Bacillus Kwashiorkori Sp Nov., a New Bacterial Species Isolated from A

Bacillus kwashiorkori sp nov., a new bacterial species isolated from a malnourished child using culturomics El Hadji Seck, Mamadou Beye, Sory Ibrahima Traore, Saber Khelaifia, Caroline Michelle, Carine Couderc, Souleymane Brah, Pierre-Edouard Fournier, Didier Raoult, Fadi Bittar

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El Hadji Seck, Mamadou Beye, Sory Ibrahima Traore, Saber Khelaifia, Caroline Michelle, et al.. Bacil- lus kwashiorkori sp nov., a new bacterial species isolated from a malnourished child using culturomics. MicrobiologyOpen, Wiley, 2018, 7 (1), pp.e00535. 10.1002/mbo3.535. hal-01780644

HAL Id: hal-01780644 https://hal.archives-ouvertes.fr/hal-01780644 Submitted on 22 May 2018

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Received: 28 April 2017 | Accepted: 15 August 2017 DOI: 10.1002/mbo3.535

ORIGINAL RESEARCH

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

1Aix-Marseille Univ, CNRS 7278, AP-HM, IRD 198, INSERM 1095, URMITE, IHU Abstract Méditerranée Infection, Marseille, France Strain SIT6T was isolated from the fecal flora of a severely malnourished child as part 2 Hôpital National de Niamey, Niamey, Niger of a broad “culturomics” study aiming to maximize the culture conditions for the in- 3King Fahd Medical Research Center, Special depth exploration of the human microbiota. An analysis of the 16S rRNA gene se- Infectious Agents Unit, King Abdulaziz University, Jeddah, Saudi Arabia quence showed that strain SIT6T shared 94.1% 16S rRNA gene sequence similarity with Bacillus thermoamylovorans DKPT (NR_029151), the phylogenetically closest type Correspondence Fadi Bittar, URMITE, IHU Méditerranée species. Colonies are creamy white, circular, 4–5 mm in diameter after cultivation at Infection, Marseille, France. 37°C for 24 hr on 5% sheep blood-enriched Colombia agar. Growth occurs at tem- Email: [email protected] peratures in the range of 25–56°C (optimally at 37°C). Strain SIT6T is a gram-positive, Funding information facultative anaerobic rod and motile by means of peritrichous flagella and sporulating; Fondation Méditerranée Infection 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).

KEYWORDS Bacillus kwashiorkori, culturomics, genome, taxonogenomics

1 | INTRODUCTION 2014; Moreno-Indias, Cardona, Tinahones, & Queipo-Ortuño, 2014). Recently, genomic and metagenomic advances have widely Although the human intestinal flora is intrinsically associated with participated in describing the human microbiota, but culture isola- the host genotype and age, many external factors can affect and tion remains the only means and the first step to characterize the modify this microbiota, such as antibiotics, probiotics, and diet physiological and genomic properties of a given bacterium and (Angelakis, Armougom, Million, & Raoult, 2012; Chen, He, & Huang, to describe a potential new species (Vartoukian, Palmer, & Wade,

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www.MicrobiologyOpen.com | 1 of 13 MicrobiologyOpen. 2018;7:e535. https://doi.org/10.1002/mbo3.535 2 of 13 | SECK et al.

2010). For this reason, in our laboratory we have developed a new with a validly published species enables identification at the species strategy called culturomics, which is based on the application of level, a score ≥1.7 but <2 enables identification at the genus level, and various culture conditions followed by rapid identification using a score <1.7 does not enable any identification. matrix-assisted laser-desorption/ionization time-of-flight mass Following three assays, unidentified colonies were identified using spectrometry (MALDI-TOF MS) to explore the bacterial composition 16S rRNA gene sequencing as described previously (Bittar et al., (Lagier et al., 2012). This new concept has allowed us to significantly 2014). The isolated colony was suspended in 200 μl distilled water for increase the bacterial species associated with the human digestive DNA extraction using an EZ1 DNA Tissue Kit with a BioRobot EZ1 tract and to find many new species (Lagier et al., 2016). Using this Advanced XL (Qiagen, Courtaboeuf, France). The amplification of the strategy (i.e., culturomics), we were able to isolate a new species be- 16S rRNA gene was performed using the universal primer pair fD1 and longing to the genus Bacillus. rP2 (Eurogentec, Angers, France) (Weisburg, Barns, Pelletier, & Lane, This new isolate was described according to the new method that 1991). The PCR product was purified and sequenced using the BigDye we have implemented (taxonogenomics) (Kokcha et al., 2012; Lagier, Terminator v1.1 Cycle Sequencing Kit (PerkinElmer, Courtaboeuf, Elkarkouri, Rivet, Couderc, & Raoult, 2013; Seck et al., 2016). In brief, it France) with the following internal primers: 536F, 536R, 800F, 800R, involves using proteomic, fatty acid, and genomic features (Ramasamy 1050F, and 1050R, and ABI Prism 3130xl Genetic Analyzer capillary et al., 2014; Welker & Moore, 2011; Seng et al., 2013), along with phe- sequencer (Applied Biosystems). 16S rRNA amplification and sequenc- notype and some conventional methods, such as 16S rRNA phylogeny ing were carried out as described previously by Morel et al. (2015). The and the G+C content. 16S rRNA nucleotide sequences were assembled and corrected using In this article, we describe the strain SIT6T (=CSUR P2452T, =DSM CodonCode Aligner software (http://www.codoncode.com). Then, the 29059T) isolated from the stool sample of a kwashiorkor patient. BLASTn searches against the GenBank NCBI database (http://blast. ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi) and EzBioCloud’s Identify Service (http://www.ezbiocloud.net/identify) (Yoon et al., 2017) were 2 | MATERIALS AND METHODS performed to determine the percentage of similarity with the closest bacteria. 2.1 | Organism information The MEGA 7 (Molecular Evolutionary Genetics Analysis) software The study and consent procedure were approved by the National (Kumar, Stecher, & Tamura, 2016) allowed us to construct a phyloge- Ethics Committee of Nigeria and the Ethics Committee of the netic tree. Sequence alignment of the different species was performed Federative Research Institute 48 (Faculty of Medicine, Marseille, using CLUSTALW and the calculation of the evolutionary distance France) under the agreement number 09-022. The stool sample was was done with the Kimura two-parameter model (Kimura, 1980; obtained from a 4-month-old Nigerian child suffering from acute mal- Thompson, Higgins, & Gibson, 1994). nutrition (kwashiorkor). The patient was not being treated with antibiotics at the time of the sample collection and the sample was stored at 2.3 | Growth conditions −80°C. The stool sample was cultured in blood culture bottles supple- mented with sheep blood (BioMérieux, Marcy l’Etoile, France). During In order to determine the ideal growth conditions for strain SIT6T, a 30-day preincubation period at 37°C in aerobic atmosphere, the liq- different growth temperatures (25°C, 28°C, 30°C, 37°C, 45°C, and uid culture is then spread on Columbia agar with 5% sheep blood COS 56°C) were tested under anaerobic and microaerophilic atmospheres medium (BioMérieux, Marcy l’Etoile, France) and the isolated colonies using GENbag Anaer and GENbag microaer systems, respectively are subsequently identified. (BioMérieux, Marcy l’Etoile, France). The strain growth was also tested T aerobically with and without 5% CO2. The growth of strain SIT6 was tested under different pH using a Columbia agar with 5% sheep blood 2.2 | Strain identification by MALDI-TOF MS and COS medium (BioMérieux, Marcy l’Etoile, France) with NaCl, MgCl , 16S rRNA sequencing 2 MgSO4, KCl, CaCl2, and glucose. The pH was modified by adding HCl MALDI-TOF MS analysis of proteins was used to identify the bacte- to the medium and measured with a pH meter. The optimal pH for ria. Each colony was deposited in duplicate on a MALDI-TOF MSP growth was determined by testing at different pH 5, 6, 6.5, 7, 7.5, 8, 96 target and then covered with 1.5 μl of a matrix solution (saturated and 8.5. Growth at various NaCl concentrations (0.5%, 5%, 7.5%, 10%, solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 2.5% 15%, and 200%) was investigated. trifluoroacetic acid) to allow the crystallization of molecules. MALDI- TOF MS was performed using the LT Microflex spectrometer (Bruker 2.4 | Morphologic, biochemical, and antibiotic Daltonics, Leipzig, Germany). All spectra were recorded in positive lin- susceptibility tests ear 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 spec- Gram staining was performed and observed using a Leica DM 2500 tra were then compared to the Bruker database, with the addition of photonic microscope (Leica Microsystems, Nanterre, France) with a new species found through the “culturomics” project. The resulting 100× oil immersion lens. A thermal shock (80°C during 20 min) was score dictates whether a tested species can be identified: a score ≥2 applied on fresh colonies in order to test sporulation. The motility of SECK et al. | 3 of 13 the strain was tested by observing fresh colonies using a DM1000 EUCAST 2015 recommendations (Matuschek, Brown, & Kahlmeter, photonic microscope (Leica Microsystems) with a 40× objective lens. 2014). The following antibiotics were tested: doxycycline (30 μg), ri- Catalase (BioMérieux) activity was determined in 3% hydrogen perox- fampicin (30 μg), vancomycin (30 μg), erythromycin (15 μg), ampicillin ide solution and oxidase activity was assessed using an oxidase rea- (10 μg), ceftriaxone (30 μg), ciprofloxacin (5 μg), gentamicin (500 μg), gent (Becton-Dickinson, Le Pont-de-Claix, France). penicillin (10 μg), trimethoprim/sulfamethoxazole (25 + 23.75 μg), Antibiotic susceptibility testing was performed using SIRscan imipenem (10 μg), metronidazole (4 μg), clindamycin (15 μg), colistin Discs (i2a, Montpellier, France) on Mueller-Hinton agar according to (50 μg), and oxacillin (5 μg).

(a)

(b)

FIGURE 1 Phylogenetic tree showing (c) the position of Bacillus kwashiorkori SIT6T (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 ge14T (EF371375) was used as out-group 4 of 13 | SECK et al.

Using the commercially available biochemical API 20NE, API ZYM, TABLE 1 Classification and general features of Bacillus T and API 50CH strips, we investigated the biochemical characteristics kwashiorkori strain SIT6 of our strain according to the manufacturer’s instructions (BioMérieux). Property Term Negative staining was done in order to visualize the cell morphol- Current classification Domain: Bacteria ogy. Cells were fixed with 2.5% glutaraldehyde in 0.1 mol/L cacodylate Phylum: Firmicutes buffer for at least 1 hr at 4°C. A drop of cell suspension was depos- Class: Bacilli ited for approximately 5 min on glow-discharged formvar carbon film on 400 mesh nickel grids (FCF400-Ni, EMS). The grids were dried on Order: Bacillales blotting paper and the cells were negatively stained for 10 s with 1% Family: Bacillaceae ammonium molybdate solution in filtered water at room temperature. Genus: Bacillus Electron micrographs were acquired with a Tecnai G20 Cryo (FEI) Species: kwashiorkori transmission electron microscope operated at 200 keV. Type strain: SIT6T Gram stain Positive 2.5 | FAME analysis by gas chromatography/mass Cell shape Rod shaped spectrometry Motility Motile Sporulation Endospore forming Cellular fatty acid methyl ester (FAME) analysis was performed by Temperature range Mesophile GC/MS. Two samples were prepared with approximately 2 mg of Optimum temperature 37°C bacterial biomass each, harvested from five culture plates. Fatty acid Optimum pH 7.5 methyl esters were prepared as described by Sasser (2006). GC/MS analyses were carried out as described previously (Dione et al., 2016). Salinity 0.0–5.0 g/L In brief, fatty acid methyl esters were separated using an Elite 5-MS Optimum salinity 0 g/L column and monitored by mass spectrometry (MS) (Clarus 500-SQ 8 Oxygen requirement Facultative aerobic S, PerkinElmer, Courtaboeuf, France). A spectral database search was performed using MS Search 2.0 operated with the Standard Reference

Database 1A (NIST, Gaithersburg, MD, USA) and the FAMEs mass 8,000 spectral database (Wiley, Chichester, UK).

2.6 | Genomic DNA preparation 6,000

After pretreatment by a lysozyme (incubation at 37°C for 2 hr), the T DNA of strain SIT6 was extracted on the EZ1 BioRobot (Qiagen) with 4,000

the EZ1 DNA tissues kit. The elution volume was 50 μl. Genomic DNA Intens. (a.u.) (gDNA) was quantified by a Qubit assay with the high sensitivity kit

(Life Technologies, Carlsbad, CA) to 55.8 ng/μl. 2,000

2.7 | Genome sequencing and assembly 0

Genomic DNA (gDNA) of B. kwashiorkori was sequenced on MiSeq 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 18,000 m/z Technology (Illumina Inc., San Diego, CA) with the mate pair strategy. The gDNA was barcoded in order to be mixed with 11 other FIGURE 2 Reference mass spectrum from Bacillus kwashiorkori SIT6T strain. Spectra from 12 individual colonies were compared and projects with the Nextera Mate Pair sample prep kit (Illumina). gDNA a reference spectrum was generated was quantified by a Qubit assay with the high sensitivity kit (Thermo Fisher Scientific, Waltham, MA) to 66.2 ng/μl. The mate pair library was prepared with 1 μg of gDNA using the Nextera mate pair on the Covaris device S2 in microtubes (Covaris, Woburn, MA). The Illumina guide. The gDNA sample was simultaneously fragmented library profile was viewed on a High Sensitivity Bioanalyzer LabChip and tagged with a mate pair junction adapter. The pattern of the (Agilent Technologies Inc.) and the final concentration library was fragmentation was validated on an Agilent 2100 Bioanalyzer (Agilent measured at 59.2 nmol/L. The libraries were normalized at 2 nmol/L Technologies Inc., Santa Clara, CA) with a DNA 7500 LabChip. The and pooled. After a denaturation step and dilution at 15 pmol/L, the DNA fragments ranged in size from 1 kb to 11 kb, with an optimal pool of libraries was loaded onto the reagent cartridge and then onto size at 3.927 kb. No size selection was performed and 505 ng of the instrument along with the flow cell. An automated cluster gen- tagged fragments were circularized. The circularized DNA was me- eration and sequencing run was performed in a single 39-hr run in chanically sheared to small fragments with an optimal size of 597 bp a 2 × 251 bp. SECK et al. | 5 of 13

FIGURE 3 Gel view comparing Bacillus kwashiorkori SIT6T 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

FIGURE 5 Transmission electron microscopy of Bacillus T T kwashiorkori SIT6 using a Morgani 268D (Philips) at an operating FIGURE 4 Gram staining of Bacillus kwashiorkori SIT6 voltage of 60 kV. The scale bar represents 500 nm

The predicted bacterial protein sequences were searched against 2.8 | Genome annotation and analysis GenBank and Clusters of Orthologous Group (COG) databases using Open reading frames (ORFs) were predicted using Prodigal (http:// BLASTP. The tRNAs and rRNAs were predicted using the tRNAScan- prodigal.ornl.gov/) with default parameters. However, the predicted SE and RNAmmer tools, respectively. Signal peptides and numbers ORFs were excluded if they spanned a sequencing gap region. of transmembrane helices were predicted using SignalP (Nielsen, 6 of 13 | SECK et al.

Engelbrecht, Brunak, & von Heijne, 1997) and TMHMM (Krogh, TABLE 2 Cellular fatty acid composition Larsson, von Heijne, & Sonnhammer, 2001), respectively. Mobile Mean genetic elements were predicted using PHAST (Zhou, Liang, Lynch, Fatty acids IUPAC name relative %a Dennis, & Wishart, 2011) and RAST (Aziz et al., 2008). ORFans were 15:0 iso 13-Methyl- tetradecanoic acid 19.6 ± 1.2 identified if their BLASTP E-value was lower than 1e-03 for align- 16:0 Hexadecanoic acid 19.5 ± 0.4 ment length >80 amino acids. If alignment lengths were <80 amino 17:0 anteiso 14-Methyl- hexadecanoic acid 16.5 ± 1.3 acids, we used an E-value of 1e-05. Such parameter thresholds have already been used in previous work to define ORFans. Artemis 18:1n12 6-Octadecenoic acid 12.7 ± 1.9 (Carver, Harris, Berriman, Parkhill, & McQuillan, 2012) and DNA 18:0 Octadecanoic acid 9.3 ± 0.4 plotter (Carver, Thomson, Bleasby, Berriman, & Parkhill, 2009) were 17:0 iso 15-Methyl- hexadecanoic acid 6.9 ± 1.7 used for data management and visualization of genomic features, 15:0 anteiso 12-Methyl- tetradecanoic acid 4.5 ± 0.1 respectively. The mauve alignment tool (version 2.3.1) was used 18:2n6 9,12-Octadecadienoic acid 4.0 ± 0.2 for multiple genomic sequence alignment (Darling, Mau, Blattner, & 16:0 iso 14-Methyl- pentadecanoic acid 3.9 ± 0.5 Perna, 2004). The mean level of nucleotide sequence similarity at 18:1n5 13-Octadecenoic acid 1.5 ± 0.1 the genome level between B. kwashiorkori and other Bacillus species 14:0 Tetradecanoic acid TR was estimated using the Average Genomic Identity of Orthologous 17:0 Heptadecanoic acid TR Gene Sequences (AGIOS) in-house software (Ramasamy et al., 15:0 Pentadecanoic acid TR 2014). This software combines the functionality of other software 14:0 iso 12-Methyl- tridecanoic acid TR programs: Proteinortho (Lechner et al., 2011) (detects orthologous a proteins between genomes compared two by two, then retrieves the Mean peak area percentage ± standard deviation (n = 3); TR, trace amounts <1%. corresponding genes) and the Needleman–Wunsch global alignment algorithm (determines the mean percentage of nucleotide sequence identity among orthologous ORFs). Finally, the Genome-to-Genome strain SIT6T relative to other closest type species with a validly pub- Distance Calculator (GGDC) web server (http://ggdc.dsmz.de) was lished name using maximum-likelihood, neighbor-joining, and maxi- used to estimate the similarity between the compared genomes mum parsimony methods, respectively. Consequently, as this 16S (Auch, Jan, Klenk, & Göker, 2010; Meier-Kolthoff, Auch, Klenk, & rRNA nucleotide sequence similarity was lower than the threshold Göker, 2013). of 98% recommended by Tindall, Rosselló-Mora, Busse, Ludwig, Average nucleotide identity at the genome level be- and Kämpfer (2010) to delineate a new species; it was classified as a tween B. kwashiorkori (CTDX00000000) and the other species new species called Bacillus kwashiorkori SIT6T (Table 1). Furthermore, B. firmus (BCUY00000000), B. shackletonii (LJJC00000000), B. smithii this percentage of similarity comprised in the range of percentage (BCVY00000000), B. aquimaris (LQXM00000000), B. thermoamylo- similarity of Bacillus species (82.7–100%), confirming the new spe- vorans (CCRF00000000), B. coagulans (CP003056), B. alveayuensis cies status (Rossi-Tamisier, Benamar, Raoult, & Fournier, 2015). The (JYCE00000000), B. sporothermodurans (LQYN00000000), B. acidicola reference spectrum for strain SIT6T was thus incremented in the (LWJG00000000), and B. ginsengihumi (JAGM00000000) was esti- URMITE database (http://www.mediterranee-infection.com/article. mated using BLASTN and a home made software, following the algo- php?laref=256&titre=urms-database) (Figure 2) and then compared to rithm described by Ouk, Chun, Lee, and Park (2016). other known species of the genus Bacillus. The differences exhibited are shown in the obtained gel view (Figure 3).

3 | RESULTS AND DISCUSSION 3.2 | Phenotypic description 3.1 | Strain identification and phylogenetic analyses Growth of strain SIT6T was observed between 25°C and 56°C on Strain SIT6T was first isolated in May 2014 after a 30-day preincu- 5% sheep blood Colombia agar and optimal growth was achieved bation in a blood culture bottle with sheep blood and cultivation on at 37°C after 24 hr incubation in aerobic conditions (37°C was the 5% sheep blood-enriched Colombia agar in an aerobic atmosphere temperature at which this strain grows most rapidly). Poor growth at 37°C. No significant MALDI-TOF score was obtained for strain occurred under microaerophilic and anaerobic conditions. Cells SIT6T against the Bruker and URMITE databases, suggesting that the were motile and sporulating. Colonies were circular, white with a isolate was not a member of a known species. Strain SIT6T shared mean diameter of 5 mm on blood-enriched Colombia agar. Gram 94.1% 16S rRNA gene sequence similarity with B. thermoamylovorans staining (Figure 4) showed gram-positive rods. Using electron mi- DKPT (NR_029151) using GenBank NCBI database (reference RNA croscopy, the rods had a mean diameter of 1.8 μm and a length of sequences). Although the 16S rRNA gene sequence of strain SIT6T 5.9 μm (Figure 5). Catalase and oxidase activities were positive for showed 94.58% similarity with Bacillus kokeshiiformis MO-04T and strain SIT6T. 94.57% similarity with Bacillus thermolactis R-6488T by EzBioCloud’s The major fatty acids are saturated species (15:0 iso, 16:0 and 17:0 identify server. Figure 1a, b, and c present the phylogenetic trees of anteiso). Of the 14 detected fatty acids, 11 are saturated, either linear SECK et al. | 7 of 13 (Continues) − + NA NA − + + + Anaerobic Facultative NA NA + 0.6–1 NA + NA NA + B. coagulans NA + + − + − − NA − NA − − − NA − + + + Anaerobic Facultative − NA NA 0.7–4.5 + NA NA NA − B. alveayuensis − − NA NA − − − − NA − − − + + − + + + Aerobic NA NA NA NA NA NA NA NA NA B. sporothermodurans NA NA NA NA NA NA − NA NA − NA NA − NA − + NA + Anaerobic Facultative NA NA NA 3.1–5.9 + NA NA NA NA B. acidicola NA − − − NA + + NA − NA a strains − + − + NA + + v Aerobic NA NA NA 0.45–1 − NA NA NA − B. aquimaris NA NA NA NA + NA − NA NA − Bacillus − − + + − + + + Anaerobic Facultative + + NA 0.5–1.1 + − + + − B. smithii − + − + − + + − + NA − + + + − + − + Anaerobic Facultative NA NA NA 0.6–10 − NA NA NA − B. thermolactis NA + NA NA − + + NA NA − with other phylogenetically close T SIT6 − + − + − + − + Anaerobic Facultative NA NA NA 0.45–4.0 − NA NA NA − B. thermoamylovorans NA − + − + + − NA NA − Bacillus kwashiorkori − − + + − + + + Anaerobic Facultative − − + 0.2–3.0 − − + − − B. kwashiorkori − − − − + − − − − + μ m) - d β - glucosamine β - mannosidase acetyl- Galactosidase Sorbitol Raffinose Sorbose glucosaminidase xylopyranose - - Ribose - Xylose - Adonitol - - - Arabinose - - Rhamnose Urease Nitrate reductase Oxidase Production of Catalase Indole Endospore formation Motility Gram stain Acid from Oxygen requirement Erythritol Inositol Alpha- N - acetyl- Cell diameter ( Glycerol Dulcitol N - Methyl- Esculin Properties d l d d d l l d d TABLE 3 Phenotypic characteristics of 8 of 13 | SECK et al. Kim, Clerck (De

(Yoon,

T T 6 Milk + B. coagulans − NA − + − + + + + 12 2- TF- Bacillus coagulans Sea + B. alveayuensis NA NA NA + − + + − − Bacillus aquimaris Bacillus and 2005), 2005), Kim, & et al., Lee, (Bae

T (Bae,

Water, milk − B. sporothermodurans NA NA NA − − + − + − T 173 FIGURE 6 Graphical circular map of the chromosome. From

NRS- outside to the center: Genes on the forward strand colored by clusters of orthologous groups (COG) categories (only gene assigned sphagnum NRRL Acidic + B. acidicola − NA NA + + + + + v to COG), genes on the reverse strand colored by COG categories

Bacillus alveayuensis TM1 (only gene assigned to COG), RNA genes (tRNAs green, rRNAs red), G+C content and G+C skew 2005), Bacillus smithii Sea − B. aquimaris NA NA NA − − + + + − TABLE 4 Nucleotide content and gene count levels of the (Albert,

T 2011), genome

et al., Attribute Value % of total

Genome size (bp) 2,784,637 100 Milk + B. smithii NA − − + + + + + + DNA coding region (bp) 2,319,082 83.28 (Coorevits

T DNA G+C content (bp) 980,134 35.19 Bacillus acidicola 105- 2

6488 Total genes 2,646 100 R- RNA genes 74 2.79 2012), tRNA genes 63 2.38 Milk − B. thermolactis NA NA NA − v − − + −

et al., Protein-coding genes 2,572 97.20 Genes with function 1,749 68.00 prediction Bacillus thermolactisBacillus

(Heyndrickx Genes assigned to COGs 1,715 66.68

T Protein associated with 487 18.93 1995), hypothetical protein et al., Oil − B. thermoamylovorans NA NA NA − − + + − − Protein associated with 156 6.06 ORFan Blanc Genes with peptide signals 250 9.72 Genes with transmembrane 720 27.99 (Combet-

helices T Genes associated with PKS 6 0.2 Human gut − B. kwashiorkori − − − + + + + + − DKP or NRPS Bacillus sporothermodurans M215 Genes associated with 1,402 54.51

2003), mobilome Genes associated with toxin/ 84 3.26 Park, antitoxin &

Oh, The total is based on either the size of the genome in base pairs or the Lyxose Fucose Glucose Fructose Lactose

- - - Mannose - mannitol - - - Maltose - total number of protein-coding genes in the annotated genome. - Arabitol Habitat Galactose Properties d d l d d d d d d Bacillus thermoamylovorans NA, not available; v, variable. et al., 2004) . Kang, TABLE 3 (Continued) a SECK et al. | 9 of 13

TABLE 5 Number of genes associated with the 25 general COG functional categories

Code Value %a Description

J 130 5.05 Translation A 0 0 RNA processing and modification K 141 5.48 Transcription L 140 5.44 Replication, recombination, and repair B 1 0.04 Chromatin structure and dynamics D 18 0.70 Cell cycle control, mitosis, and meiosis Y 0 0 Nuclear structure V 43 1.67 Defense mechanisms T 88 3.42 Signal transduction mechanisms M 81 3.15 Cell wall/membrane biogenesis N 26 1.01 Cell motility Z 0 0 Cytoskeleton W 0 0 Extracellular structures U 26 1.01 Intracellular trafficking and secretion O 87 3.38 Posttranslational modification, protein turnover, chaperones C 120 4.67 Energy production and conversion G 137 5.33 Carbohydrate transport and metabolism E 138 5.37 Amino acid transport and metabolism F 39 1.52 Nucleotide transport and metabolism H 52 2.02 Coenzyme transport and metabolism I 67 2.61 Lipid transport and metabolism P 147 5.72 Inorganic ion transport and metabolism Q 34 1.32 Secondary metabolites biosynthesis, transport, and catabolism R 241 9.37 General function prediction only S 180 6.99 Function unknown − 857 33.32 Not in COGs aThe total is based on the total number of protein-coding genes in the annotated genome.

TABLE 6 Genomic comparison of Bacillus kwashiorkori with other Bacillus spp.

Genome accession Species Strain number Genome size (Mb) GC (%) Gene content

B. kwashiorkori SIT6T CTDX00000000 2.78 35.19 2,572 B. alveayuensis TM1T JYCE00000000 6.70 38.13 6,689 B. shackletonii LMG 18435T LJJC00000000 5.29 36.70 4,727 B. coagulans 2–6T CP003056 3.07 47.29 2,971 B. ginsengihumi Gsoil 114T JAGM00000000 3.92 35.85 3,832 B. firmus IAM 12464T BCUY00000000 4.97 41.45 4,922 B. aquimaris TF-12T LQXM00000000 4.42 44.57 4,432 B. sporothermodurans M215T LQYN00000000 4.04 35.65 4,211 B. smithii NRRL NRS-173T BCVY00000000 3.38 40.75 3,619 B. acidicola 105-2 T LWJG00000000 5.13 39.39 4,876 B. thermoamylovorans DKPT CCRF00000000 3.70 37.27 3,441 or branched (iso and anteiso). The fatty acid composition of strain Bacterial cells were resistant to metronidazole, but susceptible to SIT6T is detailed in Table 2. imipenem, doxycycline, rifampicin, vancomycin, amoxicillin, ceftriax- Table 3 shows the biochemical features of B. kwashiorkori SIT6T one, gentamicin, trimethoprim/sulfamethoxazole, erythromycin, cip- and the most closely related species. rofloxacin, and gentamicin. 10 of 13 | SECK et al.

(Table 7). Moreover, B. kwashiorkori shares 455, 500, 340, 375, 541, 3.3 | Genome properties 490, 461, 283, 451, and 476 orthologous genes with B. alveayuensis, The genome is 2,784,637 bp long with 35.19% G+C content (Figure 6). B. shackletonii, B. coagulans, B. ginsengihumi, B. firmus, B. aquimaris, B. It is composed of 16 scaffolds, composed of 16 contigs. Of the 2,646 sporothermodurans, B. smithii, B. acidicola, and B. thermoamylovorans, predicted genes, 2,572 were protein-coding genes, and 74 were RNAs respectively (Table 7). Of the species with standing in nomenclature, (7 genes are 5S rRNA, 2 genes are 16S rRNA, 2 genes are 23S rRNA, ANI values ranged from 66.46% between B. coagulans and B. aquima- and 63 genes are tRNA). A total of 1,749 (68%) were assigned as ris to 72.53% between B. sporothermodurans and Bacillus shackleto- putative function (by COGs of NR blast). A total of 156 genes were nii. When comparing B. kwashiorkori to other species, the ANI value identified as ORFans (6.07%). The remaining genes were annotated ranged from 66.74% between B. kwashiorkori and B. coagulans to as hypothetical proteins (487 genes [18.93%]). Genome content is de- 69.92% between B. kwashiorkori and B. thermoamylovorans (Table 8). tailed in Table 4, while Table 5 presents the distribution of the genes The low ANI values confirmed it as a new species because ANI val- into COG functional categories. ues bigger than 95 indicated that strains belong to the same species The genome sequence has been deposited in GenBank under ac- (Konstantinidis, Ramette, & Tiedje, 2006). Finally, digital DNA–DNA cession number CTDX00000000. hybridization (dDDH) estimation of the strain SIT6T against the compared genomes confirmed its new species status, as it ranges between 18.6 and 38.3 (below the cutoff of 70%). 3.4 | Comparison with other Bacillus spp. genomes

The draft genome of B. kwashiorkori (2.78 Mb) is smaller in size than those of Bacillus alveayuensis, Bacillus shackletonii, Bacillus coagulans, 4 | CONCLUSION Bacillus ginsengihumi, Bacillus firmus, Bacillus aquimaris, Bacillus sporothermodurans, Bacillus smithii, Bacillus acidicola, and Bacillus thermoa- Based on the phenotypic properties (Table 2), phylogenetic tree mylovorans (6.70, 5.29, 3.07, 3.92, 4.97, 4.42, 4.04, 3.38, 5.13, and (Figure 1), MALDI-TOF analyses (Figure 3), and genomic comparison 3.70 Mb, respectively) (Table 6). Bacillus kwashiorkori has a lower G+C (taxonogenomics [Table 6 and Table 7] and GGDC results), we propose content (35.19%) than those of B. alveayuensis, B. shackletonii, B. co- the creation of B. kwashiorkori sp. nov. represented by the strain SIT6T. agulans, B. ginsengihumi, B. firmus, B. aquimaris, B. sporothermodurans, B. smithii, B. acidicola, and B. sporothermodurans (38.13%, 36.70%, 4.1 | Description of B. kwashiorkori sp. nov 47.29%, 35.85%, 41.45%, 44.57%, 35.65%, 40.75%, 39.39%, and 37.27%, respectively) (Table 6). The protein content of B. kwashiorkori Bacillus kwashiorkori (kwa.shi.or.ko’ri. L. adj. masc., in reference to (2,572) is lower than those of B. alveayuensis, B. shackletonii, B. co- Kwashiorkor) species are gram-positive, facultative aerobic, short agulans, B. ginsengihumi, B. firmus, B. aquimaris, B. sporothermodurans, rods, 1.8–5.9 μm in size, and motile by means of peritrichous flagella B. smithii, B. acidicola, and B. thermoamylovorans (6,689, 4,727, 2,971, and sporulating. Colonies are creamy white, circular, 4–5 mm in diam- 3,832, 4,922, 4,432, 4,211, 3,619, 4,876, and 3,441, respectively) eter after cultivation at 37°C for 24 hr on 5% sheep blood-enriched (Table 6). However, the distribution of genes into COG categories Colombia agar. Growth occurs at temperatures in the range of 25–56°C is similar in all compared genomes (Figure 7). In addition, AGIOS (optimally at 37°C). It is catalase and oxidase positive. Concerning the values ranged from 54.77% to 67.06% among the Bacillus species biochemical characteristics, the API 50CH strip showed positive reac- compared (Table 7). The range of AGIOS varied from 54.77% to tions for d-glucose, d-fructose, d-mannose, arbutin, esculin ferric cit-

65.50% between B. kwashiorkori and other compared Bacillus species rate, salicin, d-maltose, saccharose, d-trehalose, melezitose, d-raffinose,

FIGURE 7 Distribution of functional classes of predicted genes of Bacillus kwashiorkori SIT6T with 10 members of Bacillus genus SECK et al. | 11 of 13 68.28 67.43 70.06 68.50 69.06 67.80 69.39 68.33 70.47 68.17 718 640 596 676 604 862 375 700 616 838 B. ginsengihumi 100 B. ginsengihumi 3832 67.06 871 848 577 616 693 451 887 603 67.64 68.70 70.61 68.41 67.01 68.46 68.34 69.87 68.31 4876 B. acidicola 1073 B. acidicola 100 59.39 60.56 779 574 553 639 461 837 530 1026 4,211 B. sporothermodurans 68.67 68.75 72.53 68.41 68.16 67.69 68.44 68.88 B. sporothermodurans 100 63.93 58.53 65.22 583 755 484 455 839 736 525 6,689 B. alveayuensis 67.90 68.17 68.24 68.91 66.84 67.81 67.96 B. alveayuensis 100 57.12 56.32 55.77 58.03 526 508 340 529 628 466 2,971 B. coagulans 67.52 66.74 67.95 68.52 67.87 66.46 B. coagulans 100 genus Bacillus 57.09 58.45 57.46 59.13 58.01 574 476 642 656 457 66.74 69.92 67.68 67.95 66.77 3,441 B. thermoamylovorans B. thermoamylovorans 100 63.91 55.62 56.18 63.75 57.02 63.01 490 984 905 510 68.02 67.41 68.69 67.95 B. aquimaris 4,432 B. aquimaris 100 57.15 60.21 59.33 57.05 58.46 57.66 56.28 283 552 592 67.68 67.94 68.67 B. smithii 3,619 B. smithii 100 58.77 56.48 54.96 57.07 60.75 59.44 55.88 55.92 500 928 68.07 68.52 B. shackletonii 4,727 B. shackletonii 100 57.72 58.30 58.14 57.97 57.07 57.61 56.57 58.30 59.30 541 B. firmus 4,922 68.67 100 B. firmus proteins shared between genomes (upper right triangle), average percentage similarity of nucleotides corresponding to orthologous protein shared between 63.97 58.24 54.77 65.14 58.97 65.50 56.84 59.33 62.32 55.15 B. kwashiorkori 2,572 100 B. kwashiorkori B. acidicola B. thermoamylovorans B. coagulans B. alveayuensis B. sporothermodurans B. ginsengihumi B. kwashiorkori B. firmus B. shackletonii B. aquimaris B. smithii B. kwashiorkori B. firmus B. shackletonii B. smithii B. aquimaris B. ginsengihumi B. coagulans B. alveayuensis B. sporothermodurans B. acidicola B. thermoamylovorans genomes (lower left triangle), and number of proteins per genome (bold numbers) TABLE 7 Number of orthologous TABLE 8 Average nucleotide identity (ANI) pairwise comparisons among sequenced species in the ANI values are in percentages. 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