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

Introduction

The chicken gut harbors highly diverse microbes¹. 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 homeostasis². 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 the 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 the 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. 2001⁸ and amended by Zhi et al 2009⁹ and Kraatz et al 2011¹⁰. This genus has recently been reclassified as a member of the family Atopobiaceae under order Coriobacteriales, class Coriobacteria, and phylum Actinobacteria¹¹. The genus Olsenella consists of nine published species; O. uli¹², O. profusa⁸, O. umbonata¹⁰, O. scatoligenes¹³, O. urininfantis¹⁴, O. congonensis¹⁵, O. provencensis¹⁶, O. phocaeensis¹⁶, and O. mediterranea¹⁶. The members of this genus are strictly anaerobic, Gram positive, non-motile, non-spore forming rod-shaped with G+C content of DNA 62–64%^8,13,17. The main habitats of the Olsenella are the oral cavity and gastrointestinal tract of humans^18–21, animals and various anaerobic environmental sites^22–24. In the chicken cecum, many members of the genus Olsenella have been reported in the chicken microbiome in metagenomic-based studies^15,25. However, only O. uli was isolated from chicken gut²⁶.

The taxono-genomics approach uses a 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 analyses 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.

Methods

Results

Strain SW165^T was isolated from cecal contents of feral chicken in an 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°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.

Figure 1. 16S rRNA based neighbor-joining tree of SW165^T and its neighbors.

Tree shows phylogenetic position of Olsenella lakotia DSM 107283^T and closely related species in the family Atopobiaceae. GenBank accession numbers of the 16S rRNA gene sequences are given in parentheses. Black circles indicate that the corresponding branches were also recovered both by maximum-likelihood and maximum parsimony methods. Bootstrap values (based on 1000 replications) greater than or equal to 70% are shown in percentages at each node. Bar, 0.01 substitutions per nucleotide position.

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).

Figure 2. Scanning electron micrograph of O. lakotia SW165^T.

Cells were anaerobically cultured for 24 hours at 37°C in BHI-M medium. Bar, 2 μm; uncropped/unedited image.

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 the 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 67.59 mol% 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). The majority of categories included amino acid and derivatives (172 genes), carbohydrates (163 genes), and protein metabolism (132 genes) (Extended data: Supplemental Table 1⁴⁵).

Figure 3. Graphical circular map of O. lakotia SW165^T genome.

From outside to the center: coding sequences on the forward strand (CDS +), coding sequences on the reverse strand (CDS -), tRNAs, rRNAs, GC content, and GC skew.

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.

Figure 4. Average nucleotide Identity comparison of O. lakotia SW165^T and closely related strains.

Heatmap represents OrthoANI values generated using OAT software between O. lakotia and related taxa with valid taxonomy.

Figure 5. Distribution of functional features of predicted coding sequences of O. lakotia SW165^T and its neighbors.

The functional features were predicted based on the clusters of orthologous groups. Heatmap was generated from the genome annotation of individual species by RAST using Explicet software.

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 taxono-genomics as a novel microorganism.

The novelty of a prokaryotic organism is universally determined by the comparison of 16S rRNA gene sequence homology⁴⁹. The threshold values are used at distinct taxonomic levels⁴⁶. 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 the gut microbiota of humans and animals. However, O. uli is only a species that have been isolated from chicken gut²⁶. 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 distinctive 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, different bacteria. Recently, digital DNA-DNA hybridizations (dDDH) becomes a key measurement in the delineation of prokaryotic species. It is an in-silico genome-to-genome comparison inferring whole genome distance to mimic DDH⁵⁰. Besides, Average Nucleotides Identification (ANI) is another particular tool that confirms the taxonomic delineation. It measures the overall similarity between two genome sequences⁵¹. A 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 a comparison of all nucleotides in prokaryotic taxonomy and functional prediction⁵². 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 has 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.

Data availability