Characterization of the emerging zoonotic pathogen <i>Arcobacter thereius</i> by whole genome sequencing and comparative genomics

<div><p>Four <i>Arcobacter</i> species have been associated with human disease, and based on current knowledge, these Gram negative bacteria are considered as potential food and waterborne zoonotic pathogens. At present, only the genome of the species <i>Arcobacter butzleri</i> has been analysed, and still little is known about their physiology and genetics. The species <i>Arcobacter thereius</i> has first been isolated from tissue of aborted piglets, duck and pig faeces, and recently from stool of human patients with enteritis. In the present study, the complete genome and analysis of the <i>A</i>. <i>thereius</i> type strain LMG24486^T, as well as the comparative genome analysis with 8 other <i>A</i>. <i>thereius</i> strains are presented. Genome analysis revealed metabolic pathways for the utilization of amino acids, which represent the main source of energy, together with the presence of genes encoding for respiration-associated and chemotaxis proteins. Comparative genome analysis with the <i>A</i>. <i>butzleri</i> type strain RM4018 revealed a large correlation, though also unique features. Furthermore, <i>in silico</i> DDH and ANI based analysis of the nine <i>A</i>. <i>thereius</i> strains disclosed clustering into two closely related genotypes. No discriminatory differences in genome content nor phenotypic behaviour were detected, though recently the species <i>Arcobacter porcinus</i> was proposed to encompass part of the formerly identified <i>Arcobacter thereius</i> strains. The report of the presence of virulence associated genes in <i>A</i>. <i>thereius</i>, the presence of antibiotic resistance genes, verified by <i>in vitro</i> susceptibility testing, as well as other pathogenic related relevant features, support the classification of <i>A</i>. <i>thereius</i> as an emerging pathogen.</p></div

Estimation of the Average Nucleotide Identity (ANI) for the nine <i>A</i>. <i>thereius</i> strains with <i>A</i>. <i>butzleri</i> RM4018 and <i>A</i>. <i>cibarius</i> LMG21996 were included as outgroup.

<p>ANI results are based on BLAST+ analysis and expressed as [aligned nucleotides] [%].The symbol “*” is added when the same genomes are compared.</p

Estimation of <i>in silico</i> DDH for the nine <i>A</i>. <i>thereius</i> strains.

<p>The confidential interval is shown in square brackets. <i>A</i>. <i>butzleri</i> RM4018 and <i>A</i>. <i>cibarius</i> LMG21996 were included as references. The symbol “*” is added when the same genomes are compared.</p

Characteristics of the phenotypic behaviour and distribution of antimicrobial susceptibility of the nine <i>A</i>. <i>thereius</i> strains sequenced in this study.

<p>Susceptibility breakpoint (mg/L): erythromycin: ≤ 8; ciprofloxacin: ≤ 0,5; ampicillin: ≤ 2; tetracycline: ≤ 2; gentamicin: ≤ 2; streptomycin: ≤ 16; chloramphenicol: ≤ 8; spectinomycin: ≤ 32.</p

Genome architecture of <i>A</i>. <i>thereius</i> LMG24486^T.

<p>Genome architecture of <i>A</i>. <i>thereius</i> LMG24486^T.</p

General features of the <i>Arcobacter thereius</i> LMG24486^T genome sequence.

<p>The circles represent (from the outside to the inside): circle 1, position (bp); circle 2, CDS transcribed clockwise; circle 3, CDS transcribed anti-clockwise; circle 4, GC content plotted using default settings; circle 5, GC skew plotted using a window size of 25,000 and default step size. The predicted <i>oriC</i>, <i>dif</i>_<i>H</i> site, and CRISPR sequences are indicated by black arrows. The <i>difH</i> site had a consecutive 31-bp sequence match with that of <i>A</i>. <i>butzleri</i> RM4018 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180493#pone.0180493.ref061" target="_blank">61</a>].</p

Genbank accession numbers of <i>A</i>. <i>thereius</i> genomes.

<p>Genbank accession numbers of <i>A</i>. <i>thereius</i> genomes.</p

Phylogenetic analysis of the six putative virulence proteins of <i>Arcobacter thereius</i>.

<p>A Neighbor-Joining phylogenetic tree representing the six putative virulence proteins found in <i>A</i>. <i>thereius</i>. <i>Arcobacter</i>, <i>Campylobacter</i> and <i>Helicobacter</i> species are included as outgroup. The scale bar represent substitution per site.</p

The proteolytic metabolism of <i>Arcobacter thereius</i> LMG24486^T.

<p><b>(1)</b> D-3-phosphoglycerate dehydrogenase (AA347_00201); <b>(2)</b> Phosphoserine aminotransferase (AA347_00366); <b>(3)</b> Phosphoserine phosphatase (AA347_00244); <b>(4)</b> L-serine deaminase (AA347_00179); <b>(5)</b> Malate dehydrogenase (AA347_01362); <b>(6)</b> Pyruvate kinase (AA347_00705); <b>(7)</b> Phosphoenolpyruvate carboxykinase (AA347_01523); <b>(8)</b> Pyruvate dehydrogenase E1 component (AA347_01757); <b>(9)</b> Dihydrolipoyl dehydrogenase (AA347_01759); <b>(10)</b> Dihydrolipoyllysine-residue acetyltransferase (AA347_01758); <b>(11)</b> Acetyl-coenzyme A synthetase (AA347_00006); <b>(12)</b> Aldehyde dehydrogenase (AA347_00801); <b>(13)</b> NADP-specific glutamate dehydrogenase (AA347_00755); <b>(14)</b> Glutamine synthetase 1 (AA347_01959); <b>(15)</b> glucosamine 6-phosphate synthase (AA347_00043); <b>(16)</b> Carbamoyl-phosphate synthase small chain, Carbamoyl-phosphate synthase arginine-specific large chain (AA347_00362; AA347_01184); <b>(17)</b> Amino-acid acetyltransferase (AA347_01854); <b>(18)</b> Acetylglutamate kinase (AA347_00182); <b>(19)</b> N-acetyl-gamma-glutamyl-phosphate reductase (AA347_01878); <b>(20)</b> Acetylornithine aminotransferase (AA347_00770); <b>(21)</b> Arginine biosynthesis (AA347_01036); <b>(22)</b> Glutamate 5-kinase (AA347_00930); <b>(23)</b> Gamma-glutamyl phosphate reductase (AA347_01121); <b>(24)</b> Ornithine aminotransferase (AA347_01752); <b>(25)</b> Pyrroline-5-carboxylate reductase (AA347_01948); <b>(26)</b> Aspartate aminotransferase (AA347_01647); <b>(27)</b> Aspartate ammonia-lyase (AA347_00952); <b>(28)</b> L-asparaginase 2 (AA347_00953); <b>(29)</b> argininosuccinate synthase (AA347_01336); <b>(30)</b> Argininosuccinate lyase (AA347_01731); <b>(31)</b> Biosynthetic arginine decarboxylase (AA347_01873); <b>(32)</b> agmatinase (AA347_00964); <b>(33)</b> spermidine synthetase (AA347_00329); <b>(34)</b> aspartate kinase (AA347_01503); <b>(35)</b> aspartate-semialdehyde dehydrogenase (AA347_01039); <b>(36)</b> homoserine dehydrogenase (AA347_00407); <b>(37)</b> homoserine kinase (AA347_00208); <b>(38)</b> threonine synthase (AA347_00183); <b>(39)</b> Homoserine O-acetyltransferase (AA347_01182); (40) Cystathionine gamma-synthase (AA347_01684).</p

Phylogenetic tree of <i>Arcobacter genus</i>.

<p>A maximum likelihood phylogenetic tree constructed using 239 single-copy orthologs and representing the <i>A</i>. <i>thereius</i> strains sequenced in this study and all other <i>Arcobacter</i> genomes available. <i>C</i>. <i>jejuni</i> was not included in the phylogenetic tree.“White dots” = <i>A</i>. <i>thereius</i> isolated from piglet aborted foetus; “Black dots” = <i>A</i>. <i>thereius</i> isolated from pig faeces; “Yellow dots” = <i>A</i>. <i>thereius</i> isolated from cloaca content duck.</p