Species classification and novel plasmid identifications in Arcobacter cryaerophilus and Arcobacter cryaerophilus-like organisms

Abstract

The Arcobacter is a globally emerging foodborne and zoonotic pathogen that can cause diarrhea in humans. It is relatively homogenous and clearly distinguishes the group from other Epsilonproteobacteria. Arcobacter cryaerophilus (A. cryaerophilus) is a heterogeneous species and little is known about its genomic characterization in China. This study aims to determine the genetic and plasmid features of A. cryaerophilus based on whole-genome sequence (WGS). Average Nucleotide Identity (ANI) and in silico DNA–DNA hybridization (isDDH) were used for the species classification for 90 initially identified A. cryaerophilus strains. One complete genome and 42 draft genomes were obtained by whole genome sequencing. The genomic characteristics were determined using various bioinformatics software. The genomes of the strains examined were estimated to vary from 1.81 to 2.28 Mb in length, with a G + C content of around 27%. ANI and isDDH results indicated that 90 initially identified A. cryaerophilus strains should be reclassified into four new species (ANI > 96% or isDDH > 70%). Two clades (four subclades) were identified among 90 genomes with the phylogenetic analysis. The phylogenetic tree indicated these 90 genomes exhibited a high intra-species genomic diversity. No clustering was assorted with the host or geographic location among these genomes. Aminoglycoside resistance genes, such as aph(2’’)-Ih, AAC(6’)-Ie-APH(2’’)-Ia, aac(6’)-IIa, ant(6), and streptothricin resistance gene SAT-4 were detected in the chromosomes from a third of the Chinese strains. Virulence-related genes were identified in all the sequenced strains. A novel large multiple drug-resistant plasmid (named pCNAC48 with 161,992 bp in length) was identified in strain ICDCAC48. Two antibiotic-resistance islands were found in the plasmid with lengths of 7,950 and 25,137 bp and G + C content of 38.23 and 32.39%, respectively. The drug resistance genes and some transposable elements were cross-distributed among the islands in the plasmid. Antimicrobial susceptibility tests indicated these resistance genes in the plasmid were functional. Plasmid conjugation and curing experiments proved pCNAC48 was stable in strain ICDCAC48. It was the first identified multiple drug resistance plasmid in A. cryaerophilus-like.

Genomic diversity and taxonomic marker for Arcobacter species

“…As discussed in other studies, the ANI and isDDH indices have been proven to provide reliable information for the delineation of Arcobacter species and have also been included in the minimal guidelines for defining species using genomes ( Chun et al, 2018 ; Perez-Cataluna et al, 2018b ; On et al, 2021 ). For Arcobacter , ANI values >96% were the ones that better correlated with isDDH results >70% in previous studies ( Perez-Cataluna et al, 2018a ; Zhou et al, 2022 ), which was further confirmed in this study. The ANI values between genomes of most Arcobacter species were consistent at >96%, except for certain genomes in A. cloacae , A. lanthieri , A. marinus , A. skirrowii , and A. cryaerophilus that did not meet the 96% classification threshold.…”

“…Among these species, we used gene711 to verify and found that except for A. marinus and A. cryaerophilus , the remaining species met the requirement of gene711 > 96% within the species and gene711 < 96% between species. Previous studies ( Zhou et al, 2022 ) have identified four subspecies within A. cryaerophilus , and our study using gene711 for A. cryaerophilus subspecies classification further supported this conclusion. However, there were also instances of gene711 anomalies in certain strains within A. cryaerophilus , such as CNAC091.…”

“…For ANIm, intraspecies pairs generally have >96% identity, while interspecies pairs generally have <93%, with an intermediate range of 93-96% where species circumspection cannot be assured(Rossello-Mora and Amann, 2015). These findings suggested substantial genomic differences within Arcobacter species, even though they could be classified into different subspecies.Previous studies have proposed that A. cryaerophilus should be divided into four subspecies according to the species classification criteria of ANI values >96% and isDDH values >70%(Zhou et al, 2022), which was further confirmed in this study. Within the Arcobacter genus, 17 genomes potentially represented 14 new potentially species.…”

“…In this study, 371 Arcobacter genomes were used, out of which 172 were obtained from strains sequenced by our laboratory or collaborating institutions. The isolation, cultivation, genomic DNA extraction, and sequencing of these strains were described in previous publications ( Wang et al, 2021 ; Ma et al, 2022 ; Zhou et al, 2022 ). Furthermore, genomes of Arcobacter identified at the species level were investigated, 172 of which were obtained in our earlier studies (70 A. butzleri , 81 A. cryaerophilus , 19 A. skirrowii , and 2 A. lacus ), and the others from the public databases.…”

Genomic Characterization of Arcobacter butzleri Strains Isolated from Various Sources in Lithuania

“…Although the novel genus names were validated, authors of a subsequent study [ 4 ] refuted the proposal by demonstrating that Arcobacter is a phenotypically, phylogenetically, and genomically coherent taxon. Currently, the genus Arcobacter is a large group accommodating 36 recognized species that can be detected in a plethora of habitats [ 5 ].…”