In vitro antibacterial activities and molecular characterization of bacterial species isolated from farmlands against selected pathogens

Highlights • Screening bacterial isolates for antagonistic potential.• Molecular methods for identification of isolates.• Six potent antibacterial isolates against the test pathogenic species.• Pseudomonas was susceptible to antagonizing strains.• Necessity for new effective antibacterial formulation.


Introduction
Since ancient times, humans have been faced with innumerable number of diseases. Most of these diseases are caused by microorganisms [1][2][3][4]. In the treatment of diseases, various therapeutic measures have been put in place. Since the discovery of a microorganism with antibacterial activity in 1928, there have been incessant studies and use of microbes for the production of antimicrobial compounds against disease etiological agents [5].
Most of these microorganisms with antimicrobial potentials have been isolated in various habitats. The most suitable habitat of microorganisms as reported by [6] is the soil. It is a loose natural component consisting of mixtures of organic components and minerals [7].
Soil-dwelling microorganisms with antimicrobial potentials have been reported by various research findings. [8,9] reported antimicrobial activities of some fungal species such as Penicillium spp., Aspergillus spp., Ganoderma lucidium and Absidia corymbifera against the growth of Candida species, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. According to [10], these antimicrobial activities were as a result of the production of some compounds which include nigerazine B, tensidol A and so on. Research findings have also reported the antimicrobial potential of bacterial species such as Escherichia coli, Pseudomonas species, Actinobacteria and so on [11][12][13]. Etiological agents of human and plant diseases which include Salmonella species, Clostridium difficile, Fusarium verticillioides and Phaeomoniella chlamydospora, are susceptible to their antimicrobial effects [14,15]. They also produce some antimicrobial compounds such as hydrogen cyanide, monoacetylphloroglucinol (MAPG) and microcin for growth inhibition. Actinobacteria are widely known for antimicrobial activities against an enormous number of pathogens [16,17]. They produce over 60 % of antimicrobial compounds used around the world today. Two common genera of these bacteria widely studied for antimicrobial potential include Streptomyces and Micromonospora. They produce antibacterial agents belonging to the class of antibiotics which include macrolides, β-lactams, aminoglycosides, glycopeptides and so on [18][19][20]. Bacillus species are also known for antibacterial potential against the growth of common human and plant pathogens such as Staphylococcus aureus, Escherichia coli, and Rhizoctonia solani [7,8,21,22]. They are frequently studied for the production of antimicrobial compounds of varying structures and chemical properties. Such compound includes bacteriocin [23].
Most of these antimicrobial compounds have become less effective day by day due to the emergence and development of resistance to these compounds by the disease etiological agents [24]. This makes most diseases very difficult to treat [3], propelling the need to do more research findings and exploring farmland soil which could be a potential environment to discover microorganisms with appreciable antibacterial potential [9]. This study, therefore aims to investigate the antibacterial potential of bacterial isolates from Olabisi Onabanjo University farmland.

Collection of soil samples
The soil samples (50 g) were randomly collected from farmland in Olabisi Onabanjo University, Ago-Iwoye, Ogun State, Nigeria around maize and pineapple plantation. The soil top layer was collected aseptically in a container. Soil samples were taken to the laboratory immediately after collection (Fig. 1).

Source of test organisms
The test organisms were Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Bacillus subtilis and Pseudomonas aeruginosa. Stock culture of these organisms was obtained from Nigerian Institute of Medical Research (NIMR). The cultures were subcultured on solidified nutrient agar prior to use [25].

Isolation of organisms
A series of dilution were carried out in order to obtain numerable number of colony [26]. These dilutions were evenly spread on the solidified nutrient agar (NA) and incubated for 2 days at 30 C. The resulted colonies were enumerated and subjected into the subsequent purification and subculture, on the NA medium [10,27].

Standardization of the test organisms
The 0.5 McFarland Standard prepared was used to standardize the test organisms [28]. The stock cultures were firstly purified by sub-culturing on solidified nutrient agar and incubating for 24 h at 37 C. They were then inoculated into sterile nutrient broth and incubated for 4 h at 37 C. The turbidity of the bacterial suspension was compared with the turbidity of the prepared standard by placing both tubes near a white sheet of paper having black stripes to enhance easy detection of turbidity differences [11].

Preparation of cell free supernatant (CFS)
The pure isolates were inoculated into nutrient broth and incubated 48 h at 30 C. The CFS was obtained by centrifuging the broth culture at 5000 g force for 15 min and used immediately [12,29].

Screening of isolates for antibacterial activities
The antibacterial activity of the isolates CFS was determined by the employment of the well diffusion method as described by [30]. Using a sterile swab stick, standardized culture of test organisms was streaked evenly on the entire surface of the solidified agar plate. The plates were allowed to dry for 15 min. A sterile cork borer hole of 9 mm in diameter was used to create wells on the solidified agar containing streaked organisms. In each well, 0.1 mL cell free supernatant culture of isolates was dispensed. The plates were incubated for 24 h at 30 C. The diameters of inhibition were recorded in millimeter for each isolates [31].

Identification of the antibacterial potent isolates
The isolates that displayed antibacterial activities were characterized based on their biochemical characteristics. This was achieved by carrying out gram staining, catalase test, urease test and starch hydrolysis [32]. Molecular characterization of these isolates was also performed [33].
Gram staining was carried out to identify isolates if they are gram-positive or gram-negative. The isolates were smeared on a clean, grease-free and dry glass slide. It was allowed to air-dry completely and then heat fixed by passing through the flame 3 times. It was allowed to cool for 15 min. Crystal violet stain (primary stain), Lugol's iodine, Acetone-alcohol decolourizer and Safranin (counter-stain) were dispensed on the smear. Each reagent was allowed to stay on the smear for 60 s, followed by subsequent rinsing with clean water. Each stained smear was placed on a draining rack to air dry. The smear was then observed microscopically first with 40x objective to check the staining and secondly with an oil immersion objective to report the cellular morphology of the isolates and their ability to retain the primary stain [34]. Colonies were tested for the possession of catalase enzyme, whereby a pure colony was placed on a surface of clean, dry glass slide using a loop. A drop of 3% H 2 O 2 was placed onto the slide and mixed [35]. The production of bubbles was checked for.
For urease test, urease medium in test tubes was inoculated with a loopful of pure culture of the isolates. The cap was loosely fixed on the tubes. The test tubes were incubated at 35 C in ambient air for 18-24 hours. Agar slant was observed for colour change.
Isolates were inoculated on the starch agar to test for their ability to hydrolyse starch. Plates were incubated for 24 h at 37 C. After growths were observed, plates were covered with iodine. The productions of clear zones around growths were checked for, after 10 min [36].

Molecular identification
The antagonist isolates were inoculated into sterile nutrient broth and incubated at 37 C for 24 h. The resulting bacterial cells were suspended and re-suspended into 200 mL of water [37].

DNA extraction
Fifty milligram (50 mg) (wet weight) of the bacterial cells that have been re-suspended were dispensed into a ZR Bashing TM Lyses Tube. Lysis Solution (750 mL) was added. This tube was secured in a bead beater fitted with 2 mL tube holder assembly. The Mixture was processed at maximum speed for 5 min [38]. This was subjected to centrifugation using a micro centrifuge at >10,000 x g for 1 min. The resulting supernatant (400 mL) was transferred to a Zymo-Spin TM IV Spin Filter (orange top) in a collection tube and was centrifuged at 7000 x g for 1 min. The base of this Zymo-Spin TM Spin filter was snapped off before use.
Bacterial DNA Binding Buffer (1,200 mL) was added to the filtrate in the collection tube.
The mixture (800 mL) was transferred to a Zymo-Spin TM IIC Column in a collection tube and centrifuge at 10,000 x g for 1 min.
The flow was discarded through the collection tube and 800 mL of the mixture (1,200 mL of Bacterial DNA Binding Buffer and the filtrate in the collection tube from the initial centrifugation of 400 mL supernatant) was again transferred to the Zymo-Spin TM IIC Column in a collection tube. This was centrifuged at 10,000 x g for 1 min. Two hundred (200) ml DNA Pre-Wash Buffer was then added to the Zymo-Spin TM IIC Column in a new collection tube and centrifuge at 10,000 x g for 1 min. Bacterial DNA Wash Buffer (500 mL) was added to the Zymo-Spin TM IIC Column and centrifuge at 10,000 x g for 1 min. The Zymo-Spin TM IIC Column was transferred to a clean 1.5 mL Microcentrifuge tube. DNA Elution Buffer (100 mL) was added directly to the column matrix. This was centrifuged at 10,000 x g for 30 s to elute the DNA [37].

DNA amplification
The extracted DNA, which is about 900 bp was utilized as a template for 16S rRNA gene amplification. This gene was amplified using the forward and reverse primers (16SF: GTGCCAGCAGCCGCGCTAA and 16SR: AGACCCGGGAACGTATTCAC) of about 0.5 mL. Taq 5 u/ul (0.1 mL), 10x PCR buffer (1.0 mL), 25 mM Mgcl 2 (0.1 mL), 0.1 mL (DMSO), 2.5 Mm DNTPs (0.8 mL), 10 ng/ml DNA (2.0 mL) and 3.1 mL distilled water was used. Thermal cycler of Eppendorf 96AG, model 2231 was utilized in the amplification process. PCR conditions include initial denaturation at 94 C for 5 min. Another denaturation at 94 C for 30 s. Annealing was at 56 C for 30 s. Initial extension was at 72 C for 45 s. Final extension was at 72 C for 7 min. The Holding temperature of the process was 10 C. All the three processes (Denaturation, Annealing and Extension of primers) were repeated 36 times giving rise to 36 cycles [38].

PCR product purification
The PCR products, also known as amplicons were purified by adding absolute ethanol (2 volumes) to the products. This mixture was incubated at room temperature for 15 min and spin down at 10,000 rpm for 15 min. Supernatant was decant and also spin down at 10,000 g force for 15 min. Two (2) volumes (40 mL) of 70 % ethanol were added and supernatant was decant and air dry.

Agarose gel electrophoresis
The amplicons (resulting amplification products) from these processes were loaded on 1.0 % agarose gel. This was prepared by dissolving 1.0 g of agarose powder into 100 mL of 20x stock solutions SB buffer. SB buffer was prepared by dissolving 8 g of NaOH into 45 g Boric acid in 1 l of distilled water. 250 mL of this solution was added to 4.75 l of water. The gel solution was evenly mixed by stirring and placed in microwave for 2 min for homogeinity and to produce solidified gel when cooled [40].
The gel was left to cool and dispensed into gel plate having combs placed in the gel caster. This was left to solidify. Solidified gel was placed in the electrophosis chamber. 3 mL of loading dye was added to 7 mL of DNA and was dispensed into holes created by the combs. 3 mL ethidium bromide was incorporated into the gel to enhance easy visualization of the DNA under ultraviolet light. All of these were subjected to electrophoresis for 45 min at 80 V [41]. The ladder used was hyper ladder 1 from Bioloine (Fig. 2).

Data processing of the isolates' 16S rRNA sequences
The DNA was loaded on the Applied Biosystem 3500 genetic analyser from Applied Biosystems to give the sequences [42].
The BioEdit sequence alignment editor was used for editing and aligning the sequences. These edited sequences were exported and saved in FASTA format. The 16S rRNA gene sequences were compared with sequences in the Gene Bank database using basic local alignment search tool (BLAST) in the National Centre for Biotechnology Information (NCBI) to check for similarities with other existing sequences.
Accession number was assigned to each related sequence by the NCBI gene bank. The isolates' 16S rRNA gene sequences with high similarities to the sequences were compared for relatedness using Molecular Evolutionary Genetics Analysis (MEGA) version 5.05 [43]. This was used to construct a maximum likelihood phylogenetic tree.

Molecular phylogenetic analysis
The evolutionary history of isolates was inferred by using the Maximum Likelihood method based on the Tamura-Nei model [44]. The tree with the highest log likelihood (-695.55) was 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 Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis involved 5 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 511 positions in the final dataset. Evolutionary analyses were conducted in Molecular Evolutionary Genetics Analysis (MEGA) version 5.05 [45].

Statistical analysis
Chi-Square of independence test was used to test for significant association between the inhibition and the isolates [76].  (Table 2).

Results
Chi-square test result (in Tables 3 and 4) tested for significant association between the inhibitions and the isolates. The null hypothesis of no significant association was rejected based on the likelihood ratio p-value = 0.000 at 5% significance level. Hence, it was concluded that the isolate had significant association with the inhibition. It was, also, affirmed that there was significant and moderate [76] association of 51.2 % (Phi value in Tables 5 and 6). The antagonism percentage displayed by antagonistic isolates as shown in Table 7, reveals Is 35 having 50 % inhibitory activity. 20 % Table 1 Antibacterial activities of Isolates in millimeter (mm).

Escherichia coli Pseudomonas aeruginosa
Zone of Inhibition (mm)

Discussion
Farmland contains enormous microorganisms, including those with unique characteristics due to its nutritious constituents. These include organic matter, moisture, essential elemental components like nitrogen, phosphorus, potassium and so on. This facilitates their growth, multiplication and viability [46,47]. Among these unique characteristics include antibacterial potentials. According to [48], these potentials are as a result of metabolites they produced. Cell free supernatant (CFS) of bacteria     contain these metabolites, and has been recently used by researchers to elicit antimicrobial effects on pathogens. [49,50], used the CFS of Bacillus species to inhibit the growth of bacterial pathogens. [51], reported the antimicrobial activities of various antimicrobial compounds which include lactic acid, acetic acid and H 2 O 2 in the CSF of Weissella cibaria against malodor inducing bacteria like Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia.
In this study, the CFS of all the thirty five (35) isolates was utilized to examine their antibacterial potential on the pathogens using the agar well diffusion method. Among the thirty-five (35) isolates, six (6) isolates showed antibacterial activities. The Chisquare test results (in Table 3 through 5) established that there was a significant and moderate association of 51.2 % (Phi value in Table 5). This means that all the inhibitions significantly dependent on the isolate and there was significant correlation of 51.2 % between the inhibitions and the isolates.
Obtained results reveled; the antagonist isolates were rodshaped, gram-positive, catalase-producing and starch-hydrolyzing isolates. These morphological characteristics of soil isolates were similar to the characteristics reported by [52] from soil bacteria in Ngere tea catchment area of Murang'a County, Kenya. Most of the soil isolates were also rod-shaped, gram-positive, catalaseproducing, starch-hydrolyzing, oxidase and phosphate-producing isolates. These characteristics in this present study are not sufficient to identify the isolates to the genus level [53].
This phenotypic method of identification is not reliable due to different phenotypic characteristics observed in bacteria of similar genera but of different species [54]. Molecular identification is more reliable and accurate for organism identification. Conserved regions within the 16S rRNA gene of the microbes' DNA are usually utilized, which enhance sequence comparison. These provide an essential tool for the study evolutionary phylogenetic and molecular diversity [55]. Therefore, the antagonists isolates were subjected to molecular analysis for identification. Their DNA size was around 850 bp in length when subjected to Electrophoresis. According to [56], DNA size usually utilized for sequencing and comparison ranges from 500 bp to1500 bp. Therefore the utilization of 850 bp DNA size is suitable for sequencing and comparison.
According to [58], these Bacillus species are predominantly soil bacteria, though they can be found in other habitats. [  Vast numbers of research findings utilise 16S rRNA gene to identify these bacteria due to its high level of accuracy for identification. [60,61], identified Brochothrix species and Bacillus species respectively, using their 16S rRNA genes, all of which exhibit antagonizing activities. In a research study carried out by Rafiq et al. [62], an isolate from Passu glacier in Pakistan indicated as HTP6 was identified as Alcaligenes faecalis HTP6 by sequencing its 16S rRNA gene. Lysinibacillus species was also identified in a study carried out by [49] and [63] by sequencing similar genes.
The biochemical characteristics of Bacillus species in this present study, which include gram-positive, catalase-positive, urease-negative and starch hydrolyser were similar to the characteristics reported by [ [64],48]. In their research study on the identification of Bacillus species, most Bacillus species isolated were gram-positive, catalase-positive, and unable to produce urease and starch-hydrolyzing bacteria.
Brochothrix species are commonly identified as food spoilage bacteria isolated from a wide range of animal food. Their presence in the soil as reported in this present study confirms other habitat such as soil they can be isolated for study. Their biochemical characteristics which include gram-positive, catalase-positive, urea-negative and inability to hydrolyse starch conform to the report of [65]. He reported their ability to produce catalase enzymes. They are gram-positive and cannot utilise exogenous urea.
Gram-positive, catalase-positive, urea-positive and inability to hydrolyse starch are the biochemical characteristics of Lysinibacillus sphaericus reported in the study. These characteristics were also reported by [26].
In this study, in vitro antibacterial activity screening of 35 isolates, only 6 isolates were able to exhibit antibacterial activity with varying percentage of antagonism. Lysinibacillus sphearicus strain PRE16 displayed highest antibacterial activities with 50 % antagonism percentage. It inhibits the growth of Bacillus subtilis, Staphylococcus aureus and Escherichia coli with zones of inhibition 23.00 AE 2.00, 18.00 AE 2.00 and 20.00 AE 4.00 respectively. [66], also reported the inhibition of Bacillus subtilis, Staphylococcus aureus and Escherichia coli by Lysinibacillus sphearicus with a zone of inhibition of 20 mm, 21 mm and 19 mm respectively.
Bacillus sp. BCN2, Bacillus arsenicus strain CSD05, Bacillus aryabhattai strain KNUC205, Brochothrix thermosphacta strain P30C4 and Alcaligenes faecalis strain KEM24 displayed antibacterial activity against Pseudomonas aeruginosa Figure Plate 3 According to [6,47,66,[68][69][70] the antibacterial activities displayed by the antagonizing bacteria are as a result of production of antibacterial agents Surfactin and bacteriocin which include mersacidin and erisin are some of the antibacterial compounds produced by Bacillus species responsible for their antibacterial activities [23,[71][72][73][74]. Brochocin-C is a bacteriocin produced by Brochothrix species, known for its broad spectrum activity [68]. [62], reported two antibacterial compounds produced by Alcaligenes species, namely kalimantacin and tunicamycin. These compounds were reported active against a vast number of pathogenic species, including the antibiotic resistance species. Lysinnibacillus species have been observed to produce siderophores and biosurfactants which are cell destructing metabolites [46]. They have been utilized for the production of silver nanoparticles, which exhibit antimicrobial activity due to the possession of a crystallographic surface structure and large surface to volume ratios [49,66].

Conclusion
This study demonstrated that farmland is a potential habitat for the isolation of bacteria with antibacterial activity against the growth of bacteria known for antibiotic resistance. Lysinibacillus sphaericus strain PRE16 displays highest antibacterial activity, inhibiting the growth of Bacillus subtilis, Staphylococcus aureus and Escherichia coli. Pseudomonas aeruginosa was susceptible to the antibacterial activity of Bacillus species, Brochothrix thermosphacta and Alcaligenes faecalis. These bacteria species could be a potential diseasecontrol agent in eradicating most antibiotic resistance bacteria species. Further research includes characterizing the antibacterial agents responsible for the antagonizing activity, evaluating their activities and screening their expressions in different conditions. Development of effective formulation and techniques is also needed to improve the antibacterial effect of these bacteria.

Author statement
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Declaration of Competing Interest
The authors report no declarations of interest.