Table_7_Species classification and novel plasmid identifications in Arcobacter cryaerophilus and Arcobacter cryaerophilus-like organisms.XLSX

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.

螺杆菌属（Arcobacter）是一类在全球范围内新兴的食源性人畜共患病原菌，可引发人类腹泻。该类群相对均一，可与其他ε-变形菌纲（Epsilonproteobacteria）物种明确区分。嗜冷螺杆菌（Arcobacter cryaerophilus，A. cryaerophilus）是一类异质性物种，目前国内对其基因组特征的认知仍较为有限。本研究旨在基于全基因组测序（whole-genome sequence，WGS）解析嗜冷螺杆菌的遗传特征与质粒特性。研究采用平均核苷酸同源性（Average Nucleotide Identity，ANI）与计算机模拟DNA-DNA杂交（in silico DNA–DNA hybridization，isDDH），对90株初鉴定为嗜冷螺杆菌的菌株开展物种分类分析。通过全基因组测序，获得1株完成图基因组与42株草图基因组。借助多款生物信息学软件分析其基因组特征：经估算，所分析菌株的基因组长度介于1.81～2.28 Mb之间，G+C含量约为27%。ANI与isDDH分析结果显示，90株初鉴定的嗜冷螺杆菌菌株需重新划分为4个新物种（判定阈值为ANI>96%或isDDH>70%）。系统发育分析显示，90株基因组可分为2个演化支（包含4个亚支），系统发育树结果表明该类群存在较高的种内基因组多样性，且未观察到菌株按照宿主或地理来源进行聚类的现象。在三分之一的中国菌株染色体中，检测到氨基糖苷类耐药基因，包括aph(2'')-Ih、AAC(6')-Ie-APH(2'')-Ia、aac(6')-IIa、ant(6)以及链丝菌素耐药基因SAT-4。所有测序菌株均携带毒力相关基因。菌株ICDCAC48中发现1株新型大尺寸多重耐药质粒，命名为pCNAC48，全长161,992 bp。该质粒上存在2个耐药岛，长度分别为7,950 bp与25,137 bp，G+C含量分别为38.23%与32.39%；耐药基因与多种转座元件在该质粒的耐药岛内交叉分布。药敏试验证实该质粒携带的耐药基因具有功能活性。质粒接合与消除实验证明，pCNAC48在菌株ICDCAC48中具有较高稳定性。本研究是首次在嗜冷螺杆菌类群中发现多重耐药质粒。