Genome Sequence Comparisons between Small and Large Colony Phenotypes of Equine Clinical Isolates of Arcanobacterium hippocoleae

Abstract

Arcanobacterium hippocoleae is a Gram-positive fastidious bacterium and is occasionally isolated from the reproductive tract of apparently healthy mares (Equus caballus) or from mares with reproductive tract abnormalities. Apart from a few 16S rRNA gene-based GenBank sequences and one recent report on complete genome assembly, detailed genomic sequence and clinical experimental data are not available on the bacterium. Recently, we observed an unusual increase in the detection of the organism from samples associated with mare reproductive failures in Atlantic Canada. Two colony morphotypes (i.e., small, and large) were detected in culture media, which were identified as A. hippocoleae by MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Here, we report the whole genome sequencing and characterization of the morphotype variants. The genome length of the large phenotypes was between 2.42 and 2.43, and the small phenotype was 1.99 Mbs. The orthologous nucleotide identity between the large colony phenotypes was ~99%, and the large and small colony phenotypes was between 77.86 and 78.52%, which may warrant the classification of the two morphotypes into different species. Phylogenetic analysis based on 16S rRNA genes or concatenated housekeeping genes grouped the small and large colony variants into two different genotypic clusters. The UvrA protein, which is part of the nucleotide excision repair (NER) system, and 3-isopropoylmalate dehydratase small subunit protein expressed by the leuD gene were identified as potential virulence factors in the large and small colony morphotypes, respectively. However, detailed functional studies will be required to determine the exact roles of these and other identified hypothetical proteins in the cellular metabolism and potential pathogenicity of A. hippocoleae in mares.

Keywords: A. hippocoleae; NGS; Oxford Nanopore; mares; reproductive tract.

Figure 1

A circular graphical display of the distribution of the genome annotations of the Han-Large (A), Stan-Large (B), and Stan-Small (C) isolates of A. hippocoleae. This includes, from outer to inner rings, the contigs, CDS on the forward strand, CDS on the reverse strand, RNA genes, CDS with homology to known antimicrobial resistance genes, CDS with homology to known virulence factors, GC content, and GC skew. The colors of the CDS on the forward and reverse strands indicate the subsystem that these genes belong to.

Figure 2

An overview of the subsystems for the genomes of the Han-Large (A), Stan-Large (B), and Stan-Small (C) isolates of A. hippocoleae.

Figure 3

Dot-Plot analysis of the genomes of the Han-Large and Stan-Large isolates (A) and the Stan-Large and Stan-Small (B) isolates of A. hippocoleae. (C) Comparison of the genomic organization of the genomes of the Han-Large, Stan-Large, and Stan-Small isolates of A. hippocoleae by progressiveMauve alignment. Linear collinear blocks (LCB)s are shown by different colors. LCBs indicated by the arrow have an inverted orientation in the genome of the Stan-Small isolate.

Figure 4

(A) Box plot showing the distribution of the percent nucleotide identity of genes with functional annotation between the genomes of the Stan-Large and Han-Large (shown in blue) as well as the Stan-Large and Stan-Small (shown in red) isolates of A. hippocoleae. (B) Large-Scale Genome Alignment (LASTZ) graph demonstrating the pairwise alignment of the genomes of the Stan-Large and Stan-Small A. hippocoleae isolates. Blue and red lines: forward and reverse gene orientations in the Stan-Small genome in comparison to the Stan-Large genome, respectively. Green boxes: areas of deletion in the Stan-Small genomes in comparison to the Stan-Large genome.

Figure 5

(A) Percent protein sequence identity between the genomes of the Han-Large, Stan-Large, and Stan-Small isolates of A. hippocoleae using the sequence of A. hippocoleae strain DSM 15539 as a reference. List of tracks from outside to inside: A. hippocoleae strain DSM 15539, Stan-Large, Han-Large, and Stan-Small. (B) OrthoANI and (C) ANI values between A. hippocoleae strain DSM 15539 and the Han-Large, Stan-Large, and Stan-Small isolates of A. hippocoleae. The tree scale represents the percent average nucleotide identity.

Figure 6

(A) Phylogenetic tree analysis based on the concatenated sequences of twenty protein-coding regions with the highest alignment score and (B) the16S rRNA gene sequences. The large and the small colony variants of A. hippocoleae isolates are shown in blue and red fonts, respectively. The A. hippocoleae strain DSM 15539 is shown in a green font. The remainder of the light blue and pink highlighted groups show separate clusters of different A. hippocoleae isolates based on 16S rRNA gene sequences. The isolates are indicated by the GenBank sequence accession numbers. Arcanobacterium phocisimile (FN562996) was used as an outgroup.

Figure 7

Phylogenetic tree constructed by the BV-BRC codon tree pipeline using the whole genomes of eighty-two isolates of different Arcanobacterium species, including the genomes of our three isolates. The clustering of our isolates and A. hippocoleae strain DSM 15539 are highlighted in light green. Our isolates are shown in red fonts. Salmonella enterica serovar Paratyphi A Jy-2 strain and Escherichia coli J53 strain shown in blue fonts were used as outgroups.