Complete genome sequence of Bacillus cereus FORC_005, a food-borne pathogen from the soy sauce braised fish-cake with quail-egg

Classification and features

B. cereus is a Gram-positive, rod-shaped, motile, and spore-forming bacterium. It is often found in various habitats including soil, water, and even food materials (fresh vegetables and food animals). In particular, this bacterium has been well-known food-borne pathogen causing diarrhea, vomiting, and nausea by enterotoxin production. It is a facultative anaerobe that can survive in the temperature range of 10–50 °C, pH range of 4.9–9.3, and salinity of up to 7.5 % NaCl. B. cereus belongs to the family Bacillaceae , the order Bacillales, the class Bacilli, and the phylum Firmicutes. In this study, B. cereus FORC_005 was isolated from a contaminated Korean side dish, soy sauce braised fish-cake with quail-egg, which was suspected to be an original pathogen of food-borne outbreak in March 2014, by Incheon Health and Environment Institute, South Korea. Morphology observation using Transmission Electron Microscopy (TEM; JEM–2100, JEOL, Tokyo, Japan) showed that the strain FORC_005 is rod-shaped with about 4 μm long and 0.5 μm wide in diameter, and can perform motility with peritrichous flagella, suggesting that the strain FORC_005 has typical morphology of B. cereus (Fig. 1 and Table 1). In addition, the 16S rRNA sequence analysis and phylogenetic tree analysis of the strain FORC_005 and other Bacillus species revealed that this strain was identified to B. cereus (data not shown) and positioned at the group of B. cereus in the phylogenetic tree, indicating that this strain indeed belongs to B. cereus (Fig. 2).

MIGS ID	Property	Term	Evidence codea
	Classification	Domain Bacteria	TAS [7]
		Phylum Firmicutes	TAS [8–10]
		Class Firmibacteria	TAS [8, 9, 11]
		Order Bacillales	TAS [8, 9, 12, 13]
		Family Bacillaceae	TAS [8, 9, 13, 14]
		Genus Bacillus	TAS [8, 9, 13, 15]
		Species Bacillus cereus	TAS [8, 9, 13, 16]
		Strain FORC_005
	Gram stain	Positive	TAS [17, 18]
	Cell shape	Rod	TAS [17, 18]
	Motility	Motile with peritrichous flagella	TAS [17, 18]
	Sporulation	Endospore-forming	TAS [8]
	Temperature range	10 °C–50 °C	TAS [8, 17]
	Optimum temperature	28–35 °C	TAS [8]
	pH range; Optimum	4.9–9.3; 6.0–7.0	TAS [17]
	Carbon source	Glucose, Aesculin	TAS [19]
MIGS-6	Habitat	Ubiquitous; especially in soil	TAS [17, 18]
MIGS-6.3	Salinity	0–7.5 % NaCl (w/v)	TAS [17]
MIGS-22	Oxygen requirement	Facultative anaerobes	TAS [17, 18]
MIGS-15	Biotic relationship	free-living	NAS
MIGS-14	Pathogenicity	Diarrhea, emesis in human	NAS
MIGS-4	Geographic location	Incheon, South Korea	IDA
MIGS-23	Isolation	Korean soy sauce braised fish-cake with quail-egg	IDA
MIGS-5	Sample collection	March 2014	IDA
MIGS-4.1	Latitude	37.27 N	IDA
MIGS-4.2	Longitude	126.42 E	IDA
MIGS-4.4	Altitude	Not reported

^a Evidence codes - IDA inferred from direct assay, TAS traceable author statement (i.e., a direct report exists in the literature), NAS non-traceable author statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [20]

Genome project history

Growth conditions and genomic DNA preparation

B. cereus FORC_005 was aerobically cultivated at 30 °C for 12 h with Brain Heart Infusion (BHI; Difco, Detroit, MI, USA) media, and the cells were harvested by centrifugation at 16,000 x g for 1 min. Total genomic DNA was extracted and purified using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions for Gram-positive bacteria. Bacterial cells (about 2 X 10⁹ CFU) were harvested by centrifuging for 10 min at 5000 x g and pellet was resuspended with 180 μl of enzymatic lysis buffer (20 mM Tris · Cl, pH 8.0, 2 mM sodium EDTA, 1.2 % Triton X-100, and 20 mg/ml lysozyme) and this mixture was incubated at least 30 min at 37 °C. In addition to the mixture, 25 μl of proteinase K and 200 μl of Buffer AL, which are included in the kit, were mixed by vortexing before incubation at 56 °C for 30 min. And then 200 μl of absolute ethanol was added and mixed thoroughly by vortexing. The mixture was transformed to DNeasy Mini spin column in a new 2 ml collection tube and centrifuged at 6000 x g for 1 min to remove the flow-through. Then, 500 μl of Buffer AW2 was added and centrifuged for 3 min at 20,000 x g to wash the genomic DNA in the column. The column was placed into a clean 1.5 ml tube and 200 μl of Buffer AE was directly added onto the DNeasy membrane. After incubation at room temperature for 1 min, the column was centrifuged for 1 min at 6000 x g to elute the purified genomic DNA. The concentration and purity of the purified DNA was determined using a NanoVue spectrophotometer (GE Healthcare, Little Chalfont, UK).

Genome sequencing and assembly

The genome sequence was determined using a hybrid-genome sequencing approach with PacBio RS II (Pacific Biosciences, Menlo Park, CA, USA) and Illumina MiSeq (Illumina, San Diego, CA, USA). Library construction for PacBio RS II was initialized by ligating universal hairpin adaptors to both ends of DNA fragments using SMRTbell Template Prep kit 1.0 (Pacific Biosciences), followed by purification using AMPure PB bead system (Pacific Biosciences) for the removal of small fragments sized <1.5 kb. Subsequent DNA polymerase binding with template DNAs was conducted using DNA/Polymerase Binding kit P6 v2 with C2 chemistry (Pacific Biosciences), followed by loading of SMRTbells using MagBeads kit (Pacific Biosciences) for greater number of reads at longer lengths per SMRT Cell. Sequencing was conducted using DNA sequencing Bundle 2.0 (Pacific Biosciences), on the PacBio RS II platform. 300 bp paired-end library for Illumina MiSeq (Illumina) was initialized by simultaneously fragmenting template DNAs and tagging them with sequencing adapters using Nextera DNA Sample Preparation kit and Index kit (Illumina), followed by purification of prepared template DNA fragments using MinElute reaction clean up kit (Illumina) and AMPure XP bead (Beckman Coulter, Brea, CA, USA). Sequencing was conducted using MiSeq Reagent kit (600 cycle; Illumina). All kits were used according to the manufacturer’s instructions. Sequencing reads from Illumina MiSeq system were assembled using the CLC Genomics Workbench v7.0.4 (CLC bio, Aarhus, Denmark), and the reads from PacBio system were assembled using the PacBio SMRT Analysis v2.0 (Pacific Biosciences). Finally, the initially assembled scaffolds were gathered and re-assembled to obtain one contig using CLC Genomics Workbench program.

Genome annotation

Initial prediction and annotation of all open reading frames, and tRNA/rRNA gene prediction was carried out using Glimmer3 by the Rapid Annotation using Subsystem Technology server [23], and was confirmed using the GeneMarkS ORF prediction program [24]. Predicted ribosome binding sites by RBSfinder (J. Craig Venter Institute, Rockville, MD, USA) were used to confirm the predicted ORFs. The Global Annotation of Multiplexed On-site Blasted DNA-Sequences program and InterProScan5 program with conserved protein domain databases were used for the annotation of confirmed ORFs [25, 26]. Artemis16 was used for handling of genome sequence and annotated data [27]. The functional categorization and classification of all predicted ORFs were conducted using the RAST server-based SEED viewer and Clusters of Orthologous Groups -based WebMGA programs [28, 29]. Circular genome map, showed in Fig. 3, was generated using GenVision (DNASTAR, Madison, WI, USA) based on all predicted ORFs with COG information, tRNAs and rRNAs, GC-content, and gene cluster information. Detection and identification of virulence factors were carried out using BLAST search with protein sequences of VFs in the database [30]. Signal peptides, transmembrane helices, and Clustered Regularly Interspaced Short Palindromic Repeats were identified by using SignalP server v.4.1 [31], TMHMM server v.2.0 [32], and CRISPRfinder [33], respectively.

Pathogenesis and virulence factors

Frequently, B. cereus causes diarrhea and emesis after ingestion of the contaminated food. These food-borne illnesses are reported to be associated with specific toxin genes. The genome analysis of B. cereus FORC_005 revealed that there are three major toxins including cytotoxin K, hemolysin BL, and non-hemolytic enterotoxin [5]. These toxins are involved in severe diarrhea after infection of B. cereus . Cytotoxin K is encoded by a single gene, cytK (FORC5_0979). However, other two toxins are encoded by two different gene clusters, a hemolysin BL gene cluster (hblABDC; FORC5_2954 to FORC5_2957) and a non-hemolytic enterotoxin gene cluster (nheABC; FORC5_1734 to FORC5_1736)). In addition, hemolysin III (hlyIII; FORC5_2063) was detected in the genome for additional hemolysis activity. Therefore, gene expression regulation of these toxin-associate genes may be key points for control and prevention of food poisoning after pathogenic B. cereus infection.

Anthrolysin O, one of the cholesterol-dependent cytolysins, was detected in the genome (FORC5_1940), which has been suggested to be a pore-forming protein often found in many Gram-positive bacteria. This hemolytic and cytolytic protein was reported to be associated with cholesterol binding in the host cell plasma membrane, pore formation via its oligomerization, and transfer of virulence factors to the host cell cytoplasm [34, 35]. In addition, an internalin (FORC5_1206) was detected in the genome, suggesting that it may play an important role in host cell invasion. The predicted functions of these two host invasive proteins in the genome revealed that regulation and control of this initial step in the occurrence of food-borne pathogenesis and illness may be important for the host protection.

Bacillus has been known to have two kinds of bacterial protection systems from the host cell immune system, including polysaccharide capsule (PSC) and polyglutamic acid capsule [36, 37]. While PSC biosynthesis is common in B. cereus , only PSC biosynthesis gene cluster (FORC5_4952 to FORC5_4971) was detected in the genome of B. cereus FORC_005, suggesting that the strain can protect itself from the host cell immune defense system for its further pathogenesis in the host cells. Therefore, this bacterial protection system is also considered as one of the virulence factors in B. cereus .

Comparative genome analysis

To elucidate the evolutionary relationship of B. cereus FORC_005 with other complete genomes of B. cereus , 16S rRNA sequence-based phylogenetic tree analysis and whole genome-based average nucleotide identity (ANI) analysis were conducted. Comparative phylogenetic tree analysis revealed that B. cereus and B. anthracis strains formed a group including B. cereus FORC_005, suggesting that they may have been evolved from a common ancestor (Fig. 2). Subsequent ANI analysis using the complete genome sequences of strain FORC_005 and other 21 B. cereus strains revealed the closest evolutionary relationship between the strains FORC_005 and B4264 with the ANI value of 98.68 (Fig. 4). The strain B4264 was initially isolated from a male patient with a fatal pneumonia in 1969 [38], indicating that this strain is a clinical isolate. However, the strain FORC_005 was isolated from a B. cereus -contaminated Korean food, suggesting that it may have pathogenesis for potential food-borne outbreak. Therefore, the genome information of the strain FORC_005 may be important to extend our knowledge about the study of food-borne outbreak via ingestion of the contaminated foods in genomic level, even though it is a food isolate B. cereus strain.