Genomic insights and probiotic characteristics of Bifidobacterium gallinarum CACC 514 isolated from canine

Background: The genus Bifidobacterium includes common healthy gut microbes in mammals and is widely acknowledged as a probiotics with health-promoting properties. Results: Bifidobacterium gallinarum CACC 514 was isolated from canine feces and its potential probiotic properties were characterized by comparative and functional genome analyses.The complete genome of strain CACC 514 was found to be 2.4 Mb, with a G + C content of 63.9 mol%. The strain possessed factors beneficial to mammalian health based on the presence of genes related to mucosal surface adhesion proteins, stress-related genes, and extracellular polysaccharide genes. A comparative genome analysis with other Bifidobacterium species revealed the unique characteristics of this species. These functional genomics have been confirmed for its superiority by probiotic properties, acid and bile tolerance, and adhesion to mucus. Conclusions: The genome analysis and in vitro probiotics characteristics revealed its superiority in the intestine. These results add to our comprehensive understanding of B. gallinarum and suggest that this strain has potential application in mammalian probiotics.

a health benefit on the host' retaining the previous definitions by Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO, 2002) [8]. To benefit health, probiotic organisms require to have therapeutic effects, including gastric acid and bile compounds stability, adherence to the intestinal surface, and colonization to the intestinal tract [9]. Probiotics lactic acid bacteria were associated with intestinal barrier function by their metabolic products, such as shortchain fatty acids [10]. In particular, there has been a continuing report on the strong association between Bifidobacterium numbers and enhanced integrity and invasiveness of the intestinal epithelial cell barrier [11], and thus, enteric adhesion studies for this strain are important.
The genomes of several probiotic Bifidobacterium strains including B. longum subsp. infantis, B.
bifidum, and B. animalis subsp. lactis have been sequenced and analyzed in efforts to identify the genes and metabolic pathways related to their health-promoting traits [12][13][14]. However, considering the importance of biotechnology, the number of strains sequenced completely so far is still small and is largely limited to phylogenetic studies due to sampling bias for the Bifidobacterium species mentioned above [15]. The recognized taxa within the genus Bifidobacterium are divided into six main phylogenetic groups namely, B. asteroides, B. adolescentis, B. longum, B. pullorum, B. pseudolongum, and B. boum [16]. The genome of B. pullorum group species is also not completely analyzed.
In the present study, we isolated the B. gallinarum species (strain CACC 514) within the B. pullorum group from canine feces, and to the best of our knowledge, we identified its first complete genome sequences. The basic requirements of probiotic applications include resistance to gastrointestinal (GI) stress, the ability to colonize the gastrointestinal tract and to be metabolically active in the gastrointestinal tract [17]. The complete sequences of the CACC 514 genome were annotated and compared with the genomes of other Bifidobacterium strains to determine the basis of its potential and unique probiotic traits. Furthermore, in vitro experiments for survival against gastric intestinal tract confirmed the positive effects as probiotics.

General genome features of Bifidobacterium gallinarum CACC 514
The complete genome of B. gallinarum CACC 514 contained a single circular chromosome of 2,414,462 bp with a GC content of 63.9% and one plasmid (8,720 bp) with an average GC content of 64.2 % (Table 1 and Fig. 1A). In total, 1,957 protein-coding sequences (CDSs) were identified. The chromosome contained 9 rRNAs, 57 tRNAs, 3 other ncRNAs, and 58 pseudogenes. The G+C contents belong to genus Bifidobacterium (55-67 %) were similar to the values 64.2 % of B. gallinarum DSM 20670 T with 2.1 Mb genome size, 5 rRNAs and 53 tRNAs [15,18,19].

Functional classification
Each CDS in the annotated genome was grouped into the RAST subsystem category based on the predicted functional role (Additional File 1  (Fig. 1B). Most genes were classified into functional categories for carbohydrate transport and metabolism (199 genes), replication, recombination, and repair (163 genes), translation, ribosomal structure, and biogenesis (135 genes), amino acid transport and metabolism (128 genes), and transcription (112 genes).

PHAST
The PHAGER search tool identified 1 intact prophage and 2 incomplete phages in this genome (Additional file 2). One prophage region resembled Microb_Min1_NC_009603 (42.7 kb, region 1) with a GC content of 62.3%. Bacteriophages are the most abundant biological entities and are known to impact genomic evolution and the adaptive capabilities of their bacterial hosts [20]. Bifidoprophages are present in a small number in multiple bifidobacterial species, indicating their relatedness to phages infecting other Actinobacteria, Firmicutes,as well as gram-negative bacteria [21,22]. In addition, integrases are useful indicators of prophage diversity in bacterial genomes [23]. Prophage region 1 (804,476 -847,197 bp) contained 58 CDSs, with a complete prophage integrase gene (Additional file 3). Consistent with the findings reported for bifidoprophages [22], the most conserved modules of the B. gallinarum CACC 514 were DNA packaging (encoding terminase, portal, and capsid proteins), lysogeny (encoding integrase gene), and tail morphogenesis module (encoding the tape measure protein).
CRISPR-Cas Type II immune systems (II-A, II-C) are fairly rare and occur in only 5% of bacteria, but they occur at a much higher frequency in the Bifidobacterium genus [24]. Characterization of Type II elementsmay provide opportunities to use the molecular genome-editing tool for the development of next-generation probiotic bacteria [25]. The B. gallinarum CACC 514 CRISPR-Cas system may also provide a platform for various potential biotechnological and ecological uses as probiotic bacteria.

Stress-related proteins
A number of stress-related proteins can regulate the adaptability of bacteria to the gastrointestinal tract [26]. CACC 514 possesses genes involved in stress-related pathways (Table 2). These stresses include temperature, pH, and oxidative stress. CACC 514 carried various genes encoding heat shock proteins, groEL, groES, dnaJ, dnaK, grpE, clpB, clpC, and clpP, and the small heat shock proteins (sHsps), hsp20 (Table 2). These genes are induced to respond upon exposure of bifidobacterial cultures to stressful condition, and overexpression of sHsps is known to increase tolerance to heat and osmotic stress in B. breve and B. longum NCC2705 [27]. CACC 514 encodes 2 genes for sodiumproton antiporter (Na +/H +), providing evidence of tolerance to low pH in the GI environment [23]. CACC 514 contains 9 genes for antioxidative proteins that are involved in minimizing the toxicity of active oxygen species (Table 2). B. longum has NADH oxidase, NADH peroxidase, and low superoxidase dismutase, whereas CACC 514 contains an NADH peroxidase and only 1 other predicted protein anaerobilin synthase (chuW) that is not found in Bifidobacterium species [28].

Core-and pan-genomes of CACC 514 and Bifidobacterium pullorum group strains
The complete CACC 514 genome was compared with the complete and incomplete genomes of 6 Bifidobacterium strains (Additional file 6). The genome of B. pullorum group strains could not be compared in this study, are there are no complete genome data reported in NCBI. These 7 genomes composed a pan-genome of 4,526 orthologous gene families and a core genome of 1,088 orthologous gene families (Additional file 7). In total, 271 genes in CACC 514 were unique (Additional file 8), including 236 hypothetical proteins. The 35 specific genes of CACC 514 included CRISPR-Cas 2, carbohydrate metabolism, phages, and cell wall and capsular polysaccharide-related genes. These outcome proteins will provide a higher ability for environmental change adaptation and adhesion to intestinal epithelial cells.

Acid and Bile tolerances and intestinal adhesion
The acid and bile tolerance help in studying the survival of strain under low pH gastric juice condition and colonization of isolates in the small intestine [32]. The strain showed a high survival rate (%) above 80% at low pH, and 0.3 and 1% bile salt concentration after 2h exposure (Table 3). The CACC 514 with 82.80% adhesive ability to HT-29 cells was higher than the reference strain L. rhamnosus GG (Table 4).

Antibiotic susceptibility
The susceptibilities to the tested 12 antibiotics of CACC 514 was very susceptible to amoxcillin/clavulanic acid, ampicillin, clindamycin, impenem, metronidazole, tetracycline, vancomycin and erythromycin, which showed MICs ≤ 4 μg/ml (Table 5). These results was similar with the other probiotics Bifidobacterium species, B. adolescentis, B. animalis, B. bifidum, B. breve, and B. longum [33]. Bifidobacterium spp. had a high resistant to kanamycin above 500 μg/ml concentration [34], and the most have been reported as resistant to aminoglycoside antibiotic, because of the absence of cytochrome mediated drug transport system [35]. CACC 514 showed also the resistance to kanamycin (≥ 256 μg/ml) and aminoglycoside gentamycin (96 μg/ml). These antibiotic susceptibilities were evaluated comparing MIC values to breakpoints suggested by European Food Safety Authority (EFSA) [36].

Conclusions
Bifidobacterium is important in gut microbiome studies and has long been used as a probiotic to provide consistent beneficial health effects. Genomic analysis of B. gallinarum CACC 514 isolated from canine feces provided an overview of the potential mechanisms underlying the effect of the strain on host health. The specific genes of CACC 514 including CRISPR-Cas 2, carbohydrate metabolism, phages, and cell wall and capsular polysaccharide-related genes will provide higher adhesion to intestinal epithelial cells. In addition, in-vitro probiotic properties, low pH and bile resistance, and high adhesion to intestinal epithelial cells of CACC 514 validated genomic properties.
Taken together, these studies demonstrate the potential as a probiotic of B. gallinarum strains within the B. pullorum group and substantially support the safe use of CACC 514 as a probiotic in the animal industry.

Annotation
De novo assembly of the single molecule real-time sequencing reads was performed using the hierarchical genome assembly process workflow (HGAP 3.0) in PacBio's SMRT portal with subreads from PacBio. After assembly, the paired-end reads from Illumina HiSeq 2500 were mapped to the assembled contigs to improve the accuracy of the genome sequences [37]. The sequences were annotated using the combined results of the automated NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) and the RAST prokaryotic genome annotation server (http://rast.nmpdr.org/) [38]. The coding genes were predicted according to clusters of orthologous group (COG) using the WebMGA online tool [39]. Prophage insert regions were searched using the online phage search tool PHASTER (http://phaster.ca/), and clustered regularly interspaced short palindromic repeats (CRISPR) were predicted using the CRISPR web server (http://crispr.i2bc.paris-saclay.fr/) [40,41].

Phylogenetic analyses of Bifidobacterium strains
Phylogenetic analysis was performed based on the 16S rRNA gene sequences. The 16S rRNA gene sequences of the CACC 514 and related species were aligned using the multiple sequence alignment program CLUSTAL W, and phylogenetic trees were then constructed using the neighbor-joining algorithm based on 1000 bootstrap replications in MEGA version 7 [42]. The ANI and in silico DDH values between Bifidobacterium strains were calculated using a standalone software (http://www.ezbiocloud.net/sw/oat) [43] and genome-to-genome distance calculator 2.1 (GGDC, http://ggdc.dsmz.de/distcalc2.php)[44], respectively. Pan-genome Orthologous Groups (POGs) were analyzed using BIOiPLUG comparative genomics software (ChunLab Inc., Seoul, Republic of Korea), and a heat map, UPGMA dendrogram, and Venn diagram were constructed based on these data.

Quality assurance
A single colony of the B. gallinarumstrain CACC 514 was repeatedly transferred to fresh mMRS medium to obtain pure cultures and the identity of the strain was verified through 16S rRNA gene sequencing. Genomic DNA strain CACC 514 was extracted using Qiagen DNeasy UltraClean microbial kit and then confirmed through a BLAST search of the 16S rRNA gene.

Probiotic characteristics Acid and bile tolerance
Acidic pH resistance were assessed using MRS broth adjusted to pH 1.5 and 2.5 with 6N HCl and incubated at 37 °C for 30min, 1h and 2h. Bile tolerance was evaluated with MRS broth containing 0.3% (w/v) oxgall (BD Difco, USA) at 37 °C for 2h. Viable number of bacteria were enumerated using MRS agar plate.

Adhesion assay
The human colon adenocarcinoma HT-29 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100mg/ml). Cells were incubated with DMEM medium lacking antibiotics before adhesion assay. LAB cultures suspended in DMEM medium without FBS and antibiotics were added into the wells containing HT-29 cell. The plates were incubated at 37 °C for 2h under 5% CO 2 and then the adherent bacteria were counted by plating the serial 10-fold dilution of the suspensions using MRS agar plate. The L. rhamnosus GG (LGG, KCTC 5033) was used as reference strain. All probiotic activity were repeated three and results expressed as mean ±standard deviation.

Phenotypic (Fermentative) profiling
The sugar degradation and other enzyme properties of the CACC 514 strain were characterized by using an API 20A, API Rapid ID 32A, and API ZYM kit (Bio-Merieux, Marcy l'Etoile, France). The strain was grown until the logarithmic phase and then inoculated into API galleries according to the manufacturer's instructions.

Consent for publication
Not applicable.

Availability of data and materials
The complete genome of strain CACC 514 determined in this study has been deposited with the NCBI GenBank database under accession numbers CP035464 (chromosome) and CP035465 (plasmid).

Competing interests
The authors declare that they have no competing interests.

Funding:
This study was supported by the Strategic Initiative for Microbiomes in Agriculture and Food funded by the Ministry of Agriculture, Food and Rural Affairs (918002-4).

Author Contributions
MYJ and YK wrote the manuscript. MYJ, JAK, and DHK performed DNA preparation, gene annotation, and comparative genome analysis. All authors read and approved the final manuscript.

Supplementary Files
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