Campylobacter Armoricus Sp. Nov., a Novel Member of the Campylobacter Lari Group Isolated from Surface Water and Stools from Humans with Enteric Infection

Home , Campylobacter lari

International Journal Of Systematic And Evolutionary Microbiology Archimer December 2019, Volume 69 Issue 12 Pages 3969-3979 https://doi.org/10.1099/ijsem.0.003836 https://archimer.ifremer.fr https://archimer.ifremer.fr/doc/00591/70346/

Campylobacter armoricus sp. nov., a novel member of the Campylobacter lari group isolated from surface water and stools from humans with enteric infection

Boukerb Amine M. 1, Penny Christian 2, Serghine Joëlle 1, Walczak Cécile 2, Cauchie Henry-Michel 2, Miller William G. 3, Losch Serge 4, Ragimbeau Catherine 5, Mossong Joël 5, Mégraud Francis 6, 7, Lehours Philippe 6, 7, Bénéjat Lucie 7, Gourmelon Michèle 1, *

1 Ifremer, RBE-SGMM-LSEM, Laboratoire Santé Environnement Microbiologie, Plouzané, France 2 Luxembourg Institute of Science and Technology (LIST), Environmental Research and Innovation Department, Esch-sur-Alzette, Luxembourg 3 US Department of Agriculture, Produce Safety and Microbiology Research Unit, Agricultural Research Service, Albany, CA, USA 4 Laboratoire de Médecine Vétérinaire de l’Etat (LMVE), Veterinary Services Administration, Dudelange, Luxembourg 5 Laboratoire National de Santé (LNS), Epidemiology and Microbial Genomics, Dudelange, Luxembourg 6 INSERM, University of Bordeaux, UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, Bordeaux, France 7 French National Reference Centre for Campylobacter and Helicobacter, Pellegrin University Hospital, Bordeaux, France

* Corresponding author : Michèle Gourmelon, email address : [email protected]

Abstract :

During a study on the prevalence and diversity of members of the genus Campylobacter in a shellfish- harvesting area and its catchment in Brittany, France, six urease-positive isolates of members of the genus Campylobacter were recovered from surface water samples, as well as three isolates from stools of humans displaying enteric infection in the same period. These strains were initially identified as members of the Campylobacter lari group by MALDI-TOF mass spectrometry and placed into a distinct group in the genus Campylobacter, following atpA gene sequence analysis based on whole-genome sequencing data. This taxonomic position was confirmed by phylogenetic analysis of the 16S rRNA, rpoB and hsp60 (groEL) loci, and an analysis of the core genome that provided an improved phylogenetic resolution. The average nucleotide identity between the representative strain CA656T (CCUG 73571T=CIP 111675T) and the type strain of the most closely related species Campylobacter ornithocola WBE38T was 88.5 %. The strains were found to be microaerobic and anaerobic, motile, non-spore- forming, Gram-stain-negative, spiral-shaped bacteria that exhibit catalase, oxidase and urease activities but not nitrate reduction. This study demonstrates clearly that the nine isolates represent a novel species within the C. lari group, for which the name Campylobacter armoricus is proposed. Here, we present phenotypic and morphological features of the nine strains and the description of their genome sequences.

Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site. 2

The proposed type strain CA656T has a 1.589 Mbp chromosome with a DNA G+C content of 28.5 mol% and encodes 1588 predicted coding sequences, 38 tRNAs, and 3 rRNA operons.

Keywords : Campylobacter, novel species, surface water, coastal catchment, human gastroenteritis, C. lari group, whole genorne sequence

Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site.

ABBREVIATIONS: ANIb, average nucleotide identity based on BLAST; CA-SFM, Comité de l'Antibiogramme de la Société Française de Microbiologie; CFA, campyfoodagar;

EUCAST, European committee for antimicrobial and susceptibility testing; HSP, high-scoring segment pairs; isDDH, in silico DNA-DNA hybridization; LPSN, list of prokaryotic names with standing in nomenclature; MALDI-TOF, matrix assisted laser desorption ionization-time of flight; MH-F, Mueller-Hinton agar supplemented with 5% defibrinated horse blood; MIC, minimum inhibitory concentrations; MSP, main spectrum profile; NRC, French National

Reference Center for Campylobacter; TCS, trypto-casein-soy; TSA, tryptic soy agar; TSH, tryptic soy agar supplemented with 5% horse blood; TTC, triphenyltetrazolium chloride.

ACCESSION NUMBERS: The DDBJ/ENA/GenBank accession numbers for the whole genome assemblies of the type strain CA656T and strains CA592, CA594, CA639, CA658,

CA663, CA1650 (2014/2961), CA1665 (2014/0226h) and CA1670 (2016/0860h) are

SBEZ00000000, SBFC00000000, SBFB00000000, SBFA00000000, SBEY00000000,

SBEX00000000, SBEW00000000, SBEV00000000, SBEU00000000, respectively. The

GenBank accession number of the 16S rRNA gene sequence of CA656T is MK632223.

Campylobacter infections are recurrently confirmed as the world’s leading cause of bacterial gastroenteritis [1]. In the European Union, campylobacteriosis is the most frequently reported food-borne illness, with over 246000 human cases a year [2]. In addition to the major poultry- related transmission route, numerous other domestic animal and wildlife sources have been identified [3]. The number and diversity of species of the genus Campylobacter transiting through animal and environmental compartments is important [1, 4, 5], and emphasizes the need to adopt an integrated One-Health approach in Campylobacter epidemiology and risk assessment [6]. Currently, the genus Campylobacter encompasses 31 species, 11 subspecies and three biovars, according to the list of prokaryotic names with standing in nomenclature

(LPSN; http://www.bacterio.net/campylobacter.html), accessed on July 30th, 2019).

Thermotolerant Campylobacter, such as Campylobacter jejuni, Campylobacter coli and

Campylobacter lari, are the most commonly-implicated species in Campylobacter infections

[2].

The purpose of the present One-Health-based study, conducted from 2013 to 2016, was to investigate the importance of the water-related transmission and recirculation of isolates of members of the genus Campylobacter from the C. lari group [7] in the La Fresnaye coastal catchment area located in the north-eastern part of Brittany (Côtes d’Armor department,

France). The La Fresnaye catchment corresponds to a 121 km2 area, characterized by intensive livestock (cattle and swine) and poultry farming, with a human population of about 6900 inhabitants [8, 9]. Specific focus was given to the presence of the bacteria in shellfish-producing areas downstream of the coastal catchment, the diversity found in the catchment’s rivers and seawater, the faecal carriage among wildfowl and the related incidence in the French human population.

In an evaluation of the prevalence of species of the genus Campylobacter in three French coastal catchments, including the La Fresnaye catchment, species of the genus Campylobacter were recovered from 58.3% (n=228) of water samples and 26.6% (n=238) of shellﬁsh samples [9].

C. lari was the most frequently isolated species in shellﬁsh, with 26.4% of samples positive for this species, while it was isolated in 10.6% of surface water samples [9].

Among a collection of more than 250 C. lari group strains from environmental samples and human stools, special interest was given to nine isolates. Although these strains were initially identified as C. lari by mass spectrometry using a MALDI-TOF mass spectrometer (MALDI-

TOF Bruker Microﬂex), a preliminary atpA phylogenetic analysis indicated that they formed a new clade within the C. lari group. Six of these isolates were obtained from three distinct river water samples collected at the outlet of one of the four main rivers of the La Fresnaye catchment named “Le Rat” (10.5 km long and a subcatchment area of 19.2 km2) at three sampling dates in 2014 (January 6th, two isolates; February 4th, three isolates; and March 4th, one isolate).

These river water strains (CA592, CA594, CA639, CA656T, CA658, and CA663) were isolated using the ISO-10272:2016 method or by a passive filtration method. Colonies suspected to be members of the genus Campylobacter were confirmed by a qPCR assay targeting the 16S rRNA gene [9, 10]. Additionally, three clinical isolates (CA1650, CA1665, and CA1670) were recovered from human stool samples collected in 2014 and 2016 in three laboratories participating in the network of the French National Reference Centre for Campylobacters. They were located in three departments geographically distant from the La Fresnaye catchment area

[October 3, 2014 in the Ille-et-Vilaine department (69-year-old male); April 8, 2014 in the Seine department (41-year-old female); and September 14, 2016 in the Vendée department (75-year- old male)]. These nine isolates were subjected to a thorough genomic and phenotypic characterization approach, according to the minimal standards recommended for describing novel species of the genus Campylobacter [11].

The morphology, presence of flagella and cell size of C. armoricus were determined by transmission electron microscopy. Bacteria were scraped and introduced into a fixative solution of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and incubated for 1 h at room temperature. After centrifugation for 3 min at 2350 g, pellets were mixed/suspended in

500 μl of 0.1 M sodium cacodylate buffer (pH 7.4). A 10 μl sample of bacterial suspension was adsorbed onto carbon grids with negative ionization (Delta Microscopy) and negatively stained with a nano-tungsten solution at 1 drop – 1min/drop (Nano-W, Nanoprobes). Grids were examined with a transmission electron microscope (Talos F200S G2, Thermofisher) equipped with a 4×4K ONE VIEW camera (Gatan), at an operating voltage of 200 kV. Cells were spiroform, approximately 2.5 µm in length, and display an amphitrichous distribution of flagella at both ends of the cell (Fig. 1).

Phenotypic testing for the physiological and biochemical characterizations was performed as described previously [11]. Strain growth was determined on several media, including Columbia agar (Oxoid), Chocolate agar (Oxoid), tryptic soy agar (TSA; Oxoid) supplemented with 5% horse or sheep blood (Oxoid), and CFA (CampyFoodAgar, Biomérieux) supplemented with 5% horse or sheep blood (Oxoid). Microaerobic growth on TSA supplemented with 5% sheep blood

(using CampyGen, Oxoid) was evaluated at 25, 37, and 42°C for 48 h. Aerobic and anaerobic growth (using AnaeroGen, Oxoid) were evaluated at 25°C, 37°C and 42°C for 48 h. Catalase activity was evaluated by adding a 3% H2O2 solution (Sigma-Aldrich) and observing the reaction within 5s. Oxidase activity was determined with oxidase discs (Sigma-Aldrich).

Indoxyl acetate hydrolysis was determined by using indoxyl test strips (Sigma-Aldrich). Additional phenotypic tests [i.e., urease activity, nitrate reduction, hippurate hydrolysis, triphenyltetrazolium chloride (TTC) reduction and alkaline phosphatase activity] were evaluated with the API Campy identification system (bioMérieux) according to the manufacturer’s instructions. All tests were performed at least twice, with C. jejuni (LMG

6444T), C. coli (LMG 6440T), C. lari (LMG 7605T), Campylobacter subantarcticus (LMG

24377T), Campylobacter insulaenigrae (LMG 22716T), Campylobacter volucris (LMG

24380T), Campylobacter ornithocola (LMG 29815T) and Campylobacter peloridis (LMG

23910T) as controls. Also, growth in the presence of 1% glycine, 0.1% selenite, 1% bile, 2%

NaCl or 0.04% TTC on Columbia agar supplemented with 5% sheep blood was evaluated.

Haemolysis on sheep blood agar and the ability to grow in the absence of H2 were also evaluated. The isolates displayed phenotypic characteristics, specifically urease activity and inability to reduce nitrate, that were distinct from those of other current members of the genus

Campylobacter taxa (Tables 1 and S1).

The isolates were grown on tryptic soy agar supplemented with 5% horse blood (TSH, bioMérieux) under microaerobic conditions at 37°C. Antimicrobial susceptibility testing was performed, according to the European Committee for Antimicrobial and Susceptibility Testing

(EUCAST) recommendations, on in-house Mueller-Hinton agar supplemented with 5% defibrinated horse blood (MH-F) and 20 mg l-1 of β-nicotinamide adenine dinucleotide (Sigma

Aldrich, Merck), under the same atmosphere and temperature conditions as previously described [12]. Minimum inhibitory concentrations (MICs) were determined for each isolate with E-test strips (bioMérieux). For ciprofloxacin, erythromycin and tetracycline, the EUCAST breakpoints (V8.0, January 2018; http://www.eucast.org/clinical_breakpoints/) were used to classify strains as susceptible or resistant, while for the other antimicrobials (i.e. ampicillin, amoxicillin clavulanate and gentamicin) the cut-offs of the “Comité de l'antibiogramme de la Société Française de Microbiologie” (CA-SFM) [13] were considered. The reference strain C. jejuni subsp. jejuni ATCC 33560T was used as a quality control strain, according to EUCAST recommendations. In addition, resistances to nalidixic acid (tested on Muller-Hinton broth supplemented with 2.5% lysed horse blood and incubated at 41.5°C for 48 to 72 h) at a range from 1 to 64 mg l-1 (Sensititre EUCAMP2, ThermoFisher) and cephalothin (Oxoid Cephalothin

Antimicrobial Susceptibility Disks, 30 µg), on Muller-Hinton agar with 5% horse blood

(Oxoid) were also evaluated.

All the novel isolates were susceptible to first generation (nalidixic acid) and second generation

(ciprofloxacin) quinolones, macrolides (erythromycin), tetracycline, aminoglycosides

(gentamicin), penicillins (ampicillin) and the combined amoxicillin clavulanate, but resistant to cephalothin.

The nine isolates were subcultured on trypto-casein-soy (TCS) agar (bioMérieux) supplemented with 5% (v/v) defibrinated sheep blood (Oxoid, Thermo Scientific) and incubated at 42°C for 24 h in a microaerobic atmosphere generated by an Anoxomat system

(Mart Microbiology).

Bacterial DNA was extracted from these cultures using the DNA QIAamp Mini Kit 250

(Qiagen). Genomic libraries for whole-genome sequencing were prepared using the Nextera

DNA Flex library prep kit (Illumina). Sequencing was performed on an Illumina MiniSeq platform using a high output kit cartridge (FC-420-1003, Illumina) to generate 2×150 bp paired- end reads.

Illumina paired-end reads were trimmed using Trimmomatic v.0.36 [14]. Sequence data quality was checked using FastQC v.0.11.6 [15]. Assembly of paired-end reads was done de novo using

Spades v.3.12.0 [16] and Unicycler v.0.4.7 [17] with default settings. The obtained drafts were checked for consistency, e.g. the number of contigs, G+C content and total size of assembly, using Quast v.5.0.0 [18]. Annotations were done using the Prokka pipeline v.1.13.4 and the following parameters: -force -addgenes -compliant -genus Campylobacter -usegenus -rfam

[19]. The DNA G+C contents ranged from 28.3 to 28.5%, which is within the lower end range observed for members of the genus Campylobacter, and the estimated genome sizes ranged from 1.58 to 1.75 Mb (Tables 1 and S2). Details of the assembly metrics, genome annotations and GenBank accession numbers are provided in Table S2.

The high-quality genome sequences were employed to determine the taxonomic position of all nine isolates using: (i) core genome comparisons and phylogenetic comparisons based on 16S rRNA, atpA, AtpA, rpoB and hsp60 (groEL) sequences; and (ii) genome relatedness using average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) analyses.

The taxonomic position of the nine isolates was first determined by comparing their 16S rRNA gene sequences with those from representatives of type strains of species of the genus

Campylobacter, using the Arcobacter butzleri ATCC 49616T 16S rRNA gene as an outgroup.

Sequence alignment and tree construction were performed using MEGA v.10.0.5 [20]. A neighbor-joining tree and 1,000 bootstrap replicates were generated (Fig. 2). The nine isolates formed a distinct cluster within the dendrogram, which is consistent with the preliminary atpA analysis (Fig. S1). Sequence similarity within this cluster was 100%, while the 16S rRNA genes of the nine isolates were on average 98% similar to the 16S rRNA gene of the closely-related species Campylobacter peloridis strain LMG 23910T. Similar phylogenetic analyses were conducted using sequences of the full AtpA protein (Fig. 3), and portions of the rpoB (Fig. S2) and hsp60 (groEL) (Fig. S3) genes. Consistent with the 16S rRNA phylogeny, in each analysis the nine isolates, termed Campylobacter armoricus hereafter, formed a clade distinct from the other known species of the genus Campylobacter.

Additionally, a core genome analysis was conducted to provide an improved taxonomic resolution (Fig. 4). The sequences of 374 core genes, conserved between the nine C. armoricus isolates, 35 species of the genus Campylobacter and Arcobacter butzleri strain ATCC 49616T, were concatenated (composite length = 345896 bp) and aligned. Proteins included in the core gene set required a minimum 35% sequence identity for each pairwise comparison, as determined by Roary v.3.12.0 [21]. A core genome phylogeny free of correction for recombination was constructed with Iqtree v.1.6.9 [22] using model finder [23], and ultrafast bootstrap [24]. MEGA v.10.0.5 was then used to produce a maximum-likelihood phylogenetic tree. The core gene set of A. butzleri strain ATCC 49616T was used as an outgroup to root the tree. The output of this analysis confirmed the loci-based phylogenies, with C. armoricus positioned as a taxon within the larger C. lari group (Fig. 4).

Whole-genome sequence similarities, based on thresholds below 70 and 96% for DNA-DNA hybridization (DDH) and average nucleotide identity based on BLAST (ANIb), respectively, were used for the delimitation of closely-related species [25, 26]. Pyani v.0.2.7 [27, 28] was used to calculate pairwise ANI values for a strain set that included the nine C. armoricus isolates, other species of the genus Campylobacter, Wolinella succinogenes, Helicobacter pylori and Arcobacter butzleri. In silico DNA-DNA hybridization values were calculated using the Genome-to-Genome Distance Calculator (GGDC; ggdc.dsmz.de/ggdc.php [29]). The isDDH model “formula 2” was used as recommended for draft genomes. Pairwise ANI and isDDH values between the nine C. armoricus strains and the most closely related members of the genus Campylobacter are presented in Table 2, while full results are presented in Tables

S3 and S4. In both analyses, the pairwise values between the strains of C. armoricus and all other species of the genus Campylobacter are below the species thresholds described above, consistent with the establishment of C. armoricus as a novel member of the genus

Campylobacter.

We used ABRicate v.0.8.7 [30], a software package that screens draft genomes for resistance and virulence genes using the NCBI Bacterial Antimicrobial Resistance Reference Gene

Database (NCBI BARRGD; BioProject accession number PRJNA313047) and the Virulence

Factors of Pathogenic Bacteria Database (VFDB) [31], respectively. The following well-known genes associated with invasion, virulence and infection were selected for comparison and occurrence: the cdtABC operon, which encodes the cytolethal distending toxin [32]; genes encoding the secreted invasion proteins CiaB and FlaC; and the adhesion determinant-encoding genes jlpA, porA, pebA and cadF. In addition, flagella, capsule production and S-layer protein- encoding genes were screened. Except for jlpA, pebA and the S-layer protein encoding-genes

(usually restricted to Campylobacter fetus and a few other taxa), all of the widely-distributed

Campylobacter virulence determinants were present in all nine C. armoricus strains.

As with the virulence determinants, we focused on associated antibiotic resistance genes or operons that are relevant to members of the genus Campylobacter, including: the multidrug- resistant efflux pump- encoding operon cmeABC and its transcriptional regulators cmeR and cosR [33-35]; the macrolide-specific efflux locus macAB [36] and the ErmB erythromycin resistance protein; the tet(O) locus which confers resistance to tetracycline [37]; members of the class D beta-lactamase family, oxa-184 and oxa-493, that confer resistance to beta-lactam antibiotics [38]; six genes encoding aminoglycoside-modifying enzymes (aadE-sat4-aphA-3 cluster, aacA-aphD, aac and aadE) [39]; and nucleotide substitutions in gyrA that mediate resistance to fluoroquinolones [40, 41], and those in the 23S rRNA gene for high-level resistance to macrolides [42]. The following genes related to antibioresistance, cmeABC, cmeR, cosR, macAB, oxa-184 and oxa-493 were found in the nine C. armoricus isolates, while tet(O),

ErmB and the aminoglycoside-resistance genes were absent and none of the known mutations in gyrA and 23S rRNA genes were identified.

Most phenotypic susceptibilities were in agreement with the screening of resistance genes.

Although the presence of the oxa-184 and oxa-493 genes is consistent with the cephalothin- resistant phenotype, it could not explain the ampicillin susceptibility of the isolates.

As in the other members of the C. lari group, multiple genes encoding enzymes involved in amino acid/co-factor biosynthesis and energy metabolism/respiration were absent in C. armoricus, such as genes involved in the biosynthesis of acetyl-coenzyme A (acs), the ATP- binding protein of the methionine ABC transporter (metN), and the respiratory enzyme- encoding gene gltA (citrate synthase). In agreement with the inability of the strains to reduce nitrate, the napDLBGHA nitrate reductase gene cluster was absent.

MALDI-TOF mass spectrometry analysis of the C. armoricus isolates was performed at the

French National Reference Center for Campylobacter (NRC; Bordeaux, France) [43-45].

Ethanol formic acid extraction was performed on the nine strains of C. armoricus strains, five of C. lari subsp. lari, four of C. lari subsp. concheus, one of C. ornithocola, one of C. coli, one of C. upsaliensis, one of C. jejuni, and one of A. butzleri. Spectra were obtained with a MALDI

Compass Explorer (Bruker Daltonics). Each protein extract was spotted 20 times onto a 96 ground steel target plate (Bruker Daltonics). Each line included an Escherichia coli control sample provided by Bruker Daltonics, where the presence of eight specific proteins insured that the spectrometer was set properly. Measurements were determined with a Microflex mass spectrometer (Bruker Daltonics) equipped with a 200-Hz smart beam 1 laser. After extraction with formic acid, the spectra were acquired using Axima Assurance analysis (Shimadzu

Corporation). Spectra were generated using the Launchpad v2.8 software program, compared with the Saramis database (developed by Anagnostec) and were matched against the

SuperSpectra database.

First, the nine strains of C. armoricus were identified by MALDI-TOF as C. lari with a score below 1.9, indicating the absence of the corresponding spectrum in the database. Then, 20 spectra were obtained from the spots for each of the nine C. armoricus and the nine C. lari strains in the MALDI-Compass Explorer (Bruker Daltonics) to create a main spectrum profile

(MSP) of each strain. Arcobacter butzleri strain DSM 7301 was used as an unrelated control genus according to the manufacturer’s suggestions. The C. armoricus strains from human and river samples formed a distinct cluster from the C. lari strains (Figs 5 and S4). Consistent with previous results, the dendrogram indicated that the C. armoricus cluster presents a putative novel species of the genus Campylobacter, different from C. jejuni, C. coli, C. upsaliensis and

A. butzleri, and closely-related to the C. lari group.

DESCRIPTION OF CAMPYLOBACTER ARMORICUS SP. NOV

Campylobacter armoricus [ar.mo’ri.cus. L. pl. fem. n. Armoricae part of Gaul between the

Seine and the Loire including Brittany; N.L. masc. adj. armoricus pertaining to Brittany).

Cells are motile, non-encapsulated, non-spore-forming, Gram-negative curved rods, on average

0.3 µm wide and 2.5 µm long. After incubation on TSA supplemented with 5% sheep blood in a microaerobic atmosphere at 42°C for 48 h, colonies are glossy and greyish, slightly convex, and round with smooth margins (± 1 mm diameter, ± 2 mm after 72 h). Older colonies display a grey metallic surface. Coccoidal cells are observed in old cultures. Swarming on solid media was noted for the human isolates. Pigments are not produced. Growth occurs on blood agar

(Chocolate agar or TSA supplemented with 5% sheep or horse blood) at both 37 and 42°C under microaerobic culture conditions (does not require atmospheric hydrogen) and at 37°C under anaerobic conditions [good growth at 42°C under anaerobic conditions for six of the nine strains, suboptimal growth for three (CA592, CA663, and CA656T)]. No growth is observed at

37°C under aerobiosis and at 25°C under microaerobic conditions. No haemolysis is seen on blood agar. Catalase, oxidase and urease activities are present. Strains do not hydrolyse hippurate or reduce nitrate. Reduction of triphenyltetrazolium chloride (TTC) is variable.

Growth under microaerobic conditions is observed on Columbia agar supplemented with horse or sheep blood containing 1.0% glycine or 1.0% bile but not on Columbia agar containing 2.0%

NaCl. Growth is variable on Columbia agar containing 0.4% TTC or 0.1% selenite.

Three of the nine isolates originated from humans with gastroenteritis from three different departments in France, while six were recovered from river water samples in a coastal catchment area in the Côtes d’Armor department (France). The type strain is CA656T (CCUG

73571T = CIP 111675T). The DDBJ/ENA/GenBank accession numbers for the whole genome assemblies of the type strain CA656T and strains CA592, CA594, CA639, CA658, CA663,

CA1650 (2014/2961), CA1665 (2014/0226h) and CA1670 (2016/0860h) are SBEZ00000000,

SBFC00000000, SBFB00000000, SBFA00000000, SBEY00000000, SBEX00000000, SBEW00000000, SBEV00000000, SBEU00000000, respectively. The GenBank accession number of the 16S rRNA gene sequence of CA656T is MK632223. ACKNOWLEDGMENTS

We are grateful to Dr. Carlos Wetzel and Mr. Luc Ector (LIST ERIN department, Luxembourg) for their help with the specific nomenclature and etymology. We also thank Mrs. Delphine

Collard and Mrs. Laetitia Kaas (LIST ERIN department, Luxembourg) for valuable technical support, Dr Fatima M’Zali and her platform “Aquitaine Microbiologie”

(http://www.mfp.cnrs.fr/mfp/team_am.php) for her help in the MALDI-TOF analyses, and Mr

Dominique Claude for performing antibioresistance analyses (Laboratoire de Médecine

Vétérinaire de l’Etat, Luxembourg). Imaging was performed on the Bordeaux Imaging Center

(UMR 3420 CNRS – US4 INSERM), a member of the FranceBioImaging national infrastructure (ANR-10-INBS-04). We thank Sabrina Lacomme for her TEM work on this project.

The river water strains were isolated during the RiskManche project (#4163), funded by the

European Regional Development Fund Interreg IVa Program, and were characterized in the frame of the Campyshell project funded by the European Fisheries Fund (EFF).

The authors acknowledge the Pôle de Calcul et de Données Marines (PCDM) for providing

DATARMOR (storage, data access, computational resources, visualization, web-services, consultation, support services). URL: http://www.ifremer.fr/pcdm

FUNDING INFORMATION

This work was partly supported by the EFF (Campyshell project).

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

ETHICAL STATEMENT

No experiments with humans or animals were carried out. No patient information was disclosed. REFERENCES

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Phenotypic characteristics that differentiate Campylobacter armoricus sp. nov. from the most closely related members of the genus Campylobacter.

The complete comparison with the other campylobacters is displayed in Table S1.1, Campylobacter armoricus (n=9); 2, Campylobacter insulaenigrae; 3, Campylobacter lari subsp. concheus; 4, Campylobacter lari subsp. lari; 5, Campylobacter ornithocola; 6, Campylobacter peloridis; 7, Campylobacter subantarcticus; 8, Campylobacter volucris. +, positive result; -, negative result; mO2, microaerobic condition; ANO2, anaerobic condition; F, 7–27 % strains are positive; V, 29–57 % strains are positive; M, 70–95 % strains are positive; ∗, weak growth; §Urease-Positive Thermophilic Campylobacter (UPTC) variants (from On et al., 2017 [11]; Gilbert et al., 2018 [46]); NA; data not available. .** present study.

Characteristic 1 2 3 4 5 6 7 8 Growth temperature range (°C) 37-42 37 37–42 30–42 37–42 37–42 37–42 37–42 mO2, mO2, Atmospheric requirements ANO2 mO2 mO2 mO2 mO2,ANO2 mO2 mO2,ANO2 ANO ∗ (37°C) 2 Oxidase + + + + − +** + + Catalase + + + + + +** + + Nitrate reduction − + + + V +** + + Indoxyl acetate hydrolysis − − NA F − −** − − Urease + − − V§ + −** − − Alkaline phosphatase − NA NA − − −** NA − Hippurate hydrolysis − − − − − −** − − Growth on 0.1% selenite V + NA V NA NA − + Growth on 2 % NaCl − − + M NA M + − Growth on 1 % glycine + + + + + + M − Growth on 0.04 % TTC V + NA M NA NA NA − TTC reduction V + NA M F −** NA −

Resistance to: Nalidixic acid − + − V NA M + + (30 mg) Cephalothin (30 mg) + + + + NA F − +

DNA G+C content (mol%) 28.3 - 28.5 28 30 29-30 30 29 30 29

Table 2. Average nucleotide identity based on BLAST (ANIb) values (%) and pairwise in silico DNA-DNA hybridization (isDDH; using GGDC formula 2 and isDDH estimates based on identities/HSP length*) values for the nine Campylobacter armoricus sp. nov., strains and the most closely related members of the genus Campylobacter.

The numbers below the diagonal are ANI values predicted between pairwise genomes; in all ANI calculations, the alignment fraction was 71-86%. The numbers above the diagonal are DDH values between genomes. Strains: 1, Campylobacter armoricus CA656T; 2, Campylobacter armoricus CA592; 3, Campylobacter armoricus CA594; 4, Campylobacter armoricus CA639; 5, Campylobacter armoricus CA658; 6, Campylobacter armoricus CA663; 7, Campylobacter armoricus CA1650; 8, Campylobacter armoricus CA1665; 9, Campylobacter armoricus CA1670; 10, Campylobacter insulaenigrae NCTC 12927T; 11, Campylobacter lari subsp. concheus LMG 11760; 12, Campylobacter lari subsp. lari ATCC 35221T; 13, Campylobacter ornithocola WBE38T; 14, Campylobacter peloridis LMG 23910T; 15, Campylobacter subantarcticus LMG 24377T; 16, Campylobacter volucris LMG 24379.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 *** 100 90.3 91 91.1 91 93.6 90 90.6 23.6 35 33.3 36.4 31.2 31.7 25.8 2 99 *** 90.3 91 91.1 91 93.5 90 90.6 23.6 35 33.4 36.4 31.1 31.7 25.8 3 99 99.9 *** 92 92 92 90.4 90.7 91.2 23.5 35.3 33.5 36.4 31.1 31.7 25.7 4 99 98.9 98.8 *** 99.8 100 91.5 90.8 91.6 23.6 35.2 33.2 35.9 30.4 31.3 25.8 5 99.9 99 99 99.1 *** 99.9 91.3 90.8 91.5 23.6 35.2 33.2 35.9 30.4 31.3 25.8 6 99.9 99 99 99.1 100 *** 91.5 90.9 91.7 23.6 35.3 33.3 35.9 30.4 31.4 25.8 7 99 99.1 99.1 98.8 99 100 *** 90.1 90.9 23.6 35.1 33.4 36.2 31.2 32 25.8 8 98.9 98.8 98.8 98.9 98.9 98.8 100 *** 99.2 23.8 35.1 33.4 36.6 30.7 31.7 25.9 9 99 98.9 98.9 99 99 99 99.9 100 *** 23.6 35.1 33.3 36.3 30.8 31.8 25.7 10 81.5 81.5 81.5 81.5 81.5 81.5 81.6 81.5 81.5 *** 23.5 23.5 23.8 23.8 22.8 25.8 11 88.3 88.3 88.3 88.3 88.3 88.3 88.3 88.2 88.3 81.3 *** 51.3 43.4 31.4 38.8 25 12 87.5 87.5 87.5 87.5 87.5 87.5 87.5 87.5 87.5 81.5 93.2 *** 39.7 30.4 37.1 25.7 13 88.5 88.7 88.7 88.7 88.5 88.6 88.7 88.6 88.5 81.4 90.9 89.7 *** 31.6 35.8 25.1 14 86 86.5 86.5 86.5 86 86.5 86.2 86.2 86.1 81.6 86.5 86.1 86.7 *** 29.9 25.5 15 86.4 86.6 86.6 86.6 86.4 86.7 86.7 86.7 86.4 80.7 89.7 88.9 88.5 85.5 *** 24.6 16 83.3 83.3 83.3 83.2 83.3 83.3 83.4 83.2 83.3 83.2 82.7 83.1 82.6 82.9 82.2 ***

*Confidence intervals indicate inherent uncertainty in estimating DDH values from intergenomic distances based on models derived from empirical test data sets (which are always limited in size). These results are in accordance with phylogenomic analyses as well as the ANI results. GGDC: Genome-to-Genome Distance Calculator (v. 2.1; http://ggdc.dsmz.de/distcalc2.php ); HSP: high- scoring segment pairs.

Figure 1. Transmission electron microscopy of Campylobacter armoricus strains CA1650 (A) and CA656T (B) using a transmission electron microscope (Talos, Thermofisher) at an operating voltage of 200 kV. The microorganism exhibits a mean size of 2.5 × 0.3 µm. It presents a helical bacillus form with one unsheathed flagellum per pole. Bars, 500 nm.

Figure 2. Neighbor-joining phylogenetic dendrogram constructed using 16S rRNA gene sequences that highlights the position of the nine Campylobacter armoricus strains relative to other Campylobacter species. Arcobacter butzleri strain ATCC 49616T is used as an outgroup and root. Bootstrap values (≥50%) based on 1000 replications are indicated at the nodes. GenBank accession numbers (in parentheses) are provided for each type strain. The scale bar represents a 0.02% nucleotide sequence divergence.

Figure 3. Neighbor-joining phylogenetic dendrogram based on full AtpA protein sequences, highlighting the position of the nine Campylobacter armoricus strains relative to other Campylobacter species. Arcobacter butzleri strain ATCC 49616T is used as an outgroup and root. Bootstrap values (≥50%) based on 1000 replications are indicated at the nodes. GenBank accession numbers (in parentheses) are provided for each reference strain. The scale bar represents a 0.02% peptide sequence divergence.

Figure 4. Phylogenetic dendrogram based on the core genome (n=374 genes; 345,896 bp) conserved between the nine Campylobacter armoricus isolates, 35 other Campylobacter sp., and Arcobacter butzleri strain ATCC 49616T that is used as an outgroup and root. Bootstrap values are indicated at the nodes. The scale bar represents a 0.2% nucleotide sequence divergence.

Figure 5. MSP (Main Spectra Profiles) dendrogram based on Mass Spectrometry MALDI- TOF, representing Campylobacter armoricus, Campylobacter ornithocola, Campylobacter lari subsp. lari and Campylobacter lari subsp. concheus. Three other Campylobacters and Arcobacter butzleri DSM 7301 are used as rooting outgroups. (A) (B)

Figure 1.

Figure 2.

Figure 3.

Figure 4

Figure 5.

Figure S1. Neighbor-joining phylogenetic dendrogram, constructed using trimmed and aligned atpA gene sequences (489 nt), that highlights the taxonomic position of C. armoricus isolates relative to other Campylobacter species and Arcobacter butzleri strain ATCC 49616T. Bootstrap values (≥50%) based on 1000 replications are indicated at the nodes. GenBank accession numbers (in parentheses) are provided for each reference strain. The scale bar represents a 0.02% nucleotide sequence divergence.

Figure S2. Neighbor-joining phylogenetic dendrogram, constructed using trimmed and aligned rpoB gene sequences (485 nt), that highlights the taxonomic position of C. armoricus isolates relative to other Campylobacter species and Arcobacter butzleri strain ATCC 49616T. Bootstrap values (≥50%) based on 1000 replications are indicated at the nodes. GenBank accession numbers (in parentheses) are provided for each reference strain. The scale bar represents a 0.02% nucleotide sequence divergence.

Figure S3. Neighbor-joining phylogenetic dendrogram, constructed using trimmed and aligned hsp60 (groEL) gene sequences (545 nt), that highlights the taxonomic position of C. armoricus isolates relative to other Campylobacter species and Arcobacter butzleri strain ATCC 49616T. Bootstrap values (≥50%) based on 1000 replications are indicated at the nodes. GenBank accession numbers (in parentheses) are provided for each reference strain. The scale bar represents a 0.02 % nucleotide sequence divergence.

Figure S4: Reference mass spectrum (MALDI-TOF) from Campylobacter armoricus strain CA656T.

Table S1. Phenotypic characteristics that differentiate Campylobacter armoricus sp. nov. from other species of the genus Campylobacter.

1, Campylobacter armoricus (n=9); 2, Campylobacter avium; 3, Campylobacter blaseri; 4, Campylobacter canadensis; 5, Campylobacter coli; 6, Campylobacter concisus; 7, Campylobacter corcagiensis; 8, Campylobacter cuniculorum; 9, Campylobacter curvus; 10, Campylobacter fetus subsp. fetus; 11, Campylobacter fetus subsp. testudinum; 12, Campylobacter fetus subsp. venerealis; 13, Campylobacter gracilis; 14, Campylobacter hepaticus; 15, Campylobacter helveticus; 16, Campylobacter hominis; 17, Campylobacter hyointestinalis subsp. hyointestinalis; 18, Campylobacter hyointestinalis subsp. lawsonii; 19, Campylobacter iguaniorum; 20, Campylobacter insulaenigrae; 21, Campylobacter jejuni subsp. doylei; 22, Campylobacter jejuni subsp. jejuni; 23, Campylobacter lanienae; 24, Campylobacter lari subsp. concheus; 25, Campylobacter lari subsp. lari; 26, Campylobacter mucosalis; 27, Campylobacter ornithocola; 28, Campylobacter peloridis; 29, Campylobacter pinnipediorum subsp. pinnipediorum; 30, Campylobacter pinnipediorum subsp. caledonicus; 31, Campylobacter rectus; 32, Campylobacter showae; 33, Campylobacter sputorum; 34, Campylobacter subantarcticus; 35, Campylobacter upsaliensis; 36, Campylobacter ureolyticus; 37, Campylobacter volucris. + = all strains examined give a positive result; − = all strains examined give a negative result; <> = 20–30 % strains grow at this temperature; [] = 50–60 % strains grow at this temperature; () = 76–93 % grow under these conditions; O2, aerobic conditions; mO2, microaerobic conditions; ANO2, anaerobic conditions; HmO2=microaerobic atmosphere enhanced with H2; F = 7–27 % strains positive; V = 29–57 % strains positive; M = 70–95 % strains positive; ∗Weak growth.; †Biovar fecalis strains produce catalase; ‡Biovar paraureolyticus strains produce urease; §Urease-Positive Thermophilic Campylobacter (UPTC) variants (from On et al., 2017; Gilbert et al., 2018). Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 (30), 37, 30, 37, (18–22), 25– 25–37, <18–22>, Growth temperature range (°C) 25-42 37–42 25-42 37–42 30–42 25–42 37, (42) (30), 37,(42) (42) (42) 37, (42) (42) (25), 37

mO2, mO2, ANO2 mO2, HmO2, ANO2, ANO2, mO2, mO2, Atmospheric requirements mO2 ANO2 mO2 mO2 mO2, [ANO2] ANO2, [HmO2] (37°C) ANO ANO mO ∗ [HmO ] ANO (ANO ) (37°C) 2 2 2 2 2 2

Oxidase + + + + + V + + + + + + − Catalase + + + V + − + + − + + + V Nitrate reduction − + + V + F M + + + + M M Indoxyl acetate hydrolysis − + + − + − V + V − − − M Urease + − + V − − + − − − − − − Alkaline phosphatase − NA + − − M + − V − − − − Hippurate hydrolysis − + - − − − − − F − − − − Growth on 0.1% selenite V NA ND NA + F NA − − M NA F − Growth on 2 % NaCl − NA ND − − F + − V − NA − V Growth on 1 % glycine + + F V M F + − + + + F + Growth on 0.04 % TTC V NA ND NA + − − V + − + − − TTC reduction V NA ND NA + − − V V − + − −

Resistance to: Nalidixic acid (30 mg) − − − V − M + V + + NA V V Cephalothin (30 mg) + + − − + − − M − − NA − −

DNA G+C content (mol%) 28.3-28.5 35 29 NA 31 37-41 32 32 45-46 33-34 33 33-34 44-46

Continued. Characteristic 14 15 16 17 18 19 20 21 22 23 24 25 26 Growth temperature range (°C) 37–42 [30], 37 [18–25], 30–42 18–37 37 37 (30), 37-42 37–42 37–42 30–42 30–42 37–42 30–42

ANO2, mO2, Atmospheric requirements mO2 mO2 mO2 mO2,ANO2∗ mO2 mO2 mO2 mO2, ANO2∗ mO2 mO2 mO2, ANO2 [HmO2] ANO2

Oxidase + + + + + + + + + + + + + Catalase + − − + + + + M + + + + − Nitrate reduction V + V + + + + − + + + + F Indoxyl acetate hydrolysis + + − − − − − + M − NA F − Urease − − − − − − − − − − − V§ − Alkaline phosphatase NA − − − F NA NA − − + NA − M Hippurate hydrolysis M − − − − − − + M − − − − Growth on 0.1% selenite NA − − + + NA + − M NA NA V F Growth on 2 % NaCl _ F NA − − NA − − − NA + M M Growth on 1 % glycine + V + + V + + F M − + + V Growth on 0.04 % TTC + − − F − NA + V M NA NA M − TTC reduction NA − − F − NA + V M NA NA M −

Resistance to: Nalidixic acid (30 mg) V − V + + + + − − + − V M Cephalothin (30 mg) M − − F − − + − M + + + F

DNA G+C content (mol%) 28 34 32-33 35-36 31-33 36 28 31 30-31 36 30 29-30 36-38

Continued. 27 28 29 30 31 32 33 34 35 36 37 <30>, 37, Growth temperature range (°C) 37–42 37–42 37 37 (30), 37, 37–42 37–42 <42> 30–37,[42] [42] 30–37,[42] 30–37,[42] ANO2, ANO2, mO2, Atmospheric requirements mO2,ANO2 mO2 mO2,ANO2 mO2,ANO2 mO2,ANO2 mO2,ANO2 mO2 ANO2, [HmO2] [HmO2] [HmO2] ANO2∗

Oxidase − + + + + V + + + + + Catalase + + + − F V V† + − F + Nitrate reduction V NA + + + + M + + + + Indoxyl acetate hydrolysis − NA − − + V − − + F − Urease + NA − − − − V‡ − − + − Alkaline phosphatase − NA NA NA − − − NA − − − Hippurate hydrolysis − − − − − − − − − − − Growth on 0.1% selenite NA NA NA NA − − V − + − + Growth on 2 % NaCl NA M NA NA V + + + − + − Growth on 1 % glycine + + − − + V + M + + − Growth on 0.04 % TTC NA NA NA NA − − − NA V − − TTC reduction F NA NA NA − − − NA V − −

Resistance to: Nalidixic acid (30 mg) NA M − − M − M + − − + Cephalothin (30 mg) NA F − − − − − − F − +

DNA G+C content (mol%) 30 29 30 31 45-46 44-46 29-33 30 32-36 28-30 29

Table S2. Genomic features of the nine Campylobacter armoricus sp. nov. isolates. N50 is the median contig size compared to the whole sequence. DDBJ/ENA/GenBank Contigs Total length (bp) G+C (%) N50 (bp) CDS tRNA / rRNA accession CA656T 57 1,588,860 28.51 130,767 1588 38 / 3 SBEZ00000000 CA592 84 1,664,047 28.34 83,047 1644 43 / 3 SBFC00000000 CA594 85 1,662,534 28.34 94,839 1641 43 / 3 SBFB00000000 CA639 90 1,705,878 28.38 118,615 1698 42 / 3 SBFA00000000 CA658 51 1,590,434 28.51 130,760 1593 43 / 3 SBEY00000000 CA663 64 1,587,867 28.52 125,241 1584 43 / 3 SBEX00000000 CA1650 111 1,759,748 28.39 75,585 1767 41 / 3 SBEW00000000 CA1665 93 1,714,900 28.43 106,852 1720 41 / 3 SBEV00000000 CA1670 85 1,698,680 28.34 118,193 1689 40 / 3 SBEU00000000

Table S3. Average nucleotide identity based on BLAST (ANIb) values (%) for the nine Campylobacter armoricus sp. nov., strains and other Campylobacter species and distant taxa (Wolinella, Helicobacter and Arcobacter). Strains: 1, Campylobacter armoricus CA656T; 2, Campylobacter armoricus CA592; 3, Campylobacter armoricus CA594; 4, Campylobacter armoricus CA639; 5, Campylobacter armoricus CA658; 6, Campylobacter armoricus CA663; 7, Campylobacter armoricus CA1650; 8, Campylobacter armoricus CA1665; 9, Campylobacter armoricus CA1670; 10, Campylobacter avium LMG 24591T; 11, Campylobacter blaseri LMG 30333T; 12, Campylobacter coli ATCC 33559T; 13, Campylobacter concisus ATCC 33237T; 14, Campylobacter corcagiensis LMG 27932T; 15, Campylobacter cuniculorum LMG 24588T; 16, Campylobacter curvus ATCC 35224T; 17, Campylobacter fetus subsp. fetus ATCC 27374T; 18, Campylobacter fetus subsp. testudinum ATCC BAA-2539T; 19, Campylobacter geochelonis LMG 29375T; 20, Campylobacter gracilis ATCC 33236T; 21, Campylobacter helveticus ATCC 51209T; 22, Campylobacter hepaticus NCTC 13823T; 23, Campylobacter hominis ATCC BAA-381T; 24, Campylobacter hyointestinalis subsp. hyointestinalis ATCC 35217T; 25, Campylobacter hyointestinalis subsp. lawsonii LMG 14432T; 26, Campylobacter iguaniorum LMG 28143T; 27, Campylobacter jejuni subsp. doylei LMG 8843T; 28, Campylobacter jejuni subsp. jejuni ATCC 33560T; 29, Campylobacter lanienae NCTC 13004T; 30, Campylobacter mucosalis ATCC 43264T; 31, Campylobacter pinnipediorum subsp. caledonicus LMG 29473T; 32, Campylobacter pinnipediorum subsp. pinnipediorum LMG 29472T; 33, Campylobacter rectus ATCC 33238T; 34, Campylobacter showae ATCC 51146T; 35, Campylobacter sputorum bv. sputorum RM3237; 36, Campylobacter upsaliensis ATCC 43954T; 37, Campylobacter ureolyticus DSM 20703T; 38, Helicobacter pylori ATCC 43504T; 39, Sulfurimonas denitrificans ATCC 33889T; 40, Sulfurospirillum barnesii ATCC 700032T; 41, Wolinella succinogenes ATCC 29543T; 42, Arcobacter butzleri ATCC 49616T. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 1 *** 99.0 98.9 99.1 99.9 99.9 99.0 98.9 99.0 72.3 71.6 74.9 70.8 71.0 73.7 69.2 71.0 71.4 70.8 69.8 73.4 75.1 70.9 71.5 71.4 71.2 75.3 74.9 71.9 70.4 71.9 71.9 69.1 69.4 71.6 73.3 71.4 69.5 70.1 70.4 69.8 71.2 2 99.0 *** 99.9 98.9 99.0 99.0 99.1 98.9 99.0 72.3 71.6 75.0 70.8 71.1 73.9 69.3 71.1 71.5 70.8 69.8 73.4 75.1 70.9 71.5 71.4 71.2 75.3 75.1 71.8 70.5 71.9 72.0 69.3 69.4 71.7 73.3 71.3 69.4 70.1 70.3 70.0 71.2 3 98.9 99.9 *** 98.8 99.0 99.0 99.1 98.9 98.9 72.3 71.6 75.0 70.8 71.1 73.9 69.3 71.1 71.4 70.8 69.8 73.4 75.1 70.9 71.5 71.4 71.2 75.3 75.1 71.8 70.5 71.9 72.0 69.3 69.4 71.7 73.3 71.3 69.4 70.1 70.3 70.0 71.2 4 99.1 98.9 98.8 *** 99.1 99.1 98.8 98.9 99.0 72.3 71.6 75.3 70.8 71.0 73.9 69.2 71.1 71.5 70.9 69.9 73.4 75.1 70.8 71.5 71.3 71.2 75.3 75.1 71.8 70.4 71.9 71.9 69.2 69.5 71.7 73.3 71.4 69.5 70.1 70.4 70.0 71.3 5 99.9 99.0 99.0 99.1 *** 99.9 99.0 98.9 99.0 72.3 71.6 74.9 70.8 71.0 73.7 69.2 71.0 71.4 70.8 69.8 73.4 75.1 70.9 71.5 71.4 71.2 75.3 74.9 71.9 70.4 71.9 71.9 69.1 69.5 71.6 73.3 71.4 69.5 70.1 70.4 69.8 71.2 6 99.9 99.0 99.0 99.1 99.9 *** 99.0 99.0 99.0 72.3 71.6 74.9 70.8 71.0 73.7 69.2 71.0 71.4 70.8 69.8 73.4 75.1 70.9 71.5 71.4 71.2 75.3 74.9 71.9 70.4 71.9 71.9 69.1 69.5 71.6 73.3 71.4 69.5 70.1 70.4 69.8 71.2 7 99.0 99.1 99.1 98.8 99.0 99.0 *** 98.9 99.0 72.3 71.6 75.0 70.8 71.0 73.9 69.2 71.1 71.5 70.9 69.8 73.4 75.1 70.9 71.5 71.4 71.1 75.4 75.2 71.8 70.5 71.9 71.9 69.4 69.4 71.7 73.4 71.3 69.6 70.0 70.3 70.1 71.2 8 98.9 98.9 98.9 98.9 98.9 99.0 98.9 *** 99.9 72.3 71.6 75.3 70.8 71.0 73.7 69.3 71.1 71.4 70.8 69.9 73.3 75.1 71.0 71.5 71.4 71.2 75.3 75.0 71.7 70.5 71.9 71.8 69.3 69.3 71.7 73.4 71.3 69.2 70.0 70.2 69.8 71.3 9 99.0 99.0 98.9 99.0 99.0 99.0 99.0 99.9 *** 72.3 71.6 75,0 70.8 71.0 73.8 69.3 71.1 71.4 70.8 70.0 73.4 75.2 70.9 71.5 71.4 71.2 75.3 75.0 71.8 70.5 72.0 71.9 69.3 69.4 71.7 73.4 71.3 69.4 70.1 70.4 70.1 71.3 10 72.3 72.3 72.3 72.3 72.3 72.3 72.3 72.3 72.3 *** 70.7 72.2 70.7 70.6 72.2 69.7 70.5 71.3 70.3 70.0 72.3 72.2 70.3 71.1 70.8 71.0 72.5 72.2 71.2 70.4 71.5 71.3 69.9 69.9 70.9 72.3 70.7 68.9 69.4 69.7 69.8 70.2 11 71.6 71.6 71.6 71.6 71.6 71.6 71.6 71.6 71.6 70.7 *** 71.0 70.6 72.9 70.9 69.1 71.4 71.7 73.7 69.2 70.9 71.3 71.4 71.7 71.7 71.9 71.5 71.0 71.6 70.8 72.7 73.8 69.2 69.0 72.9 71.1 73.6 69.3 70.1 69.9 69.6 71.2 12 74.9 75.0 75.0 75.3 74.9 74.9 75.0 75.3 75,0 72.2 71.0 *** 71.0 70.9 76.4 69.4 70.8 71.1 70.8 70.1 75.5 80.9 71.0 71.4 71.4 71.3 84.4 84.4 71.8 70.5 71.7 71.5 69.4 69.7 71.2 75.5 71.0 69.8 70,0 70.3 70.0 71.1 13 70.8 70.8 70.8 70.8 70.8 70.8 70.8 70.8 70.8 70.7 70.6 71.0 *** 70.7 70.0 74.8 71.2 71.8 71.6 71.4 70.7 70.4 70.8 71.7 71.6 72.0 70.8 70.2 71.7 73.2 72.5 72.6 73.4 74.1 70.9 70.6 70.5 69.8 69.8 69.9 69.7 70.3 14 71.0 71.1 71.1 71.0 71.0 71.0 71.0 71.0 71.0 70.6 72.9 70.9 70.7 *** 70.5 69.3 71.0 71.6 72.6 69.6 70.9 71.0 71.6 71.7 71.7 72.0 71.2 70.8 71.6 70.7 71.7 72.0 69.3 69.3 72.9 70.9 74.1 69.3 70.2 69.8 69.5 70.6 15 73.7 73.9 73.9 73.9 73.7 73.7 73.9 73.7 73.8 72.2 70.9 76.4 70.0 70.5 *** 69.0 70.4 70.6 70.0 69.6 75.1 76.1 70.3 70.7 70.5 70.5 76.5 76.9 70.9 70.1 70.9 70.8 69.1 69.0 70.8 75.0 70.5 69.3 69.2 70.0 69.1 70.2 16 69.2 69.3 69.3 69.2 69.2 69.2 69.2 69.3 69.3 69.7 69.1 69.4 74.8 69.3 69.0 *** 70.5 70.7 70.5 72.7 70.2 69.6 69.8 70.7 70.9 71.1 70.1 69.5 70.9 72.5 71.0 70.9 74.3 74.4 69.8 70.6 69.1 69.8 69.3 68.9 69.4 70.2 17 71.0 71.1 71.1 71.1 71.0 71.0 71.1 71.1 71.1 70.5 71.4 70.8 71.2 71.0 70.4 70.5 *** 91.9 71.8 70.3 71.1 71.0 71.4 79.1 78.6 76.2 70.9 70.8 73.9 71.0 71.8 71.7 70.2 70.4 71.8 70.8 71.1 68.7 69.5 69.5 69.2 69.9 18 71.4 71.5 71.4 71.5 71.4 71.4 71.5 71.4 71.4 71.3 71.7 71.1 71.8 71.6 70.6 70.7 91.9 *** 72.0 70.3 71.0 71.1 71.5 80.4 79.4 76.7 71.1 70.9 74.2 71.2 72.0 72.1 70.2 70.6 72.0 70.9 71.4 69.4 69.8 69.8 69.8 70.3 19 70.8 70.8 70.8 70.9 70.8 70.8 70.9 70.8 70.8 70.3 73.7 70.8 71.6 72.6 70.0 70.5 71.8 72.0 *** 70.7 71.3 71.0 71.8 72.6 72.3 72.9 71.2 71.0 72.1 71.4 72.2 72.1 70.1 70.5 72.6 71.0 72.9 69.4 70.0 70.1 69.7 70.5 20 69.8 69.8 69.8 69.9 69.8 69.8 69.8 69.9 70.0 70.0 69.2 70.1 71.4 69.6 69.6 72.7 70.3 70.3 70.7 *** 70.5 69.5 70.6 70.4 70.2 70.5 70.1 69.0 70.4 70.3 69.9 69.5 74.9 75.1 69.6 70.3 68.6 69.6 69.7 68.7 69.6 70.8 21 73.4 73.4 73.4 73.4 73.4 73.4 73.4 73.3 73.4 72.3 70.9 75.5 70.7 70.9 75.1 70.2 71.1 71.0 71.3 70.5 *** 74.8 70.6 70.7 70.6 70.8 75.6 75.6 71.3 70.6 70.9 70.9 69.9 69.7 70.8 85.4 70.5 69.5 69.8 69.8 69.7 70.5 22 75.1 75.1 75.1 75.1 75.1 75.1 75.1 75.1 75.2 72.2 71.3 80.9 70.4 71.0 76.1 69.6 71.0 71.1 71.0 69.5 74.8 *** 70.8 71.1 71.1 71.0 84.0 84.1 71.4 70.0 71.4 71.2 69.0 69.3 71.3 74.7 70.9 69.1 69.9 70.2 69.6 70.8 23 70.9 70.9 70.9 70.8 70.9 70.9 70.9 71.0 70.9 70.3 71.4 71.0 70.8 71.6 70.3 69.8 71.4 71.5 71.8 70.6 70.6 70.8 *** 71.6 71.2 71.5 70.9 70.8 71.0 70.4 71.2 71.5 69.7 69.8 72.1 71.1 72.6 69.6 69.7 69.6 69.6 70.3 24 71.5 71.5 71.5 71.5 71.5 71.5 71.5 71.5 71.5 71.1 71.7 71.4 71.7 71.7 70.7 70.7 79.1 80.4 72.6 70.4 70.7 71.1 71.6 *** 94.6 76.6 71.1 70.8 74.4 71.2 71.8 71.8 70.4 70.8 71.9 71.1 71.3 69.3 70.1 70.0 69.8 70.4 25 71.4 71.4 71.4 71.3 71.4 71.4 71.4 71.4 71.4 70.8 71.7 71.4 71.6 71.7 70.5 70.9 78.6 79.4 72.3 70.2 70.6 71.1 71.2 94.6 *** 76.6 71.1 70.9 76.5 71.4 72.0 71.9 70.4 70.5 72.1 71.2 71.2 69.1 69.8 69.6 69.2 70.4 26 71.2 71.2 71.2 71.2 71.2 71.2 71.1 71.2 71.2 71.0 71.9 71.3 72.0 72.0 70.5 71.1 76.2 76.7 72.9 70.5 70.8 71.0 71.5 76.6 76.6 *** 71.1 70.8 74.1 71.4 71.9 71.8 70.5 70.7 71.7 71.0 71.2 69.6 70.0 70.0 70.1 70.6 27 75.3 75.3 75.3 75.3 75.3 75.3 75.4 75.3 75.3 72.5 71.5 84.4 70.8 71.2 76.5 70.1 70.9 71.1 71.2 70.1 75.6 84.0 70.9 71.1 71.1 71.1 *** 96.1 71.8 70.5 71.5 71.5 69.5 69.6 71.3 75.7 71.0 69.3 70.0 70.5 69.8 70.6 28 74.9 75.1 75.1 75.1 74.9 74.9 75.2 75.0 75.0 72.2 71.0 84.4 70.2 70.8 76.9 69.5 70.8 70.9 71.0 69.0 75.6 84.1 70.8 70.8 70.9 70.8 96.1 *** 71.8 70.4 71.6 71.5 69.4 69.6 71.3 75.6 71.0 69.7 70.2 70.5 69.7 70.7 29 71.9 71.8 71.8 71.8 71.9 71.9 71.8 71.7 71.8 71.2 71.6 71.8 71.7 71.6 70.9 70.9 73.9 74.2 72.1 70.4 71.3 71.4 71.0 74.4 76.5 74.1 71.8 71.8 *** 71.5 71.9 71.7 70.2 70.6 71.7 71.5 71.2 69.7 69.8 69.9 69.9 70.5 30 70.4 70.5 70.5 70.4 70.4 70.4 70.5 70.5 70.5 70.4 70.8 70.5 73.2 70.7 70.1 72.5 71.0 71.2 71.4 70.3 70.6 70.0 70.4 71.2 71.4 71.4 70.5 70.4 71.5 *** 73.3 73.3 71.6 72.0 71.0 71.2 70.5 69.9 69.7 70.1 69.5 70.1 31 71.9 71.9 71.9 71.9 71.9 71.9 71.9 71.9 72.0 71.5 72.7 71.7 72.5 71.7 70.9 71.0 71.8 72.0 72.2 69.9 70.9 71.4 71.2 71.8 72.0 71.9 71.5 71.6 71.9 73.3 *** 94.4 69.8 70.2 72.2 70.9 71.7 69.3 70.2 70.3 69.5 70.7 32 71.9 72.0 72.0 71.9 71.9 71.9 71.9 71.8 71.9 71.3 73.8 71.5 72.6 72.0 70.8 70.9 71.7 72.1 72.1 69.5 70.9 71.2 71.5 71.8 71.9 71.8 71.5 71.5 71.7 73.3 94.4 *** 69.9 70.2 72.2 71,0 71.8 69.1 70.2 70.3 69.4 70.7 33 69.1 69.3 69.3 69.2 69.1 69.1 69.4 69.3 69.3 69.9 69.2 69.4 73.4 69.3 69.1 74.3 70.2 70.2 70.1 74.9 69.9 69.0 69.7 70.4 70.4 70.5 69.5 69.4 70.2 71.6 69.8 69.9 *** 89.1 69.4 70.2 68.9 69.4 68.8 68.7 69.6 70.4 34 69.4 69.4 69.4 69.5 69.5 69.5 69.4 69.3 69.4 69.9 69.0 69.7 74.1 69.3 69.0 74.4 70.4 70.6 70.5 75.1 69.7 69.3 69.8 70.8 70.5 70.7 69.6 69.6 70.6 72.0 70.2 70.2 89.1 *** 69.5 70.6 69.1 70.0 69.8 69.1 70.0 71.1 35 71.6 71.7 71.7 71.7 71.6 71.6 71.7 71.7 71.7 70.9 72.9 71.2 70.9 72.9 70.8 69.8 71.8 72.0 72.6 69.6 70.8 71.3 72.1 71.9 72.1 71.7 71.3 71.3 71.7 71.0 72.2 72.2 69.4 69.5 *** 70.8 73.0 69.6 69.8 69.6 69.4 70.5 36 73.3 73.3 73.3 73.3 73.3 73.3 73.4 73.4 73.4 72.3 71.1 75.5 70.6 70.9 75.0 70.6 70.8 70.9 71.0 70.3 85.4 74.7 71.1 71.1 71.2 71.0 75.7 75.6 71.5 71.2 70.9 71,0 70.2 70.6 70.8 *** 70.5 69.6 69.6 69.9 69.5 70.3 37 71.4 71.3 71.3 71.4 71.4 71.4 71.3 71.3 71.3 70.7 73.6 71.0 70.5 74.1 70.5 69.1 71.1 71.4 72.9 68.6 70.5 70.9 72.6 71.3 71.2 71.2 71.0 71.0 71.2 70.5 71.7 71.8 68.9 69.1 73.0 70.5 *** 69.0 69.9 70.0 69.6 70.8 38 69.5 69.4 69.4 69.5 69.5 69.5 69.6 69.2 69.4 68.9 69.3 69.8 69.8 69.3 69.3 69.8 68.7 69.4 69.4 69.6 69.5 69.1 69.6 69.3 69.1 69.6 69.3 69.7 69.7 69.9 69.3 69.1 69.4 70.0 69.6 69.6 69.0 *** 70.2 69.4 70.4 70.9 39 70.1 70.1 70.1 70.1 70.1 70.1 70.0 70.0 70.1 69.4 70.1 70,0 69.8 70.2 69.2 69.3 69.5 69.8 70.0 69.7 69.8 69.9 69.7 70.1 69.8 70.0 70.0 70.2 69.8 69.7 70.2 70.2 68.8 69.8 69.8 69.6 69.9 70.2 *** 69.5 69.0 70.9 40 70.4 70.3 70.3 70.4 70.4 70.4 70.3 70.2 70.4 69.7 69.9 70.3 69.9 69.8 70.0 68.9 69.5 69.8 70.1 68.7 69.8 70.2 69.6 70.0 69.6 70.0 70.5 70.5 69.9 70.1 70.3 70.3 68.7 69.1 69.6 69.9 70.0 69.4 69.5 *** 69.8 70.1 41 69.8 70.0 70.0 70.0 69.8 69.8 70.1 69.8 70.1 69.8 69.6 70.0 69.7 69.5 69.1 69.4 69.2 69.8 69.7 69.6 69.7 69.6 69.6 69.8 69.2 70.1 69.8 69.7 69.9 69.5 69.5 69.4 69.6 70.0 69.4 69.5 69.6 70.4 69.0 69.8 *** 71.1 42 71.2 71.2 71.2 71.3 71.2 71.2 71.2 71.3 71.3 70.2 71.2 71.1 70.3 70.6 70.2 70.2 69.9 70.3 70.5 70.8 70.5 70.8 70.3 70.4 70.4 70.6 70.6 70.7 70.5 70.1 70.7 70.7 70.4 71.1 70.5 70.3 70.8 70.9 70.9 70.1 71.1 *** Table S4. Pairwise in silico DNA-DNA hybridization (isDDH) comparison of the nine Campylobacter armoricus strains and strains representing other Campylobacter and epsilonproteobacterial taxa, using GGDC formula 2 (isDDH estimates based on identities/HSP length*) CA656 CA59 CA59 CA63 CA65 CA66 CA165 CA166 CA167 T 2 4 9 8 3 0 5 0 Campylobacter armoricus CA656T *** 91 91 92 99.8 100 91.5 90.8 91.6 Campylobacter armoricus CA592 91 *** 100 90.3 91.1 91 93.6 90 90.6 Campylobacter armoricus CA594 91 100 *** 90.3 91.1 91 93.5 90 90.6 Campylobacter armoricus CA639 92 90.3 90.3 *** 92 92 90.4 90.7 91.2 Campylobacter armoricus CA658 99.8 91.1 91.1 92 *** 99.9 91.3 90.8 91.5 Campylobacter armoricus CA663 100 91 91 92 99.9 *** 91.5 90.9 91.7 Campylobacter armoricus CA1650 91.5 93.6 93.5 90.4 91.3 91.5 *** 90.1 90.9 Campylobacter armoricus CA1665 90.8 90 90 90.7 90.8 90.9 90.1 *** 99.2 Campylobacter armoricus CA1670 91.6 90.6 90.6 91.2 91.5 91.7 90.9 99.2 *** Campylobacter avium LMG 24591T 18 18 18 18.1 18.1 18.1 18 18.1 18.1 Campylobacter blaseri LMG 30333T 18.9 18.4 18.4 18.4 18.9 18.9 18.4 18.8 18.8 Campylobacter coli ATCC 33559T 18.6 18.7 18.7 19.4 18.6 18.6 18.9 19.2 18.6 Campylobacter concisus ATCC 33237T 19.7 20.5 20.5 20.4 19.7 19.7 20.2 19.6 19.6 Campylobacter corcagiensis LMG 27932T 18.6 18.5 18.5 18.2 18.6 18.6 18.6 18.6 18.5 Campylobacter cuniculorum LMG 24588T 17.7 17.7 17.7 18 17.7 17.7 17.7 17.5 17.6 Campylobacter curvus ATCC 35224T 19 19 19 19 19 19 19 19 19 Campylobacter fetus subsp. fetus ATCC 27374T 20.8 21.2 21.2 20.8 20.8 20.8 20.8 21.2 21.1 Campylobacter fetus subsp. testudinum ATCC BAA- 20.3 20.2 20.2 21 20.3 20.3 20.3 20.8 20.7 2539T Campylobacter geochelonis LMG 29375T 18.4 18.1 18.1 18.4 18.5 18.5 18.1 18.2 18.1 Campylobacter gracilis ATCC 33236T 19.8 19.8 19.8 19.8 19.8 19.8 19.8 19.9 19.8 Campylobacter helveticus ATCC 51209T 18.8 18.7 18.7 19 18.9 18.9 18.8 18.8 18.8 Campylobacter hepaticus NCTC 13823T 18.6 18.5 18.5 18.7 18.6 18.6 18.5 18.8 18.7 Campylobacter hominis ATCC BAA-381T 18.8 19.3 19.3 18.8 18.9 18.9 18.8 19.3 19.2 Continued.

CA656T CA592 CA594 CA639 CA658 CA663 CA1650 CA1665 CA1670 Campylobacter hyointestinalis subsp. hyointestinalis ATCC 21.2 21.2 21.2 21.2 21.2 21.2 21.2 21.3 21.2 35217T Campylobacter hyointestinalis subsp. lawsonii LMG 14432T 20.8 20.8 20.8 21.1 20.8 20.8 20.8 20.8 20.8 Campylobacter iguaniorum LMG 28143T 22.4 21.1 21.1 21.2 22.4 22.4 21.7 21.6 21.7 Campylobacter insulaenigrae NCTC 12927T 23.6 23.6 23.6 23.5 23.6 23.6 23.6 23.8 23.6 Campylobacter jejuni subsp. doylei LMG 8843T 18.8 18.5 18.5 18.6 18.8 18.8 18.5 18.7 18.7 Campylobacter jejuni subsp. jejuni ATCC 33560T 18.5 18.7 18.7 18.8 18.5 18.5 18.7 18.5 18.5 Campylobacter lanienae NCTC 13004T 20.2 20.4 20.4 20.5 20.2 20.2 20.1 21.5 20.1 Campylobacter lari subsp. concheus LMG 11760 35.2 35 35 35.3 35.2 35.3 35.1 35.1 35.1 Campylobacter lari subsp. lari ATCC 35221T 33.2 33.3 33.4 33.5 33.2 33.3 33.4 33.4 33.3 Campylobacter mucosalis ATCC 43264T 20.5 20.5 20.5 20.4 20.5 20.5 20.5 20.5 20.5 Campylobacter ornithocola WBE38T 35.9 36.4 36.4 36.4 35.9 35.9 36.2 36.6 36.3 Campylobacter peloridis LMG 23910T 30.4 31.2 31.1 31.1 30.4 30.4 31.2 30.7 30.8 Campylobacter pinnipediorum subsp. caledonicus LMG 20.6 20.9 20.9 20.1 20.6 20.6 20.8 20.9 20.8 29473T Campylobacter pinnipediorum subsp. pinnipediorum LMG 22.6 21.7 21.7 21.1 22.6 22.6 21.6 21.8 21.7 29472T Campylobacter rectus ATCC 33238T 19.7 19.7 19.7 19.7 19.7 19.7 30.4 19.8 19.7 Campylobacter showae ATCC 51146T 19.7 19.7 19.7 19.6 19.7 19.7 19.8 19.8 19.6 Campylobacter sputorum bv. sputorum RM3237 19.1 19.1 19.1 19 19.1 19.1 18.7 19.3 19.4 Campylobacter subantarcticus LMG 24377T 31.3 31.7 31.7 31.7 31.3 31.4 32 31.7 31.8 Campylobacter upsaliensis ATCC 43954T 19.7 19.8 19.8 19.8 19.7 19.7 19.8 19.9 19.9 Campylobacter ureolyticus DSM 20703T 18.9 18.8 18.8 18.7 18.9 18.9 18.9 18.9 18.9 Campylobacter volucris LMG 24379 25.8 25.8 25.8 25.7 25.8 25.8 25.8 25.9 25.7 Continued.

CA656T CA592 CA594 CA639 CA658 CA663 CA1650 CA1665 CA1670 Helicobacter pylori ATCC 43504T 25 25 25 25 25 25 25 25.1 25 Sulfurimonas denitrificans ATCC 24.1 24.1 24.1 24.1 24.1 24.1 24.1 25.5 25.4 33889T Sulfurospirillum barnesii ATCC 23.5 24.1 24.1 23.2 23.2 23.2 24.2 24.3 24 700032T Wolinella succinogenes ATCC 29543T 22.4 22.5 22.5 22.7 22.5 22.5 22.5 22.6 22.5 Arcobacter butzleri ATCC 49616T 21.8 21 21 22.5 21.8 21.8 22.5 24.6 25

GGDC: Genome-to-Genome Distance Calculator (v. 2.1; http://ggdc.dsmz.de/distcalc2.php); HSP: high-scoring segment pairs. *Confidence intervals indicate inherent uncertainty in estimating DDH values from intergenomic distances based on models derived from empirical test data sets (which are always limited in size). These results are in accordance with phylogenomic analyses as well as the ANI results.