Bacillus kwashiorkori sp. nov., a new bacterial species isolated from a malnourished child using culturomics

Abstract Strain SIT6T was isolated from the fecal flora of a severely malnourished child as part of a broad “culturomics” study aiming to maximize the culture conditions for the in‐depth exploration of the human microbiota. An analysis of the 16S rRNA gene sequence showed that strain SIT6T shared 94.1% 16S rRNA gene sequence similarity with Bacillus thermoamylovorans DKPT (NR_029151), the phylogenetically closest type species. Colonies are creamy white, circular, 4–5 mm in diameter after cultivation at 37°C for 24 hr on 5% sheep blood‐enriched Colombia agar. Growth occurs at temperatures in the range of 25–56°C (optimally at 37°C). Strain SIT6T is a gram‐positive, facultative anaerobic rod and motile by means of peritrichous flagella and sporulating; it is catalase and oxidase positive. The 2,784,637‐bp‐long genome, composed of 16 contigs, has a G+C content of 35.19%. Of the 2,646 predicted genes, 2,572 were protein‐coding genes and 74 were RNAs. The major fatty acids are saturated species (15:0 iso, 16:0 and 17:0 anteiso). Of the 14 detected fatty acids, 11 are saturated, either linear or branched (iso and anteiso). Digital DNA–DNA hybridization (dDDH) estimation and average genomic identity of orthologous gene sequences (AGIOS) of the strain SIT6T against genomes of the type strains of related species ranged between 18.6% and 38.3% and between 54.77% and 65.50%, respectively. According to our taxonogenomics results, we propose the creation of Bacillus kwashiorkori sp. nov. that contains the type strain SIT6T (=CSUR P2452T, =DSM 29059T).

2010). For this reason, in our laboratory we have developed a new strategy called culturomics, which is based on the application of various culture conditions followed by rapid identification using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to explore the bacterial composition . This new concept has allowed us to significantly increase the bacterial species associated with the human digestive tract and to find many new species . Using this strategy (i.e., culturomics), we were able to isolate a new species belonging to the genus Bacillus. This new isolate was described according to the new method that we have implemented (taxonogenomics) (Kokcha et al., 2012;Lagier, Elkarkouri, Rivet, Couderc, & Raoult, 2013;Seck et al., 2016). In brief, it involves using proteomic, fatty acid, and genomic features (Ramasamy et al., 2014;Welker & Moore, 2011;Seng et al., 2013), along with phenotype and some conventional methods, such as 16S rRNA phylogeny and the G+C content.
In this article, we describe the strain SIT6 T (=CSUR P2452 T , =DSM 29059 T ) isolated from the stool sample of a kwashiorkor patient.

| Organism information
The study and consent procedure were approved by the National Ethics Committee of Nigeria and the Ethics Committee of the Federative Research Institute 48 (Faculty of Medicine, Marseille, France) under the agreement number 09-022. The stool sample was obtained from a 4-month-old Nigerian child suffering from acute malnutrition (kwashiorkor). The patient was not being treated with antibiotics at the time of the sample collection and the sample was stored at −80°C. The stool sample was cultured in blood culture bottles supplemented with sheep blood (BioMérieux, Marcy l'Etoile, France). During a 30-day preincubation period at 37°C in aerobic atmosphere, the liquid culture is then spread on Columbia agar with 5% sheep blood COS medium (BioMérieux, Marcy l'Etoile, France) and the isolated colonies are subsequently identified.

| Strain identification by MALDI-TOF MS and 16S rRNA sequencing
MALDI-TOF MS analysis of proteins was used to identify the bacteria. Each colony was deposited in duplicate on a MALDI-TOF MSP 96 target and then covered with 1.5 μl of a matrix solution (saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 2.5% trifluoroacetic acid) to allow the crystallization of molecules. MALDI-TOF MS was performed using the LT Microflex spectrometer (Bruker Daltonics, Leipzig, Germany). All spectra were recorded in positive linear mode for the mass range from 2,000 to 20,000 Da (parameters: ion source 1 [ISI], 20 kV; IS2, 18.5 kV lens, 7 kV). The generated spectra were then compared to the Bruker database, with the addition of new species found through the "culturomics" project. The resulting score dictates whether a tested species can be identified: a score ≥2 with a validly published species enables identification at the species level, a score ≥1.7 but <2 enables identification at the genus level, and a score <1.7 does not enable any identification.
Following three assays, unidentified colonies were identified using 16S rRNA gene sequencing as described previously (Bittar et al., 2014). The isolated colony was suspended in 200 μl distilled water for DNA extraction using an EZ1 DNA Tissue Kit with a BioRobot EZ1 Advanced XL (Qiagen, Courtaboeuf, France). The amplification of the 16S rRNA gene was performed using the universal primer pair fD1 and rP2 (Eurogentec, Angers, France) (Weisburg, Barns, Pelletier, & Lane, 1991). The PCR product was purified and sequenced using the BigDye Terminator v1.1 Cycle Sequencing Kit (PerkinElmer, Courtaboeuf, France) with the following internal primers: 536F, 536R, 800F, 800R, 1050F, and 1050R, and ABI Prism 3130xl Genetic Analyzer capillary sequencer (Applied Biosystems). 16S rRNA amplification and sequencing were carried out as described previously by Morel et al. (2015). The 16S rRNA nucleotide sequences were assembled and corrected using  (Yoon et al., 2017) were performed to determine the percentage of similarity with the closest bacteria.
The MEGA 7 (Molecular Evolutionary Genetics Analysis) software (Kumar, Stecher, & Tamura, 2016) allowed us to construct a phylogenetic tree. Sequence alignment of the different species was performed using CLUSTALW and the calculation of the evolutionary distance was done with the Kimura two-parameter model (Kimura, 1980;Thompson, Higgins, & Gibson, 1994).

| Morphologic, biochemical, and antibiotic susceptibility tests
Gram staining was performed and observed using a Leica DM 2500 photonic microscope (Leica Microsystems, Nanterre, France) with a 100× oil immersion lens. A thermal shock (80°C during 20 min) was applied on fresh colonies in order to test sporulation. The motility of the strain was tested by observing fresh colonies using a DM1000 photonic microscope (Leica Microsystems) with a 40× objective lens.
F I G U R E 1 Phylogenetic tree showing the position of Bacillus kwashiorkori SIT6 T (red) relative to other phylogenetically close members of the family Bacillaceae. GenBank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALW, and phylogenetic inferences were obtained using (a) the maximum-likelihood method, (b) the neighbor-joining method and (c) the maximum parsimony method within the MEGA software. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 1,000 times to generate a majority consensus tree. Only values >70% were displayed. Bhargavaea ginsengi ge14 T (EF371375) was used as out-group Using the commercially available biochemical API 20NE, API ZYM, and API 50CH strips, we investigated the biochemical characteristics of our strain according to the manufacturer's instructions (BioMérieux).
Negative staining was done in order to visualize the cell morphology. Cells were fixed with 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer for at least 1 hr at 4°C. A drop of cell suspension was deposited for approximately 5 min on glow-discharged formvar carbon film on 400 mesh nickel grids (FCF400-Ni, EMS). The grids were dried on blotting paper and the cells were negatively stained for 10 s with 1% ammonium molybdate solution in filtered water at room temperature.
Electron micrographs were acquired with a Tecnai G20 Cryo (FEI) transmission electron microscope operated at 200 keV.

| FAME analysis by gas chromatography/mass spectrometry
Cellular fatty acid methyl ester (FAME) analysis was performed by GC/MS. Two samples were prepared with approximately 2 mg of bacterial biomass each, harvested from five culture plates. Fatty acid methyl esters were prepared as described by Sasser (2006). GC/MS analyses were carried out as described previously .

| Genomic DNA preparation
After pretreatment by a lysozyme (incubation at 37°C for 2 hr), the DNA of strain SIT6 T was extracted on the EZ1 BioRobot (Qiagen) with the EZ1 DNA tissues kit. The elution volume was 50 μl. Genomic DNA (gDNA) was quantified by a Qubit assay with the high sensitivity kit (Life Technologies, Carlsbad, CA) to 55.8 ng/μl.  Intens. (a.u.) 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 18,000 m/z

| Genome annotation and analysis
Open reading frames (ORFs) were predicted using Prodigal (http:// prodigal.ornl.gov/) with default parameters. However, the predicted ORFs were excluded if they spanned a sequencing gap region.
The predicted bacterial protein sequences were searched against GenBank and Clusters of Orthologous Group (COG) databases using BLASTP. The tRNAs and rRNAs were predicted using the tRNAScan-SE and RNAmmer tools, respectively. Signal peptides and numbers of transmembrane helices were predicted using SignalP (Nielsen, Gel view comparing Bacillus kwashiorkori SIT6 T spectra with other members of the genus Bacillus. The gel view displays the raw spectra of all loaded spectrum files arranged in a pseudo-gel-like look. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a gray scale scheme code. The color bar and the right y-axis indicate the relation between the color a peak is displayed with and the peak intensity in arbitrary units. Displayed species are indicated on the right F I G U R E 4 Gram staining of Bacillus kwashiorkori SIT6 T F I G U R E 5 Transmission electron microscopy of Bacillus kwashiorkori SIT6 T using a Morgani 268D (Philips) at an operating voltage of 60 kV. The scale bar represents 500 nm Engelbrecht, Brunak, & von Heijne, 1997) and TMHMM (Krogh, Larsson, von Heijne, & Sonnhammer, 2001), respectively. Mobile genetic elements were predicted using PHAST (Zhou, Liang, Lynch, Dennis, & Wishart, 2011) and RAST (Aziz et al., 2008   php?laref=256&titre=urms-database) ( Figure 2) and then compared to other known species of the genus Bacillus. The differences exhibited are shown in the obtained gel view (Figure 3).
T A B L E 6 Genomic comparison of Bacillus kwashiorkori with other Bacillus spp.

| Genome properties
The genome is 2,784,637 bp long with 35.19% G+C content ( Figure 6).
It is composed of 16 scaffolds, composed of 16 contigs. Of the 2,646 predicted genes, 2,572 were protein-coding genes, and 74 were RNAs (7 genes are 5S rRNA, 2 genes are 16S rRNA, 2 genes are 23S rRNA, and 63 genes are tRNA). A total of 1,749 (68%) were assigned as putative function (by COGs of NR blast). A total of 156 genes were identified as ORFans (6.07%). The remaining genes were annotated as hypothetical proteins (487 genes [18.93%]). Genome content is detailed in Table 4, while Table 5 presents the distribution of the genes into COG functional categories.
The genome sequence has been deposited in GenBank under accession number CTDX00000000.
The low ANI values confirmed it as a new species because ANI values bigger than 95 indicated that strains belong to the same species (Konstantinidis, Ramette, & Tiedje, 2006). Finally, digital DNA-DNA hybridization (dDDH) estimation of the strain SIT6 T against the compared genomes confirmed its new species status, as it ranges between 18.6 and 38.3 (below the cutoff of 70%).
The G+C content of the genome is 35.19%. The 16S rRNA gene sequence and whole-genome shotgun sequence of B. kwashiorkori SIT6 T are deposited in GenBank under accession numbers LK985393 and CTDX00000000, respectively. The strain SIT6 T (=CSUR P2452 T , =DSM 29059 T ) was isolated from the fecal flora of a Nigerian 4-month-old child suffering from acute malnutrition (kwashiorkor).
Habitat is the human gut.