Bacillus species are Gram positive rods often arranged in pairs or chains with rounded or square ends and usually have a single endospore . The Bacillus cereus group is a subdivision of the genus Bacillus. It comprises more than 20 closely related species including diverse Gram-positive heterotrophic aerobic and facultative anaerobic bacilli with the ability to form environmentally resistant and metabolically inert spores [2-4]. The B. cereus group is ubiquitously present in various environments including many types of soils, sediments, plants, water, and food [5,6]. Members of the B. cereus group are known to produce numerous enzymes and metabolites and serve as probiotics for animal and plant growth; however, most studies on this group have been focused on the aspects of epidemiology and pathogenesis [3,7,8].
The endospores are generally oval or sometimes round or cylindrical and are very resistant to adverse conditions Bacillus wiedmannii cells are Facultative anaerobe, Gram positive rod shaped bacteria with an average length of 2.8 µm and average width of 1.2 µm. Due to its shape and character, the new microbe is part of the Bacillus cereus group. For people who ingest the microbe, it has the potential to cause food poisoning, according to postdoctoral researcher Jasna Kovac and Rachel Miller, doctoral candidate in food science and the announcement’s lead author. While pasteurization kills the pathogen, making milk safe to drink, the spores although inert can survive in refrigerated conditions. B. wiedmannii creates toxins in the human gut that form cellular pores, making people sick with diarrhea. Due to its shape and character, the new microbe is part of the Bacillus cereus group. For people who ingest the microbe, it has the potential to cause food poisoning, according to postdoctoral researcher Jasna Kovac and Rachel Miller, doctoral candidate in food science and the announcement’s lead author. While pasteurization kills the pathogen, making milk safe to drink, the spores although inert can survive in refrigerated conditions. B. wiedmannii creates toxins in the human gut that form cellular pores, making people sick with diarrhea. Collected from raw milk stored at a dairy powder processing plant silo, the microbe was detected by graduate students and staff using a new whole-genome research tool at Cornell’s Food Safety Laboratory and Milk Quality Improvement Program [9,10].
The issue of food losses is of high importance in the efforts to combat hunger, raise income and improve food security in the world’s poorest countries. Food losses have an impact on food security for poor people, on food quality and safety, on economic development and on the environment. The exact causes of food losses vary throughout the world and are very much dependent on the specific conditions and local situation in a given country. In broad terms, food losses will be influenced by crop production choices and patterns, internal infrastructure and capacity, marketing chains and channels for distribution, and consumer purchasing and food use practices. Irrespective of the level of economic development and maturity of systems in a country, food losses should be kept to a minimum. The real economic cost of food spoilage is difficult to estimate. It is generally considered that circa 30 per cent of manufactured food product is spoiled, microbial food spoilage being the major cause [9,11,12]. In this study, we reported identification of a B. wiedmannii, analyzed the biological, genomic, and potential effective features of the isolate. 16S rRNA gene sequences has been useful in phylogenetic studies at the genus level, its use has been questioned in the case of closely related species groups such as Bacillus, where insufficient divergence in 16S rDNA prevented the resolution of strain and species relationships . Subsequent use of housekeeping genes that are essential and therefore not lost from genomes, but that evolve more quickly than 16S rDNA, has proven to be useful for taxonomic classification . Although such approaches are useful for single isolates studied intensively in the laboratory, 16S rDNA remains the gold standard for environmental sequencing projects due to its ubiquity and ease of amplification from divergent species48. One lineage that suitably illustrates the disagreement between molecular and phenotypic/ecological methods of classification in Bacillus is the B. cereus group. This group, also called B. cereus sensu lato, contains six very closely related species according to current taxonomy: B. cereus, B. thuringiensis, B. anthracis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis .
Materials and Methods
Soil sample was aseptically collected from the rhizospheric soils (10-15cm depth) of rice from farm sites where there has been no previous documented record of Bacillus wiedmannii. The sample was collected at a depth of 2cm to 5cm below soil layer, after scrapping off the topmost soli layer according to the procedure earlier documented by , using a sterile cutlass to clear the highest soil and use sterile meter rule to dig. Whole plant after chopping off the shoots, was carefully uprooted (along with the adhering soil; without breaking the secondary and tertiary roots), picked up the soil using spatula each sample to different sterile petri dish, and labeled was transported to the laboratory on ice bag for the isolation and identification of probable bacterial isolates. Serial dilution was performed on the soil samples. Bacillus wiedmannii were culturally identified using morphological and physiological method using culture media.
Preparation of media
The media used were prepared according to the manufacturer specifications, they were sterilized in an autoclave pressured at 121oc for quarter-hour. The culture media used include: Nutrient agar, Sabouraud Dextrose Agar, Salmonella Shigella Agar, MRS Agar, Centrimid Agar, MacConkey Agar. The plates were incubated at 350C overnight. The number of colonies on the surfaces of the agar plates were counted, and expressed as colony forming units per 1 gram of soil sample (CFU/g).
Catalase test: This test is usually carried out to differentiate between those organism that produce the enzyme catalase such as staphylococci and non-catalase. The principle of this is that catalase acts as catalyst in the breakdown of hydrogen peroxide to oxygen and water. The inoculum was picked using inoculating loop and was placed on a glass slide. A drop of three peroxide (H2O2) was added to the isolate. The assembly of gas bubbles or foaming indicated positive result while no bubble showed a negative result .
Indole test: Indole is a nitrogen-containing compound produced when the amino acid tryptophan is hydrolyzed by bacteria that have tryptophanase. Ten milliters (10ml) of sterile peptone broth was aseptically inoculated with isolate using wire loop leaving one tube un-inoculated to function control. The tubes were incubated at 30̊ c for twenty-four hours. After incubation. 1.0ml of Kovac’s reagent was added. The tubes were then allowed to face for about 20 minutes. Red color at the highest layer indicated positive (+) result implying production of indole whiles a negative (-) result gave a yellow coloration .
Citrate Utilization Test: This was done by streaking sterile and solidified Simon’s citrate agar plates with the isolates leaving one plate un-inoculated to function control and incubated at 30̊C for 48 hours. Color change from greet to blue indicated positive result while original greet indicated negative result .
Starch hydrolysis Test: Using a sterile inoculating loop, the bacterial isolates were streaked on the prepared and solidified starch agar plates and incubated at 37oc for 24 hours. The starch agar plates were covered with iodine solution after incubation. The formation of transparent zone round the colony indicated positive reaction for the test while negative is indicated as a reaction that's otherwise.
Gram staining Techniques
A thin smear of then isolate was made on clean glass slide and was allowed to air-dry before it was gram stained and viewed under 1000X objective lens according to earlier studies .
Motility Test: Motility test was performed using candle wax when loopful sample was introduced onto a slide-covered withy glass slide was sealed with candle wax and observed under wet preparation under 10X and later 40X objectives of light microscope .
Antibiotics Sensitivity and Resistivity
Disc diffusion method was used for the assessment of antibiotics sensitivity and resistivity:
The antimicrobial sensitivity and resistivity test was done using Disc diffusion method according to , on Mueller-Hinton agar. Antibiotics disc containing Gentamicin (GEN) 10, Ofloxacin (OFL)5, Augmentin (AUG)30, Cefuroxime (CRX)30, Erythromycin (ERY)5, Ceftazidime (CAZ)30, Cloxacillin (CXC)5, Ceftrizone (CTR)30, was used. This antibiotics are chosen because they are either used in both animal medicine and human medicine. Sterile cotton swab was use to take broth culture of the test bacteria aseptically. A prepared and sterile Muller Hinton plate is swabbed with the test culture. Swabbing is completed to hide the whole plate. With a sterile forceps, the antibiotics disc is placed on the swabbed agar surface. Disc is gently pressed to make sure contact. Plate is inverted and test culture details were noted on the plate and incubated at 37℃ for twenty-four hours. After incubation, plate was taken out and examined. Diameter of growth inhibition zone is measured employing a ruler. If the antibiotics is effective i.e. bacteria are sensitive to the antibiotics, a transparent (clear) ring, or area of inhibition, is seen round the wafer indicating poor growth. The result obtained were used to classify isolates as being resistant, intermediate resistant or susceptible to a particular antibiotic using standard reference values according to National Committee for Clinical Laboratory Standard.
Bacteria DNA Preparation Kit designed by JENA bioscience for easy and fast isolation of genomic DNA from both gram-positive and gram-negative bacteria samples. The solution based system reduces DNA fragmentation that may be problematic in spin-column or filtration based techniques, because phenol or chloroform is not used it is safe and does not produce any harmful waste. Solution based genomic DNA purification kits guarantee minimal DNA fragmentation and yield DNA sized up to 150 kb. Expected yield of genomic DNA will vary from sample to sample depending on the amount, quality and type of material processed. An amount of approximately 40 μg purified DNA per preparation can be expected.
PCR amplification and sequencing total genomic
Isolates was carried out on the basis of 16SrRNA sequencing. For this, the isolates were sent to Humanizing Genomics MACROGEN laboratory, United States of America. As per the details shared the16rRNA sequence was obtained using Sanger sequencing method. Amplification of the16SrRNA gene was performed usingaT1-Thermocycler PCR machine with 1492R primers pA (5′-TAC GGYTAC CTT GTT ACG ACT T-3′) and 27F primers pH (5′-AGA GTT TGA TCM TGG CTC AG-3'). In Sanger sequencing, a DNA primer complementary to the template DNA (the DNA to be sequenced) is used to be a starting point for DNA synthesis. In the availability of the four deoxy nucleotides triphosphates (dNTPs: A, G, C, and T), the polymerase stretched the primer by the addition of the complementary dNTP to the template DNA strand. So as to know which nucleotide is associated into the chain of nucleotides, four dideoxynucleotides triphosphates (ddNTP: ddATP, ddGTP, ddCTP, and ddTTP) labeled with a distinct fluorescent dye are used to terminate the synthesis reaction. Compared to dNTPs, ddNTP has an oxygen atom dismissed from the ribonucleotide, hence cannot form a link with the next nucleotide. Following synthesis, the reaction products are loaded into four lanes of a single gel based on the diverse chain-terminating nucleotide and subjected to gel electrophoresis. According to their sizes, the sequences of the DNA is then determined (Figure 1,2).
The Sanger sequencing method consists of 6 steps:
1.The double-stranded DNA (dsDNA) is denatured into two single-stranded DNA (ssDNA).
2. A primer that equivalent to one end of the sequence is attached
3. Four polymerase solutions with four types of dNTPs but only one type of ddNTP was added.
4. The DNA synthesis reaction initiates and the chain extends until a termination nucleotide is randomly incorporated.
5. The resulting DNA fragments are denatured into ssDNA.
6. The denatured fragments are separated by gel electrophoresis and the sequence is determined.
For phylogenetic analysis, 16S rRNA genes was sequenced from the Bacillus strains were obtained. Phylogenetic tree was constructed via the neighbor-joining (NJ) methods and the maximum likelihood method [22,23], for 16S rRNA gene with 1,000 bootstrap replicates using Mega X. The average nucleotide identity (ANI) was calculated using EzBiocloud web service (https://www.ezbiocloud.net/net/tools/ani).
Total genomic DNA of strain ABC was extracted using MACROGEN biolabs techniques bacterial Kit. The DNA was subjected to agarose gel electrophoresis by using standard control and quantified. Genome classification was performed with NCBI genome annotation cited at www.ncbi.nlm.gov/genome/annotation using BLAST (Gardner et al., 2009).
Bacillus wiedmannii strain ABC 123 were hemolytic on sheep’s blood, were Gram-stain-positive and were positive for catalase activity, as determined according to the FDA BAM protocols . This strain were oxidase-negative, hydrolyzed starch and casein, as determined by plating on starch using standard methods . All strains were facultative anaerobes, as determined by the BAM method (U.S. Food and Drug Administration, 2015). The tested strain was motile. Out of eight antibiotics (Ceftriaxone, Gentamicin, Ceftazidime, Ofloxacin, Augmentin, Cefuroxime, Erythromycin, Cloxacillin) screening using disc diffusion method the isolates resists all of the antibiotics except Ofloxacin and ciprofloxacin.
Conventional characterization was performed on the isolate by the use of morphological appearances on agar plates, microscope and biochemical. Based on this, Gram positive cells with rod shapes seeming endospore forming were seen under the digital Light microscope which was projected on HP laptop. Colonies with off white appearance was observed under Chocolate Agar and Brain Heart Infusion medium; this of-white appearance disappeared with increasing temperature from 250C ± 420C. All the biochemical test carried out on the isolate turned positive except Indole test which came out negative.
Molecular identification and Phylogeny
Based on 16S rRNA sequencing it was identified as B. weidmanii. The resultant nucleotides sequences from sequencing result was submitted at GenBank for assignment of accession number. The webpage displayed at NCBI was as shown in figure 3,4 below. Further classification of the 1483 bp 16S rRNA was performed using Mega X software to construct phylogenetic tree at alignment using the MUSCLE algorithm; pairwise distance matrices were calculated in MEGA version of X [24,25]. The 16S rRNA genetic phylogeny confirm the close relatedness of B. wiedmannii with other member of Bacillus cereus as shown in the phylogenetic tree.
Evolutionary analysis by Maximum Likelihood method
The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model. The tree with the highest log likelihood (-2066.60) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-model, and then selecting the topology with superior log likelihood value. Data associated with this study has been deposited at GenBank under the accession number MW 362293.1.
In this study, we reported the first isolation from soil and molecular characterization of Bacillus wiedmannii as being reported in earlier studies around the world . The results of the conventional morphological and biochemical characterization was in conformity with earlier studies , which obtained similar results. The identification and characterization according to biochemical and phylogenetic relatedness to Bacillus cereus was earlier confirmed by earlier studies [27,28]. Further characterization by the use of housekeeping genes, confirmation of cytotoxic of the extracellular products, Intracellular infection potential, toxicity to teleost and mice, putative virulence-associated genes in the genome and the use of whole genome sequencing method [2,7], would need until much later when the isolate will be subjected to verify its full genomic features for comparative genomics . Also, further future work is needed to document the impacts of Bacillus wiedmannii in food security in the study area. However, this study could serve as the baseline study for surveillance of probable emergence of potential food spoilage and food poisoning , by the emerging new bacteria strain in the study area.