoprim Resistance Gene, dfrA36 , Characterized from Escherichia coli from Calves

“…DNA was sequenced on an Illumina HiSeq platform (2 × 150 paired ends) (Eurofins, Constance, Germany) and on a R9.4 SpotON flow cell and library kit (Oxford Nanopore Technologies, Oxford, United Kingdom) to obtain long reads and facilitate genome assembly. Genome assembly and read mapping of the Illumina reads against the MinION scaffolds were performed as previously described (12). Analyses of the complete genome using RESFinder 3.1 (Center for Genomic Epidemiology, DTU, Denmark) (20% coverage, 30% identity) confirmed the absence of any known acquired dfr gene in both strains MF2156 and PF9285.…”

RTX Toxins of Animal Pathogens and Their Role as Antigens in Vaccines and Diagnostics

“…This finding, together with small residual 5' apxIIB fragments found on the apxIICA loci in most A. pleuropneumoniae, indicates that apxIIBD genes in A. pleuropneumoniae were most likely deleted due to the presence of the very similar apxIBD. This most likely occurred since the apxIBD-encoded T1SS is able to secrete both ApxI and AxpII [75][76][77][78]. Surprisingly, TolC which also belongs to T1SS, is not annotated on any of the A. pleuropneumoniae genomes available at GenBank/NCBI.…”