Genome sequence and description of Blautia brookingsii SG772 sp. nov., a novel bacterial species isolated from human faeces

An anaerobic isolate SG772 belonging to the genus Blautia was isolated from a healthy human faecal sample. When compared using 16s rRNA sequence identity, SG772 showed only 94.46% similarity with its neighbour species Blautia stercoris. As strain SG772 showed both phenotypic and genomic differences from other members of the type species within the genus Blautia, we propose the designation of SG772 as novel species ‘Blautia brookingsii SG772T’.


Introduction
The human gut microbiome of the European and North American population is typically dominated by Bacteroides whereas Prevotella dominates in Asian and African populations [1]. To determine the cultivable microbial diversity of the Prevotella-dominant human gut microbiota, faecal samples were cultured from six healthy donors using a high-throughput culturomics approach [2]. A new species of a bacterium belonging to the genus Blautia was isolated during this study. We characterized this strain using the recently proposed taxonogenomics strategy which uses the combination of phenotypic and genomic characterization of the strain to define new species. Herein, we describe strain Blautia brookingsii SG772 T (DSM 107275 = CCOS 1888) that was isolated from the healthy human gut microbiota.

Isolation and growth conditions
The strain was isolated on a brain-heart infusion (BHI) agar medium after 48 hours of incubation at 37°C in strict anaerobic conditions (85% nitrogen, 10% carbon dioxide, 5% hydrogen). The strain grew in the pH range 5.5-7.5 with optimum growth at 6.8. No growth of was observed after 20 minutes of thermal shock at 80°C, suggesting its non-spore-forming nature.

Phenotypic characteristics
The colonies grown in agar plates appeared to be round, whitish, convex and smooth after 48 hours of anaerobic incubation on BHI plates. Cells were Gram-positive and like a coccobacillus in shape with no flagellum, suggesting a non-motile nature. An overnight culture of the strain SG772 T grown in BHI broth was centrifuged at 10 000 × g. The supernatant was then removed to obtain the pellet, which was mixed with 30% glycerol solution and frozen to -80°C and sent to Bowling Green State University Center for Microscopy (Bowling Green, OH, USA) for scanning electron microscopy. The size of the bacterium when imaged using scanning electron microscopy was between 0.5 and 0.8 by between 1.8 and 2.5 μm (Fig. 1).
Using API ZYM strip, enzymatic activities were determined. Positive enzymatic reactions were observed for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, α-galactosidase, β-galactosidase, α-glucosidase and N-acetylβ-glucosaminidase. Carbon-source utilization of the strain SG772 T was determined using a BIOLOG AN plate. The strain SG772T was grown on BHI agar plate for 48 hours and the cells were scooped using sterile cotton swab and mixed in BIOLOG AN fluid until the optical density at 600 nm reached 0.01. Then, 100 μL of the mixture was pipetted into 96 wells of the BIOLOG AN plate and incubated for 24 hours. The initial and final optical densities were measured and compared with control. Those wells with optical density >20% compared with control were considered as showing positive nutrient utilization. Results from this test revealed that the strain used 21 substrates with maximum preference for propionic acid followed by L-alanyl-Lglutamine (Table 1). Furthermore, it was able to use complex carbohydrates like palatinose, rhamnose and arbutin, along with organic acids such as α-ketovaleric acid, urocanic acid, glycoxylic acid, fumaric acid, galacturonic acid and α-hydroxybutyric acid. Comparison of the substrate utilization of strain SG772 T with other neighbouring Blautia strains is given in Table 1. Additionally, cellular morphology and optimum pH for growth varied among the different species (Table 1). Antibiotic sensitivity testing using the disc diffusion technique showed that the strain SG772 T was resistant to tetracycline and streptomycin. However, it was susceptible to chloramphenicol, ampicillin, erythromycin and novobiocin.

Strain identification
After initial isolation of the strain SG772 T , matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed, but without identification (score <1.7). Therefore, DNA of the strain was isolated and 16S rRNA gene was amplified and sequenced using Applied Biosystems 3500xL Genetic Analyzer (Thermo Fisher Scientific, UK). The 16S rRNA sequences were trimmed and assembled using GENEIOUS 10.2.3 (https://www.geneious.com). A continuous stretch of 1440 bp of the 16S rRNA gene of strain SG772 T was obtained and searched against EzTaxon e-server [3] for identification. This comparison showed that the closest neighbour for strain SG772 T is Blautia stercoris GAM6-1 sharing only 94.46% sequence similarity (Fig. 2).

Genome sequencing and comparison
The genomic DNA was isolated using an E.Z.N.A DNA isolation kit (Omega Bio-tek Inc, GA, USA) following the manufacturer's protocol. The sequencing was performed using Illumina MiSeq (Illumina Inc., San Diego, CA, USA), 2 × 250 paired end chemistry. The reads were assembled using SPADES 3.9.0 [4] and the contigs were validated using QUAST [5].