Genotypic Profiling of Bacillus cereus Recovered from Some Retail Foods in Ogun State, Nigeria, and Their Phylogenetic Relationship

Identifying Bacillus cereus with conventional methods is neither specific nor rapid because of the close relatedness of the B. cereus group, hence the need for molecular methods. Genotypic profiling of B. cereus isolates from food was obtained by Random Amplified Polymorphic DNA-polymerase chain reaction (RAPD-PCR) using OPR13 primer. A dendrogram was drawn with the Numerical Taxonomy System of Statistic (NTSYS) software. Thirty of the isolates were subjected to molecular identification by 16S rDNA sequencing. The thirty sequences were deposited in GenBank for accession number. Phylogenetic relationship of the 16S rDNA sequence obtained was carried out with the Multiple Alignment using Fast Fourier Transform (MAFFT) software version 7.0. The evolutionary tree was drawn using the Molecular Evolutionary Genetics Analysis (MEGA 6) software. The dendrogram generated for the RAPD profile showed that all the strains are closely related, with a similarity coefficient of 70%. The isolates were confirmed with 16S rDNA sequencing as B. cereus. The thirty sequences deposited in GenBank were given accession numbers: KX574760–KX574769, KX610811–KX610820, MT757957-MT757963, and MT772282-MT772284. By comparing the phylogenetic relationship, eleven of the strains did not cluster with the reference strains from the GenBank but form distinct clades, which means they are likely to be of different ancestors. Conventional methods rarely differentiate bacteria of the same species into clade, neither can it describe their ancestral lineage. Therefore, it is important to employ molecular methods in identifying bacteria to give detailed information about them.


Introduction
Bacillus cereus is Gram-positive rod, motile with peritrichous flagella, and ubiquitous. It can multiply in soil [1] and has also been found in food [2][3][4]. It survives adverse conditions through endospore formation [2,5]. Spores are more resistant to dry heat and radiation than vegetative cells, and resistance to heat is more considerable in reduced water activity food [6]. e B. cereus group is now made up of seven members [7] in which B. cereus, B. anthracis, and B. thuringiensis are the most significant and are closely related [8]. erefore, identifying members of the B. cereus group with biochemical or physiological characteristics is difficult because these characteristics complicate their accurate peculiarity [9]. Biochemical tests do not give distinct differentiation among the groups, but molecular techniques provide fast and accurate identification of the microorganisms [10]. Desai and Varadaraj [3] used polymerase chain reaction (PCR) to confirm twelve isolates as B. cereus out of twenty-six isolates previously characterized as B. cereus with the conventional method.
RAPD, which is a form of PCR, is used to study the genetic diversity of an individual. e short RAPD primers of 10 nucleotides are used to amplify the random sequences of the targeted DNA with low temperature during the annealing stage. It is a widely used tool in the study of genetic diversity, population and evolutionary genetics, animalplant-microbe relationships, plant and animal breeding, pesticide/herbicide resistance, and forensic studies [17][18][19].
Phylogenetics has made it possible to study the evolutionary history and associations that exist among individuals and groups of organisms, e.g., species, population, or genes. Phylogenetic inference is used to ascertain such association, and the outcome is represented with a phylogenetic tree. e three main inference methods for deducing molecular phylogenies are maximum parsimony, maximum likelihood, and pairwise distances [20]. Phylogeny is presently used in many disciplines such as molecular biology, epidemiology, genetics, ecology, conservation biology, evolution, and forensics.
In Nigeria, foods are retailed by people with little or no knowledge about personal hygiene. Vegetables are sometimes put on the bare floor and handled with bare hands before being sold to consumers. Cooked foods like rice and spaghetti sometimes had contact with the hands of the seller when the spoons and fork used in taking the food are supported with bare hands that have been used in handling money laden with pathogenic bacteria. Smoked fish and hides are exposed to dust, flies and sold with bare hands to consumers. Fried meats are sold inside old newspapers, which might have been contaminated. Knowing fully well that B. cereus has been described as a volatile human pathogen, there is need to confirm the isolates recovered from these retailed foods as B. cereus using 16S rDNA sequencing, to carry out genotypic profiling of the isolates to confirm if strains in retailed foods are the same, and to determine the relationship of some of the genes with closely related genes in GenBank.

Materials and Methods
2.1. Extraction of Genomic DNA. One hundred B. cereus isolates from some retailed foods previously identified with biochemical tests were randomly selected for further confirmation with molecular methods. Each bacterial isolate was subcultured on Luria-Bertani Agar (Merck, South Africa) and incubated at 37°C for 18-24 hours. Genomic DNA was extracted using the Zymo Soil DNA kit (Zymo Research, USA) by following the manufacturer's instructions written in the manual.

Genetic Typing of Bacillus cereus with RAPD-PCR.
e OPR13 primer was used for the amplification of random segments of the genomic DNA of Bacillus strains. e RAPD-PCR assay was performed in a reaction volume of 25 μl containing 12.5 μl Master mix, 11 μl nuclease-free water, 0.5 μl primer, and 1 μl DNA template. e PCR cycles consisted of an initial denaturation step at 94°C for 4 min, then 40 cycles comprising DNA denaturation at 94°C for 1 min, primer annealing at 35°C for 1 min, and DNA extension at 72°C for 2 min, then a final extension step at 72°C for 5 min [23]. RAPD-PCR bands were separated by electrophoresis in a 2.0% (w/v) agarose gel (LASEC) containing SYBR safe stain (Life Technologies, ermofisher), immersed in Tris/Acetic acid/EDTA (TAE) (BioRad, USA) buffer, run at 80 V for 90 min. Molecular Ruler ( ermofisher) 1kb base pairs (bp) ladder was used as a molecular weight marker. After the migration of DNA bands, the gel was photographed on the Gel Doc 2000 Image analyzer (BioRad, USA).

Analysis of RAPD-PCR Profile.
Each gel was examined, and the presence or absence of polymorphic bands in individual lanes was scored 1 and 0, respectively. e scored bands were subjected to the Numerical Taxonomy System of Statistic (NTSYS) software. In NTSYS analysis, the scatter diagram of the scored bands is useful in revealing a grouping.

16S rDNA Identification.
e primer set F1/R2 was used for the identification. ey were performed in reaction mixtures containing 25 μl Master mix, 22 μl nuclease-free water, 1.0 μl primer, and 2 μl template DNA making a final volume of 50 μl. e PCR amplification was performed in a thermal cycler (C1000 Touch, BioRad) with the following conditions: initial denaturation at 96°C for 5 minutes, followed by 30 cycles at 96°C for 45 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 2 minutes, and a final extension at 72°C for 5 minutes.
e PCR product was analyzed and viewed as described above.

Sequencing of the PCR Product.
irty PCR products from 16S rDNA were submitted to Inqaba Biotechnical Industrial (Pty) Ltd, Pretoria, South Africa, for sequencing. e PCR products were sequenced in the forward and reverse directions on the ABI PRISM ™ 3500xl Genetic Analyser. Purified sequencing products were analyzed using CLC Main Workbench 7 followed by a BLAST search in the National Centre for Biotechnology Information (NCBI). e thirty sequences were deposited in GenBank for accession number.

Phylogenetic Analysis.
16S rDNA gene of the B. cereus was also used for the phylogenetic analyses in order to establish the relationship among them. e search for possible reference nucleotide sequences in the NCBI Gen-Bank database was achieved by using the partial sequences from the 16S rDNA of Bacillus cereus [24]. Multiple alignments of nucleotide sequences were obtained with the software Multiple Alignment using Fast Fourier Transform (MAFFT) version 7.0 [25], while two main methods distance-based and character-based were employed in drawing the tree using Molecular Evolutionary Genetics Analysis 6 (MEGA 6) [26].

Genetic Profiling.
Results from Agarose gel electrophoresis using Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR) for the genetic profiling of the isolates showed that many of the isolates expressed multiple bands characteristic of RAPD-PCR (Figures 1(a)-1(c)). Some showed a single band while others did not show any band at all. Some of the isolates on the lanes SP 5 Figure 1). e dendrogram generated from computer analysis of the RAPD-PCR using the NTSYS software revealed that all the bacteria are closely related while those grouped together, that is, MP 75 and SW 89 , only showed single band and SW 79 and WR 123 did not produce any band while GP 27 and WR 30 , MP 104 and SW 134 are the same strain ( Figure 2). e OPR13 primer used for the genetic profiling gave a good representation. Many of the isolates showed multiple bands characteristic of RAPD-PCR. Oh et al. [11] employed RAPD-PCR using the OPR13 primer SG5  WR147  RB84  MT154  MP113  SG139  WR94  JR189  SG99  WR176  WR193  RB8  WR39  RB155  MP75  SW89  MP117  SW79  WR123  SG166  CB72  WR88  PM112  WR71  SW151  SW136  PM60  SG61  WR150  SP102  SP100  MP104  SW134  TT120  SG129  SG127  MT22  SW131  PM62  JR168  SW68  WR91  SP96  WR130  MP149  WR163  TT158  SW124  JR170  GP137  CR10  WR13  TT11  CB26  CB135  PN32  TT35  WR140  TT143  MT200  MT167  WR36  GP114  TT106  WR126  GP87  SG118  CR122  SG93  RB29  SW98  MP34  SG38  MP111  RB31  MT181  SG187  WR165  WR186  MT191  SW86  PN142  MT146  GP153  SG169  RB157  SP160  SG192  WR90  SP6  SG17  GP27  WR30   for typing of Bacillus isolates. eir B. cereus strains were classified into 19 banding patterns. ey reported that the RAPD patterns obtained with OPR13 distinguished better than OPA3. RAPD and Pulsed-Field Gel Electrophoresis (PFGE) were used to assess the similarity between emetic B. cereus strains. Seventeen (17) distinct banding patterns were obtained, while 10 strains did not give any banding pattern with PFGE [27]. Also, in this research, five strains SW 79 , MP 117 , WR 123 , RB 155 , and SG 166 did not give any bands, but the ImageLab software was able to detect bands for MP 117 and SG 166 . Strains WR 123 and SW 79 did not produce any band based on the analysis, while the remaining three clustered very close to them. Ghelradi et al. [28] also used RAPD-PCR to identify B. cereus to the strain level and concluded that the method is useful in describing intraspecific changes among organisms.

3.2.16S rDNA Identification of B. cereus.
e universal couple of primers F1/R2 was able to detect the 16S region of the isolates with a molecular size of 1500 bp. e Basic Local Alignment Search Tool (BLAST) result that has the highest similarity with the biological sequence in the National Centre for Biotechnology Information (NCBI) database was recorded as the identity of the isolates. e sequences deposited in GenBank with their accession numbers and source of each isolate are presented in Table 1. e isolates were identified as Bacillus cereus. Oh et al. used the universal primer F27/R1492 and reported that their strains have high similarity with members of the B. cereus group in the GenBank.

Phylogenetic Relationship of B. cereus.
e phylogenetic relationship between B. cereus and the very closely related strains from the GenBank were analyzed using NeighbourJoining (NJ) and MaximumLikelihood (ML) trees (Figures 3 and 4). e percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. All positions greater than 50% are shown. e two trees were employed to establish the proven resolution and statistical significance of the various treeing algorithms [29,30]. e NJ tree revealed that RB 8   is high bootstrap value expressed by the aforementioned Bacillus spp. is beyond 70% borderline of the degree of relatedness proposed by [31]. Furthermore, SG 5 , RB 29 , RB 31 , GP 87 , MP 111 , MP 113 , SG 129 , JR 168, and SG 169 form distinct clades with a bootstrap value lower than 50% but with closest relative to B. cereus. ey did not cluster with any strains as a result of their nucleotide signature pattern peculiarity, which is in line with the findings of [32]. is distinctiveness calls for their novelty as reported by [30]. is NJ result was based on a cluster-based algorithm utilized in calculating the pairwise distance between sequences and group sequences that are most similar. For clearness, the character-based method (Maximum Likelihood) chooses the best model for the variation pattern of the sequences by correlating a set of data against the set of models of evolution [33]. high similarity percentage greater than 90% with B. cereus of the reference sequences from the GenBank.
is compares well with the result obtained from the NJ with the exception of RB 8 and RB 155 that exhibited lower than 50% homology with their Bacillus relatives. is may be as a result of mutation.Hence, the relationship between these strains and their Bacillus spp. relatives has been wiped out [30]. ML deduced that RB 29 and JR 168 have very low similarity percentage with their relative, which is almost the same with the NJ tree.
is is not reliable because their DNA reassociation is above the threshold level based on the result depicted by the ML tree [34]. ML tree also showed that some B. cereus from this research did not align with any of the reference taxa based on their uniqueness. ese include SG 5

Conclusions
is research has shown that some of the isolates are the same as revealed by the dendrogram generated for the RAPD profile. Eleven of the strains form a distinct clade that can never be deduced with conventional methods. e B. cereus isolates are associated with foods such as runner bean and  Figure 4: Maximum Likelihood phylogenetic tree based on partial 16S rDNA gene sequence, showing the phylogenetic relationships between the Bacillus cereus and the most closely related strains from GenBank. Sequences obtained in this study higher than 90% are denoted with the yellow triangle, greater than 50%, but lower than 80% with the blue rectangle and less than 50% with a red circle. green pea, which are used in the preparation of fried rice, while meat pie is one of the snacks consumed by people to satisfy hunger. Extra caution must be taken when preparing foods with these ingredients, and snacks such as meat pie should be consumed when hot to prevent food poisoning.

Data Availability
e data used to support the findings of this study were deposited at NCBI GenBank.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.