Description of a new member of the family Erysipelotrichaceae: Clostridium fusiformis sp. nov., isolated from healthy human feces

A Gram-positive, non-motile, rod-shaped facultative anaerobic bacterial strain SG502T was isolated from the healthy human fecal samples in Brookings, SD, USA. The comparison of the 16S rRNA gene placed the strain within the Clostridium cluster XVI, where, Clostridium innocuum ATCC 14501T, Longicatena caecimuris strain PG-426-CC-2, Eubacterium dolichum DSM 3991T and Eubacterium tortuosum DSM 3987T were its closest taxa with 95.15%, 94.49%, 93.28%, and 93.20% sequence identities respectively. The optimal growth temperature and pH for the strain SG502T were 37°C and 7.0 respectively. Acetate was the major short-chain fatty acid product of the strain SG502T when grown in BHI-M medium. The major cellular fatty acids produced by the strain SG502T were C18:1 ω9c, C18:0 and C16:0. The DNA G+C content of the strain was 34.34 mol%. The average nucleotide identity of the genome of the strain SG502T and its closest neighbor C. innocuum ATCC 14501T was 63.48%. Based on the polyphasic analysis, the type strain SG502T (=DSM 107282T), represents a novel species of the genus Clostridium for which the name Clostridium fusiformis sp. nov. is proposed.


Introduction
The family Erysipelotrichaceae is an emerging group of bacteria originally described by Verbarg et al (1). The members of family Erysipelotrichaceae includes Gram-positive, filamentous rods and were originally described as facultative anaerobes but later amended by previously recognized as the "walled relatives" of mycoplasma (4) and later classified under Clostridial cluster XVI (5). With major changes in the taxonomy of Erysipelotrichaceae, recently some members have been reclassified into new families Coprobacillaceae (5) and Turicibacteraceae (3) but the placement of cluster XVI is still debated.
The members of Erysipelotrichaceae have been isolated from the intestinal tracts of mammals (6)(7)(8) and insects (9) and are associated with host metabolism and inflammatory diseases (10,11). Since there only few cultured species available from this family, their function in the gut ecosystem is not yet well understood (11).
Here, we describe the isolation, physiology and genome characteristics of a new member of the family Erysipelotrichaceae isolated from healthy human feces. The strain SG502 T clustered with clostridial cluster XVI and revealed genetic and phenotypic differences from C. innocuum strain the closest strain with valid taxonomy. Therefore, we propose the novel species Clostridium fusiformis sp. nov within family Erysipelotrichaceae.

Isolation and Ecology
Strain SG502 T was isolated from the healthy human fecal sample. After transferring the fresh fecal samples into the anaerobic chamber (85% nitrogen, 10% hydrogen and 5% carbon dioxide) within 10 minutes of voiding, the sample was diluted 10 times with anaerobic PBS and stored with 18% DMSO in -80 0 C. The samples were cultured in modified BHI medium (BHI-M) containing 37g/L of BHI, 5 g/L of yeast extract, 1 ml of 1 mg/mL menadione, 0.3 g of L-cysteine, 1 mL of 0.25 mg/L of resazurin, 1 mL of 0.5 mg/mL hemin, 10 mL of vitamin and mineral mixture,1.7 mL of 30 mM acetic acid, 2 mL of 8 mM propionic acid, 2 mL of 4 mM butyric acid, 100 μ l of 1 mM isovaleric acid, and 1% of pectin and inulin. After isolation, the strain was subjected to MALDI-ToF (Bruker, Germany). Since MALDI-TOF did not identify a species, , 16S rRNA gene sequencing was performed for species validation.

Physiology and Chemotaxonomy
The strain was cultivated in BHI-M medium in anaerobic conditions at 37 0 C at pH 6.8±0.2 for morphological characterization. Bacterial colony characteristics were determined after streaking the bacteria on BHI-M agar plates followed by 48 hours of anaerobic incubation.
Gram staining was performed using Gram staining kit (Difco) according to the manufacturer's instruction. During the exponential growth of the bacterium, cell morphology was examined by scanning electron microscopy (SEM). SG502 T was grown separately under aerobic and anaerobic conditions to determine the aerotolerance. Further, the strain was grown at 4, 20, 30, 40 and 55 0 C to determine the range of growth under anaerobic conditions. The BHI-M media was adjusted to pH range of 4-9 with 0.1N HCl and 0.1N NaOH to determine the growth of the strain at different pH levels. BHI-M medium was supplemented with triphenyltetrazolium chloride (TTC) (12) to determine the motility of the strain in which red color indicated positive motility.
Individual cells of the strain SG502 T was gram-positive rod-shaped with 1.5×0.35μ in dimensions (Fig 1 and Table 1). The colonies appeared to be white, smooth and convex with entire edges. The strain SG502 T grew from a pH range of 6.0-7.5 with optimal growth at pH 7.0 at 37 0 C anaerobically. The bacterium could grow anaerobically over the temperature range of 25-45 0 C with optimal growth at 37 0 C.
The strain grew well in BHI-M broth under anaerobic conditions but under aerobic conditions, the growth was comparatively lower and prolonged confirming that the strain was a facultative anaerobe. The utilization of various carbon sources and enzymatic activities were determined using AN MicroPlate (Biolog) and API ZYM (bioMerieux) according to the manufacturer's instructions. After growing the strain SG502 T and ATCC 14501 T in BHI-M medium at 37 0 C for 24 hours, cells were harvested for cellular fatty acid analysis. Fatty acids were extracted, purified, methylated and identified and analyzed using GC (Agilent 7890A) according to manufacturer's instructions (MIDI) (13). Short-chain fatty acids were determined using gas chromatography after cells were grown in BHI-M medium. For SCFAs, the culture was maintained in 25% meta-phosphoric acid before collecting the supernatant for analysis to detect volatile fatty acids.
Since SG502 T was found to be 95.15% identical to C. innocuum, it was classified within genus Clostridium. With the differences in physiological, biochemical, and 16s rRNA sequence dissimiliarities of SG502 T with C. innocuum, we propose classifying it as a new species under genus Clostridium.

Genome Features
The whole-genome sequencing of strain SW165 was performed using Illumina MiSeq using 2x 250 paired-end V2 chemistry. The reads were assembled using Unicycler that builds an initial assembly graph from short reads using the de novo assembler SPAdes 3.11.1 (19). The quality assessment for the assemblies was performed using QUAST (20).
Genome annotation was performed using Prokka (21). The draft genome of the strain SG502 consisted of 146 contigs and was 2,387,606 bps long with 34.34 mol% G+C content. The total number of predicted coding sequences, tRNAs, rRNAs, and tmRNAs was 2348, 63, 2 and 1 respectively. The average nucleotide identity between the two type strains SG502 T and C. innocuum (NZ_AGYV00000000.1) was found to be 63.48% (22). This delineates the two strains from one another as the proposed cut off for ANI for a new species is 95-96% (23).  Table 2). The type strain, SG502 T (=DSM 107282 T ), was isolated from a healthy human fecal sample. The DNA G+C content of the strain SG502 T is 34.34 mol%.

Protologue
The GenBank BioProject ID number for the draft genome sequence of the strain SG502 T is PRJNA494608.    "ND" not determined. Those fatty acids which were not separated using MIDI system were considered as summed features. Summed feature 5 contains C 15:0 DMA or C 14:0 3-OH; summed feature 8 contains C 17:1 cis 9 or C 17:2 and summed feature contains C 18:1 c11/t9/t6 or UN17.83Q.