Taxono-genomics description of Olsenella lakotia SW165 T sp. nov., a new anaerobic bacterium isolated from cecum of feral chicken

Background: The microbial community residing in the animal gastrointestinal tract play a crucial role in host health. Because of the high complexity of gut microbes, many microbes remain unclassified. Deciphering the role of each bacteria in health and diseases is only possible after its culture, identification, and characterization. During the culturomics study of feral chicken cecal sample, we cultured a possible novel strain SW165 T. Methods: For the possible novel strain SW165 T, phenotypic characterization was performed using colony morphology, Gram staining, growth in different temperature and pH and motility. Biochemical assays included carbon source utilization, enzymatic activity, cellular fatty acids and short chain fatty acid production. 16S rRNA sequencing and whole genome sequencing and comparison was performed for genetic analysis. Results: This strain was isolated from cecal content of feral chickens in Brookings, South Dakota, USA. Phylogenetic analyses based on 16S rRNA gene sequence revealed that the closest valid neighbor was Olsenella profusa DSM 13989 T (96.33% similarity) within the family Atopobiaceae. Cells were Gram-strain-positive and obligately anaerobic bacilli in chains. The optimum temperature and pH for the growth of the microorganism were 37-45 oC and pH 6.0-7.5 respectively. This strain produced acetic acid as the primary fermentation product. Major fatty acids were C 12:0, C 14:0, C 14:0 DMA and summed feature 1 (C 13:1 at 12-13 and C 14:0 aldehyde). Strain SW165 T exhibited a genome size of 2.43 Mbp with a G+C content of 67.59 mol%, which is the second highest G+C content among members of the genus Olsenella. The digital DNA-DNA hybridization and OrthoANI values between SW165 T and DSM 13989 T were only 17.6 ± 5.3 and 74.35%, respectively. Conclusion: Based on the phenotypic, biochemical, and genomic analyses, we propose the new species of the genus Olsenella, and name it Olsenella lakotia SW165 T sp. nov., (=DSM 107283 =CCOS 1887) as the type strain.


Introduction
The chicken gut harbors highly diverse microbes 1 . The gut microbes are known for their nutritional benefits by producing short chain fatty acids, enzymes, amino acids along with their ability to resist pathogens, immunity development and maintain homeostasis 2 . Even though culture independent methods have highlighted the functional capability of gut microbes, validation of these functions requires their cultivation, identification, and characterization. Most of the intestinal bacteria have been never isolated in the laboratory 3,4 thus hindering the understanding of their ecological and functional roles in the gut. Recently, "culturomics" strategy drives discovery of previously uncultured species based on modified culture conditions, such as media, temperature, pH and atmosphere and rapid identifying methods; matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing 5-7 . We employed culturomics to isolate bacteria from cecum of feral chickens. Based on bacterial isolation and identification, strain SW165 T was found as a new species within the genus Olsenella.
The members of the genus Olsenella are strictly anaerobic, Gram-positive, non-motile, non-spore-forming bacilli or cocci. This genus was first named by Dewhirst et al. The taxono-genomics approach uses combination of phenotypic and genotypic characterization to describe new bacteria 27,28 . Phenotypic investigation includes morphological, physiological, and biochemical assays. Genome-based and 16S-based analysis are used in genotypic characterization. In this study, strain SW165 T was described using taxono-genomics and compared to its closely related phylogenetic neighbors. Following analysis, we found that strain SW165 T belongs to a novel species for which the name Olsenella lakotia SW165 T sp. nov. is proposed.

Strain isolation
The cecal content of feral chickens was collected in Brookings, South Dakota, USA. For cultivation, the samples were transferred into an anaerobic workstation (Coy Laboratory) containing 85% nitrogen, 10% hydrogen and 5 % carbon dioxide and plated on a modified Brain Heart Infusion (BHI-M) agar containing 37 g/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 µl of 1 mM isovaleric acid, and 1% of pectin and inulin. After 3 days of incubation at 37°C under anaerobic conditions, single colony of strain SW165 was identified by MALDI-TOF mass spectrometry using a Microflex spectrometer (Bruker Daltonics, Bremen, Germany). The strain was maintained in BHI-M medium and stored with 10% (v/v) Dimethyl Sulfoxide (DMSO) at -80°C.

Phenotypic and biochemical tests
For morphological characterization, the strain SW165 T was anaerobically cultivated in BHI-M medium, pH 6.8-7.2, at 37°C. Colony morphologies were examined after 2-3 days of incubation on BHI-M agar plates. Gram-staining was performed using a Gram-Staining kit set (Difco), according to the manufacturer's instructions. Cell morphologies were examined by scanning electron microscopy (SEM) of cultures during exponential growth. Aerotolerance was examined by incubating cultures for 2 days separately under aerobic and anaerobic conditions. Growth of strain SW165 T at 4, 20, 30, 37, 40 and 55°C was determined. For determining the range of pH for growth of SW165 T , the pH of the medium was adjusted to pH 4.0-9.0 with sterile anaerobic stock solutions of 0.1 M HCl and 0.1 M NaOH. Motility of this microorganism was determined using motility medium with triphenyltetrazolium chloride (TTC) 29 . The growth was indicated by the presence of red color, reduced form of TTC after it is absorbed into bacterial cell wall.
Biochemical tests to determine standard taxonomic characteristics for strain SW165 T were performed in triplicate. The utilization of various substrates as sole carbon and energy sources and enzyme activities were performed using the AN MicroPlate (BIOLOG) and API ZYM (bioMérieux) according to the manufacturer's instructions. Reference strain, DSM 13989 T purchased from the DSMZ culture collection and isolated strains SW165 T were simultaneously cultured in BHI-M medium at 37°C for 24 h under anaerobic condition before cell biomass were harvested for cellular fatty acid analysis. The fatty acids were extracted, purified, methylated and analyzed using gas chromatography (Agilent 7890A) based on the instruction from Microbial Identification System (MIDI) 30 . Metabolic end-products such as short-chain fatty acids of strain SW165 T and DSM 13989 T grown in BHI-M were determined using a gas chromatography. The cultures were deproteinized with 25% metaphosphoric acid before supernatant collection. The supernatant was analyzed for the presence of acetic acid, butyric acid, isovaleric acid and propionic acid using GC (Themo Scientific TM TRACE TM 1310 GC equipped with a TraceGOLD TM TG-WaxMS A GC column.).

Genome sequencing and analysis
The whole genome sequencing of strain SW165 T was performed using Illumina MiSeq sequencer using 2x 300 paired end V3 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 39 . The quality assessment for the assemblies was performed using QUAST5.0.2 40 . Genome annotation was performed using Rapid Annotation using Subsystem Technology (RAST) server 41 . The digital DNA-DNA hybridization (dDDH) was performed using Genome-to-Genome Distance Calculator (GGDC) web server (http://ggdc.dsmz.de) to estimate the genomic similarity between strain SW165 T and the closest phylogenetic neighbor. Average nucleotide identity (ANI) between strain SW165 T and the closely related strains was also calculated using the OrthoANI software 42 . Distribution of functional categories of strain SW165 T was compared to Olsenella species and was presented in a heatmap generated using Explicet version 2.10.5 43 .

Results
Strain SW165 T was isolated from cecal contents of feral chicken in anaerobic chamber (Coy Laboratory Product, MI, USA). Colonies of SW165 T on BHI-M agar were 0.2-0.5 cm in diameter, appeared white, smooth, and umbonate with entire circular edges when grown at 37 o C anaerobically after 48 hours of incubation. After cultivation, the colonies of this strain were subjected to identification by MALDI-TOF using a Microflex spectrometry (Bruker Daltonics, Bremen, Germany). MALDI-TOF did not identify the strain as the scores obtained were < 1.70. Thus, full length 16S rRNA gene was sequenced using Sanger sequencing method. The 16S rRNA of the strain SW165 T showed 96.33% identity with O. profusa DSM 13989 T (GenBank accession no. AF292374), the validly closest species within phylogenetical nomenclature ( Figure 1). The current cut off for species delineation from its nearest neighbor based on 16S rRNA is 98.7% 44 . As the identity of 16S rRNA of strain SW165 T was lower than threshold, it was considered as a representative of putatively novel species within the genus Olsenella in the family Atopobiaceae. Phylogenetically, the strain was found to cluster together with other members of genus Olsenella, as shown in Figure 1, validating that SW165 T belongs to genus Olsenella taxonomically.
Phenotypic growth of strain SW165 T was observed on modified BHI-M agar after 2-3 days of incubation at temperature between 37°C and 45°C and pH between 6.0-7.0. The optimum temperature and pH for the growth were at 45°C and pH 7.0, respectively. Strain SW165 T grew only under anaerobic conditions, suggesting obligate anaerobic nature. Bacterial cells were Gram-stain-positive bacilli (0.5-2.0 µm), growing in pairs or as short chains and were non-motile ( Figure 2).
To further analyze the biochemical properties of the strain, we performed the carbon source utilization assay using BIOLOG AN microplate and compared it to closely related taxa. Strain SW165 T consumed various carbon sources for the growth, which differed from related strains in the utilization of D-fructose, L-fucose, D-galactose, maltose, D-melibiose and D-raffinose, and in the non-utilization of dulcitol. Based on enzymatic activity test, the strain produced several enzymes, including alkaline phosphatase, leucine arylamidase, cysteine arylamidase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, and β-glucosidase. Interestingly, alkaline phosphatase α-galactosidase, β-galactosidase and β-glucuronidase are not reported from its closest neighbors (Table 1). Furthermore, the dominant cellular fatty acids of the strain SW165 T were saturated, including C 12 : 0 (25.5%) and C 14 : 0 (22.83%). Moreover, other dominant fatty acids were C 14 : 0 DMA (15.61%) and summed feature 1 [C 13 : 1 and/or C 14 : 0 aldehyde; 13.94%]. However, there were distinct quantities of some fatty acids between SW165 T and the relative strains ( Table 2). The major short chain fatty acids produced by SW165 when cultivated in BHI-M were acetic acid (3.74 mM) followed by propionic acid (0.53 mM).
We examined the genome of the strain SW165 T to investigate its differentiation from the neighbors. The genome size of strain SW165 T was 2,427,227 bp with 67.59mol% G+C content. The draft genome was assembled into 33 contigs with 2,228 protein-coding sequences and 52 RNAs (Table 3) and is visualized as Figure 3. The genomes sizes for the Olsenella species were comparable to one another except for O. urininfantis whose was only 1.75 Mbps. However, the G+C contents strain SW165 T and O. mediterranea were the highest but comparable to other neighboring Olsenella species (Table 3). The genome of SW165 T possessed a total of 1, 230 genes with putative function and 998 genes as hypothetical proteins. Among 1,230 genes, 823 were classified as features in subsystem, following functional categories (COGs). Majority of categories included amino acid and derivatives (172 genes), carbohydrates (163 genes), and protein metabolism (132 genes) (Extended data: Supplemental Table 1 45 ).
Furthermore, we compared the genome of SW165 T to its neighbor using OrthoANI as shown in Figure 4. The genome of SW165 was only 73.41% identical to its nearest neighbor O. profusa DSM 13989 T . Also, the OrthoANI values for SW165 T and closely related strains ranged from 65.40 to 74.18 % (Figure 4) indicating that the genome of SW165 T is unique compared to its neighbors. The proposed cut off for OrthoANI  for the species delineation is 95-96% identity 46,47 . In addition, dDDH between SW165 T and the closest neighbor, O. profusa DSM 13989 T was only 17.6 ± 5.3. These values were lower than threshold of ANI and dDDH for delineating prokaryotic species, suggesting that these strains are distinct species. Also, the gene distribution into COGs was comparable in all eight compared Olsenella genomes ( Figure 5). Hence, the phenotypic and genetic discrepancy of the SW165 T with its close neighbor apparently supports that strain SW165 T represents a new species of the genus Olsenella.

Discussion
Culturomics of the gut microbiota has evolved as a strong tool to increase the isolation of diverse previously uncultured bacteria from the gut 5, 48 . The cultivation of the gut microbiota enables to improve the health through an enhanced understanding of their roles in the gut ecosystem and finally to the host. Thus, using the culturomics strategy, we were able to isolate previously uncultured bacterium SW165 T from cecal content of feral chicken and finally characterize and describe it using taxonogenomics as a novel microorganism. The novelty of a prokaryotic organism is universally determined by the comparison of 16S rRNA gene sequence homology 49 .
The threshold values are used at distinct taxonomic levels 46 .
In this context, the newly discovered bacterium was initially validated using the full-length 16S rRNA gene sequences, which were thereafter used for taxonomic classification. Phylogenetic analysis of 16S rRNA gene showed that the novel strain SW165 T clustered with closely related taxa in the genus Olsenella within the family Atopobiaceae (Figure 1). This genus composes of nine species, most of which are members of gut microbiota of humans and animals. However, O. uli is only a species that have been isolated from chicken gut 26 . Remarkable, this study revealed a new member of Olsenella from gut microbiota of chicken.
Phenotypic analyses are performed to differentiate closely valid bacteria. Based on phenotypic tests, strains SW165 T appeared several distinct properties compared to other members of the genus Olsenella (Table 1 and Table 2). The obvious distinct phenotypic features were observed in biochemical tests including enzymatic activity and carbon source utilization, thereby they might be important parameters for discriminating closely related species. These differences suggested the novelty of this microorganism belonging to Olsenella.
In addition to 16S based comparison, whole genome can be used for distinguishing, distinct bacteria. Recently, digital DNA-DNA hybridizations (dDDH) becomes a key measurement in delineation of prokaryotic species. It is an in-silico genome-to-genome comparison inferring whole genome distance to mimic DDH 50 . Besides, Average Nucleotides Identification (ANI) is another particular tool that confirm the taxonomic delineation. It measures the overall similarity between two genome sequences 51 . Recent publication of novel bacteria trend to perform genome-based analysis to support the results of 16S rRNA gene-based analysis. The strengths of genome-based analyses include comparison of all nucleotides in prokaryotic taxonomy and functional prediction 52 . Based on genomic evidence, strain SW165 T showed low similarity in terms of OrthoANI with Olsenella species of the family Atopobiaceae ( Figure 4). Further, genome features and distribution of predicted functional categories of strain SW165 T was corresponded to all other Olsenella species ( Figure 5 and Table 3). Thus, we proposed the strain SW165 T as a new species Olsenella lakotia SW165 T sp. nov., within the family Atopobiaceae.

Mohamed Seleem
Virginia Polytechnic Institute and State University, Blacksburg, USA This manuscript describes the isolation and characterization of a proposed new species "lakotia" within the genus Olsonella. The isolate has been characterized using the combination of genome sequencing and phenotypic assays. The phenotypic characterization includes nutrient utilization using Biolog plates, enzyme profiling using API zyme, and cellular membrane fatty acid profiling using FAME. At the phenotypic level, the proposed new species show clear differences with Olsonella umbonata, the closest taxonomic neighbor. At the genomic level, the proposed new species is only 74% similar to other species in the genus Olsonella. Therefore, the proposed species meets the genomic and phenotypic requirements of being designated as a new species. However, some minor changes could improve the clarity of the manuscript:

Is the study design appropriate and is the work technically sound? Yes
Are sufficient details of methods and analysis provided to allow replication by others? Yes