Isolation and Genomic Characteristics of Cat-Borne Campylobacter felis sp. nov. and Sheep-Borne Campylobacter ovis sp. nov

Nine novel bacterial strains were isolated from the feces of cats and sheep in 2019 and 2020 in Beijing, China. Cells were 1–3 μm long and ≤0.5 μm wide, Gram-stain negative, microaerobic, motile, oxidase positive, and urease negative. Phylogenetic analyses based on 16S rRNA gene sequences indicated that these nine isolates belong to the genus Campylobacter but formed two robust clades that were clearly separate from the currently recognized species and, respectively, isolated from the cat and sheep. Both these strains shared low 16S rRNA gene sequence similarity, dDDH relatedness, and ANI values with their closest species C. upsaliensis CCUG 14913T and C. lanienae NCTC 13004T, and against each other, which are below the cut-off values generally recognized for isolates of the same species. The genomic DNA G + C contents of type strains XJK22-1T and SYS25-1T were 34.99 mol% and 32.43 mol%, respectively. Electron microscopy showed that these cells were spiral shaped, with bipolar single flagella. Based on results from genotypic, phenotypic, phylogenetic, and phylogenomic analyses, these nine strains represent two novel species within the genus Campylobacter, for which the names Campylobacter felis sp. nov. (Type strain XJK22-1T = GDMCC 1.3684T = JCM 35847T) and Campylobacter ovis sp. nov. (Type strain SYS25-1T = GDMCC 1.3685T) are proposed.


Introduction
The genus Campylobacter belongs to the family Campylobacteraceae and the order Campylobacterales, which currently contains 37 and 13 validly described species and subspecies, respectively, and 2 not validly described species (https://lpsn.dsmz.de/genus/ campylobacter) (accessed on 15 February 2023). Members of the Campylobacter genus are morphologically diverse, and can be spiral, curved, or rod shaped. These bacteria are nutritionally fastidious and grow under strictly anaerobic or microaerobic conditions. They naturally colonize humans, other mammals, birds, reptiles, shellfish, etc. [1][2][3].
Campylobacter is among the four main causes of gastroenteritis worldwide [4]. Most reported Campylobacter infections are caused by C. jejuni, which is a leading cause of bacterial gastroenteritis in humans worldwide [2] and whose antecedent infection could trigger a Guillain-Barré Syndrome (GBS) outbreak [5], and, to a lesser extent, C. coli, which accounts for 1-25% of all Campylobacter-related diarrheal diseases [2]. However, the other emerging Campylobacter pathogens are gaining increasing recognition as important pathogens in humans and animals [6]. In addition, with the continuous progress of isolation and culture technology, more and more novel Campylobacter species have been identified in recent years [7][8][9][10][11][12][13][14]. Domestic animals, especially pets, have close contact with humans.

Morphological, Physiological and Biochemical Characteristics
For the study of the morphological and biochemical characteristics, cells were cultivated and harvested in the late-exponential growth phase. Gram staining was conducted using a Gram-staining kit (Baso, Zhuhai, China) [23] and observed under a light microscope. The morphological characteristics of these two putative novel species' type strains were determined using transmission electron microscopy. Fresh cells were gently suspended in 0.1 M phosphate-buffered saline (PBS) to an OD600 of 1 and collected via gentle centrifugation. The pellet was gently resuspended in a 2% (v/v) glutaraldehyde solution for fixation. Fixation was completed by incubating strains for 1 h on the grid. All samples were stained with 2% (w/v) uranyl acetate for 1 min and examined using a Hitachi H7700 transmission electron microscope (Eclipse Ci-L, NIKON, Tokyo, Japan) at 80 kV.
The catalase activity was evaluated using a 3% (v/v) H 2 O 2 solution for bubble production. The general phenotypic traits of Campylobacter spp., oxidase, catalase, hydrolysis of hippurate, and indoxyl acetate were evaluated using the Campylobacter Biochemical Assay kit (ZC-CAMPY-010, Qingdao Sinova Biotechnology Co., Ltd., Qingdao, China). Further biochemical characteristics were obtained using the identification system of API Campy, strictly following the manufacturers' instructions (bio-Mérieux, Lyon, France). Biochemical tests were carried out to characterize the physiology and chemotaxonomy of the isolates. C. jejuni ATCC 33560 T , C. coli ATCC 33559 T , and C. upsaliensis CCUG 14913 T were used as controls.

Antimicrobial Susceptibility Testing
The minimum inhibitory concentrations (MICs) for 11 antimicrobials (erythromycin, azithromycin, nalidixic acid, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, florfenicol, tetracycline, telithromycin, and clindamycin) at concentrations ranging from 0.02 to 256 µg mL −1 were determined for all isolates using the agar dilution method (ZC-AST-001, Qingdao Sinova Biotechnology Co., Ltd., Qingdao, China) and the gradient strip diffusion method (E-test, bio Mérieux, Nürtingen, Germany) following the manufacturer's instructions, as previously reported [24,25]. The MIC was read as the lowest concentration without visible growth. Type strain C. jejuni ATCC 33560 T was used as a control.

Species-Specific PCR
To define a diagnostic method for the rapid detection and identification of these two putative new species, specific TaqMan real-time PCR primers targeting different genes were designed. The exact primer and probe sequences are listed in Table 1. The PCR conditions were as follows: initial denaturation at 94 • C for 30 s, followed by 40 cycles of 94 • C for 5 s and 60 • C for 30 s. The positive and negative results refer to the cycle threshold (Ct) value of the real-time PCR that, according to the manual, Ct < 35 with the typical S curve, is determined as positive; Ct > 40 or no typical S curve is determined as negative, and 35 ≤ Ct ≥ 40 should be repeated 3 times and determined as weakly positive after the third repeat. To assess the sensitivity and specificity of this qPCR method, some species of Campylobacter strains which were stored in our laboratory, including C. coli, C. concisus, C. fetus subsp. fetus, C. gracilis, C. helveticus, C. hyointestinalis subsp. hyointestinalis, C. jejuni subsp. doylei, C. jejuni subsp. jejuni, C. lari subsp. lari, C. mucosalis, C. rectus, C. showae, and C. upsaliensis, were used as reference strains. Several kinds of other genus bacteria stored in our laboratory were used as negative controls, such as Arcobacter butzleri, Arcobacter skirrowii, Helicobacter pylori, Escherichia coli, etc.

Genome Extraction and Sequencing
After culturing, the DNA for genome sequence was extracted using the QIAamp DNA Mini Kit (Qiagen, German) according to the manufacturer's instructions for sequencing. Then, the NanoDrop 2000 spectrophotometer (Thermo Fisher, MA, USA) was used to measure the concentration and purity of DNAs. The quality requirements were a concentration ≥ 20 ng/µL and a total amount > 2 µg. The purity requirement was as follows: OD260/OD280 value should be between 1.6 and 1.8. The DNA sequencing was performed by an Illumina PE150 platform (Illumina Inc., San Diego, CA, USA) at the Novogene Corporation (Beijing, China) with a depth of 100× coverage. To sequence the genomes, a 350 bp paired-end library was constructed and then 150 bp reads were generated. FastQC v0.11.8 and fastp v0.23.2 were applied to evaluate and improve the quality of the raw sequence data, respectively. Low-quality reads were removed if the quality scores of ≥3 consecutive bases were ≤Q30. The clean reads were assembled by SOAPdenovo v2.40.

Phylogenetic and Phylogenomic Analysis
To determine the phylogenetic positions of strains, 16S rRNA gene PCR amplification was performed with primers 27F and 1492R, as previously reported. Each almost-complete sequence of the 16S rRNA gene PCR product was purified, sub-cloned into the pMD18-T vector for 30 min at 16 • C, transformed into Escherichia coli DH5α, and the inserted 16S rRNA gene fragment was obtained from a single colony after lysis and sequenced. The newly generated 16S rRNA gene sequences were compared with other Campylobacter species by EzBioCloud's identification service to locate their taxonomic position [33]. Multiple sequence alignment of the 16S rRNA gene sequences of the type strains in the genus Campylobacter was performed using the MAFFT 7.471 software [34] and phylogenetic analysis using the software package MEGA X [35], by the neighbor-joining (NJ) [36], maximum parsimony (MP) [37] and maximum likelihood (ML) [38] algorithms with a bootstrap analysis of 1000 replicates [39] and strain Arcobacter butzleri ATCC 49616 T was used as an outgroup.
The protein sequences of core genes of genomes from the isolates and the other Campylobacter species were extracted using the CD-HIT v4.8.1 [40] based on 40% protein sequence similarity and aligned to reconstruct a phylogenomic tree using FastTree v2.1.11 [41]. The multiple sequence alignment of the core genomes of the genus Campylobacter was also performed using the MAFFT software. Then, the phylogenomic tree was visualized by Dendroscope 3.8.3 [42], and modified with Interactive Tree of Life (https://itol.embl.de/) (accessed on 28 February 2023).

Accession Numbers
The GenBank/EMBL/DDBJ accession numbers for the nearly full-length 16S rRNA gene and the draft genome sequences of these 9 isolates were submitted to NCBI (https: //www.ncbi.nlm.nih.gov/) (accessed on 9 August 2022). In addition to these 9 isolates, the other 10 temporarily undefined genomes in NCBI which belonged to Campylobacter ovis sp. nov. and genomes from the other type strains of Campylobacter genus were downloaded from NCBI. There were no genomes found in NCBI belonging to Campylobacter felis sp. nov. More information about the genomes of novel species used in this study was listed in Table 2.
These Gram-negative, microaerobic, motile, spiral-shaped cells with bipolar single flagellum ranged from 1.8 to 2.2 µm for type strain XJK22-1 T and 2.1 to 2.5 µm for type strain SYS25-1 T (Figure 1). Colonies were circular, 2-3 mm in diameter, smooth, and gray after 2 days of growth on Karmali agar with 5% defibrinated sheep blood. The cells appeared coccoid after 5-6 days of incubation or when exposed to air.
These Gram-negative, microaerobic, motile, spiral-shaped cells with bipolar single flagellum ranged from 1.8 to 2.2 μm for type strain XJK22-1 T and 2.1 to 2.5 μm for type strain SYS25-1 T (Figure 1). Colonies were circular, 2-3 mm in diameter, smooth, and gray after 2 days of growth on Karmali agar with 5% defibrinated sheep blood. The cells appeared coccoid after 5-6 days of incubation or when exposed to air. Like most other Campylobacter species, the isolates were positive for oxidase and negative for urease activities. The catalase was negative for species of type strain XJK22-1 T and positive for species of type strain SYS25-1 T . While most Campylobacter species are unable to hydrolyze hippurate and able to hydrolyze indoxyl acetate and reduce nitrate, two-thirds (n = 6, XJK22-1 T , XJK33-1, XJK49-2, XJK56-3, XJK62-3, and XJK7-1) of the new species isolates were able to hydrolyze hippurate and indoxyl acetate positive and could Like most other Campylobacter species, the isolates were positive for oxidase and negative for urease activities. The catalase was negative for species of type strain XJK22-1 T and positive for species of type strain SYS25-1 T . While most Campylobacter species are unable to hydrolyze hippurate and able to hydrolyze indoxyl acetate and reduce nitrate, two-thirds (n = 6, XJK22-1 T , XJK33-1, XJK49-2, XJK56-3, XJK62-3, and XJK7-1) of the new species isolates were able to hydrolyze hippurate and indoxyl acetate positive and could reduce nitrate, and the other one-third (n = 3, S13-1, SYS25-1 T , and SYS28-3) could not hydrolyze hippurate and indoxyl acetated, among which only two isolates could reduce nitrate (n = 2, S13-1 and SYS25-1 T ). There are only five other species of the genus Campylobacter, C. avium, C. curvus, C. jejuni, C. hepaticus, and C. geochelonis that can hydrolyze hippurate [11].
All nine isolates were initially identified as C. upsaliensis and C. lanienae according to the results of the sequencing of the 16S rRNA gene. Thus, it was not unexpected that the results of the standard biochemical of strain XJK22-1 T and SYS25-1 T showed a strong similarity between the composite phenotypic profile observed from the isolates and the phenotypic profile reported previously for C. upsaliensis CCUG 14913 T and C. lanienae NCTC 13004 T , respectively (Table 3). Nevertheless, these six strains (XJK22-1 T , XJK33-1, XJK49-2, XJK56-3, XJK62-3, and XJK7-1) could be unambiguously distinguished from C. upsaliensis by the ability to hydrolyze hippurate, which may challenge the discriminability of C. jejuni to distinguish from other Campylobacter species by hydrolyzing hippurate. Meanwhile, the other three strains (S13-1, SYS25-1 T , and SYS28-3) could be distinguished from C. lanienae by the phenotypic characteristic of positive for GGT (gamma-glutamyltransferase) and negative for reduction of TTC (Triphenyltetrazolium chloride). These partial results preliminarily supported the theory that these nine strains were two novel Campylobacter species.
Based on 40% protein identity, orthologous groups of 332 core genes shared by our 9 isolates and all available genomes of the genus Campylobacter were extracted and used to build a phylogenomic tree (Figure 3).

Genome Characteristics
The draft genome of strain XJK22-1 T (1.70 Mb) was predicted to contain 1747 coding genes and carried 2 rRNA genes and 43 tRNA genes, whereas SYS25-1 T (1.58 Mb) was predicted to contain 1567 coding genes and carried 1 CRISPR/Cas loci, which were obviously different between two type strains, 1 rRNA gene, and 38 tRNA genes. The genomic DNA G + C content of type strain XJK22-1 T was 34.99 mol%, which is slightly higher than the most closely related bacterium, C. upsaliensis CCUG 14913 T (34.73%). Meanwhile, strain SYS25-1 T was 32.43 mol%, which is lower than the most closely related bacterium, C. lanienae NCTC 13004 T (34.60%), and within the range of DNA base compositions previously reported for the members in the genus Campylobacter (29-47 mol% G + C) [44]. This further confirms that these nine strains are two novel Campylobacter species. More genomic characteristics (G + C content, CDS, size, etc.) are listed in Table 4.   Based on 40% protein identity, orthologous groups of 332 core genes shared by our 9 isolates and all available genomes of the genus Campylobacter were extracted and used to build a phylogenomic tree (Figure 3).  The dDDH scores within each strain pair were 73.80-94.00% (XJK22-1 T , XJK33-1, XJK49-2, XJK56-3, XJK62-3, and XJK7-1) and 75.00-99.60% (strains S13-1, SYS25-1 T , SYS28-3, and the other 10 strains), which were well above 70%, the threshold for species demarcation. In contrast, the scores of these 19 strains with their closest species were below 70%. Meanwhile, the ANI values within each strain pair were 96.92-99.26% (strains XJK22-1 T , XJK33-1, XJK49-2, XJK56-3, XJK62-3, and XJK7-1) and 97.09-99.91% (strains S13-1, SYS25-1 T , SYS28-3, and the other 10 strains), in contrast to below 95%, the cutoff for species demarcation, between our isolates and all established species of Campylobacter (Tables 5 and S1). Based on the gold standard for the delineation of bacterial species [45], these results suggested that strains XJK22-1 T and SYS25-1 T represented two novel species of the genus Campylobacter.
In genomes of these strains, numerous Campylobacter virulence-associated genes were detected, which could encode genes related to adherence, colonization and immune evasion, invasion, motility and export apparatus, secretion system, and toxins. The species of type stains of XJK22-1 T and SYS25-1 T had significantly different virulence-associated gene profiles. The species of type stain XJK22-1 T have more Campylobacter virulence-associated genes than the species of SYS25-1 T . The species of XJK22-1 T have the complete cytolethal distending toxin, while the species of type strain SYS25-1 T only have an incomplete type IV secretion system (T4SS). The details of the virulence genes were presented in Figure 4.

Specific Real-Time PCR
These nine strains were simultaneously tested using Taqman real-time PCR to identify the species of Campylobacter. Amplification of six strains, XJK22-1 T , XJK33-1, XJK49-2, XJK56-3, XJK62-3, and XJK7-1, was positive for Campylobacter felis sp. nov. specific qPCR, whereas the other Campylobacter species and blank control were qPCR negative. Similarly, S13-1, SYS25-1 T, and SYS28-3 were positive for Campylobacter ovis sp. nov. specific qPCR, and the other Campylobacter species and blank control were qPCR negative. This suggests that these two specific real-time PCR are robust and can be used to rapidly discriminate between these two novel species and other Campylobacter species strains.
were detected, which could encode genes related to adherence, colonization and immune evasion, invasion, motility and export apparatus, secretion system, and toxins. The species of type stains of XJK22-1 T and SYS25-1 T had significantly different virulenceassociated gene profiles. The species of type stain XJK22-1 T have more Campylobacter virulence-associated genes than the species of SYS25-1 T . The species of XJK22-1 T have the complete cytolethal distending toxin, while the species of type strain SYS25-1 T only have an incomplete type Ⅳ secretion system (T4SS). The details of the virulence genes were presented in Figure 4.

Specific Real-Time PCR
These nine strains were simultaneously tested using Taqman real-time PCR to identify the species of Campylobacter. Amplification of six strains, XJK22-1 T , XJK33-1, XJK49-2, XJK56-3, XJK62-3, and XJK7-1, was positive for Campylobacter felis sp. nov. specific qPCR, whereas the other Campylobacter species and blank control were qPCR negative. Similarly, S13-1, SYS25-1 T, and SYS28-3 were positive for Campylobacter ovis sp. nov. specific qPCR, and the other Campylobacter species and blank control were qPCR negative. This suggests that these two specific real-time PCR are robust and can be used to rapidly discriminate between these two novel species and other Campylobacter species strains.

Conclusions
A polyphasic approach, including DNA sequencing and analysis (16S rRNA and whole-genome sequencing), electron microscopy, and a wide range of biochemical tests, as suggested by On et al. [47], provided sufficient evidence to distinguish these nine isolates from their closest related type strains and to confirm that they represent two novel

Conclusions
A polyphasic approach, including DNA sequencing and analysis (16S rRNA and whole-genome sequencing), electron microscopy, and a wide range of biochemical tests, as suggested by On et al. [47], provided sufficient evidence to distinguish these nine isolates from their closest related type strains and to confirm that they represent two novel species. With XJK22-1 T and SYS25-1 T as the type strains, we suggest the names Campylobacter felis sp. nov. and Campylobacter ovis sp. nov. for the two novel members of the genus Campylobacter. The cells are Gram negative, mesophilic, motile, and spiral shaped with sizes between 1.8-2.2 µm after 48 h of growth on Karmali or Columbia agar with 5% defibrinated sheep blood in a microaerophilic atmosphere at 37 • C. The colonies are wet, flat, gray, circular, and smooth, with sizes between 1.0 and 1.3 mm after 2 days of growth, but they may vary in size and morphology after a long incubation. No hemolysis on blood agar is observed. Fresh cells are motile and have long bipolar single flagella.
These six strains are negative for catalase, urease, GGT, TTC, and pyroglutamylpeptidase I, and cannot produce H 2 S. On the other hand, they are positive for oxidase, reduced nitrate, L-arginine arylamidase, L-aspartic acid arylamidase, and alkaline phosphatase, and all can hydrolyze indoxyl acetate and hippurate. All six strains are sensitive to chloramphenicol, florfenicol, and tetracycline and resistant to erythromycin, azithromycin, nalidixic acid, and streptomycin.
The type strain XJK22-1 T (=GDMCC 1.3684 T = JCM 35847 T ), which was isolated from the feces of cats in 2019 and 2020 in Beijing, China, has a DNA G + C content of 34.99 mol%. The other strains XJK33-1, XJK49-2, XJK56-3, XJK62-3, and XJK7-1 are also classified in this species. The cells are Gram negative, mesophilic, motile, and spiral or S-shaped with sizes between 2.1-2.5 µm after 48 h of growth on Karmali or Columbia agar with 5% defibrinated sheep blood in a microaerophilic atmosphere at 37 • C. The colonies are wet, flat, gray, circular, and smooth, with sizes between 1.0 and 1.3 mm after 2 days of growth, but they may vary in size and morphology after a long incubation period. No hemolysis on blood agar is observed. Fresh cells are motile and have long bipolar single flagella.
These three strains are negative for urease, TTC, pyroglutamyl-peptidase I, and Laspartic acid arylamidase and cannot hydrolyze indoxyl acetate and hippurate or produce H 2 S. On the other hand, they are positive for catalase, oxidase, GGT, L-arginine arylamidase, and alkaline phosphatase and are variable for the reduction of nitrate. All three strains are resistant to nalidixic acid and sensitive to erythromycin, azithromycin, gentamicin, streptomycin, chloramphenicol, florfenicol, tetracycline, telithromycin, and clindamycin.
The type strain SYS25-1 T (=GDMCC 1.3685 T ), which was isolated from the feces of sheep in 2019 and 2020 in Beijing, has a DNA G + C content of 32.43 mol%. The other strains, S13-1 and SYS28-3, are also classified in this species.

Limitations
In this study, we utilized a polyphasic approach to isolate and identify two novel Campylobacter species, which we have named Campylobacter felis sp. nov. and Campylobacter ovis sp. nov. Although their pathogenicity is currently unknown, studies indicate that further investigation into the potential health implications of these novel bacteria is necessary. Such research may aid in the management of diseases that could potentially be caused by these novel Campylobacter species.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms11040971/s1. Figure S1: Maximum-likelihood phylogenetic tree based on nearly complete 16S rRNA gene showing the relationships between our isolates and the type strains of the genus Campylobacter. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes, with Arcobacter butzleri ATCC 49616 T as an outgroup. Novel strains are highlighted in bold. Figure S2: Maximum-parsimony phylogenetic tree based on nearly complete 16S rRNA gene showing the relationships between our isolates and the type strains of the genus Campylobacter. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes, with Arcobacter butzleri ATCC 49616 T as an outgroup. Novel strains are highlighted in bold. Table S1: ANI (lower diagonal) and dDDH (upper diagonal) among the novel Campylobacter strains and other Campylobacter species.