Characterization of the antimicrobial activity produced by Bacillus sp. isolated from wetland sediment

: Bacteria of the genus Bacillus sp. present the potential for inhibiting various pathogens, making them a promising starting point in the search for new antimicrobial substances. In this study, bacteria were isolated from sediment samples from humid areas of a Natural Conservation Unit in the state of Rio Grande do Sul, Brazil. The isolate Bacillus sp. sed 1.4 was selected for production of antimicrobial activity, and was characterized by MALDI-TOF and 16S rDNA sequencing. Phylogenetic analysis showed that Bacillus sed 1.4 was closely related to Bacillus altitudinis and Bacillus pumilus . The cell-free supernatant was partially purifi ed using ammonium sulfate precipitation, gel fi ltration chromatography (Sephadex G-200) and an ultrafi ltration membrane. Partial purifi cation resulted in specifi c activity of 769.23 AU/mg, with a molecular mass of approximately 148 kDa. This antimicrobial substance showed stability at 100°C for 5 min, and was inactivated by proteolytic enzymes. An antimicrobial effect against Listeria species was observed. Considering the importance of the Listeria genus in the area of food safety, this antimicrobial activity should be further explored, specifi cally in the fi eld of dairy products and with a focus on food biopreservation studies.


INTRODUCTION
In recent decades, new challenges have arisen for the pharmaceutical and food industries regarding the fight against pathogenic microorganisms and spoilage.In the clinical area, the challenges are even greater due to the emergence of antimicrobial resistance, which due to selective pressure, causes microorganisms to no longer respond to the usual antimicrobial drugs (Devatkal et al. 2014, Choi et al. 2019).In this view, exploring new antimicrobial substances has become an object of intensive investigation.The sporulation and rapid growth, which are characteristic of the genus Bacillus, mean signifi cant advantages in terms of survival in different habitats.This is also the reason for the research on their ability to produce bioactive substances (Ebrahimipour et al. 2014, Dimkic et al. 2017, Beltran et al. 2018, Zhao et al. 2018).Bacillus also has industrial applications due to its easy genetic manipulation, favorable cultivation characteristics on a large scale and the ability to secrete GRAS status proteins ("Generally Recognized As Safe") (Zhang et al. 2020).
Bacillus can produce a diversity of antimicrobial substances, including several antimicrobial peptides (Gebhardt et al. 2002, Stein 2005).Bacteriocins, for example, are antimicrobial peptides that can potentially be used as food preservatives.Nisin, produced by Lactococcus lactis, is a bacteriocin that has granted a safe status and has been approved for use as natural food preservative in several countries (Garsa et al. 2014, Bali et al. 2016).
Other studies have shown that antimicrobial lipopetides produced by Bacillus also present relevant inhibitory properties.Surfactin can reduce Salmonella enterica and Legionella pneumophila biofilm formation in urinary catheters.The extract obtained from the Bacillus subtilis I1a strain exhibited an inhibitory effect against planktonic and sessile forms of E. coli, Serratia marcescens, Enterobacter cloacae, Proteus mirabilis, Citrobacter freundii and E. faecalis (Bernat et al. 2016).
It is important to highlight that environments such as soil and sediment, due to the multiplicity of metabolic activities they accommodate, are viable and sustainable paths for the isolation of microorganisms and the obtaining of new antimicrobial compounds (Zhou et al. 2017, Quintero et al. 2018).Thus, the objective of this research was to characterize a substance produced by a bacterium isolated sediment samples from a conservation unit (CU) in southern Brazil in order to verify its antimicrobial activity against clinical and foodborne pathogens.

Identification of the microorganism
The bacterial isolate was obtained from sediment samples from the wetlands of a Conservation Unit (CU), at a park called the Parque Natural Municipal Imperatriz Leopoldina (S 29º45'374"/ W 51º07'992), located in São Leopoldo, Brazil (unpublished data).For identification, the isolate was subjected to analysis by Matrix Associated Laser Desorption-Ionization -Time of Flight (Bruker MALDI-TOF/MS system), MALDI-TOF Biotyper (software v 4.0).From the identification results, a dendrogram was constructed through Principal Component Analysis (PCA).The identification of the bacteria was also performed through the amplification and sequencing of 16S rDNA, run on a Thermal Cycler model 2720 (Applied Biosystems by Life Technologies ®).Primers FC27 (5'-AGAGTTTGATCCTGGCTCAG-3') and R530 (5'-CCGCGGCTGCTGGCACGTA -3') were used (Gontang et al. 2007).The PCR was run for 5 min at 94 °C followed by 35 cycles of 1 min at 95 °C, 1 min at 58 °C and 1 min at 72 °C, with a final extension of 10 min at 72 °C.The sequencing of the samples was performed at the ACTGene Análises Moleculares Ltda company (Porto Alegre, Brazil).The AB 3500 Genetic Analyzer automated sequencer (Applied Biosystems) was used.Sequencing data were collected with the Data Collection 2 program (Applied Biosystems).Results were analyzed using the Chrome program version 2.6.4 (Technelysium Pty Ltd) and compared to the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/).The construction of the phylogenetic tree was performed using the MEGA X software.

Growth curve and production of the antimicrobial substance
The microorganism was cultivated in tryptic soy broth (Kasvi) at 30 °C for 48 h in an orbital shaking incubator at 50 rpm.Aliquots were taken at regular 2-h intervals and evaluated for bacterial growth, which was expressed as colonyforming units per milliliter (CFU/ml) (Miles et al. 1938).The production of the antimicrobial substance was determined by the serial dilution method to measure activity units per milliliter (AU/ml) (Motta & Brandelli 2002).The pH of the culture was evaluated in triplicate using a pH meter (DM-22A Digimed).

Partial purification of the antimicrobial substance
After cultivation, precipitation with ammonium sulfate at 0-60% was performed according to Scopes (1994).The precipitated material was suspended in 10 mM sodium phosphate buffer pH 6.0, and partially purified by liquid gel-filtration column chromatography (Sephadex G-200) eluted with the same buffer.Fractions were collected and the absorbance at 280 nm was determined using a spectrophotometer.The antimicrobial activity of the collected fractions was assessed according to Motta & Brandelli (2002).The fractions presenting antimicrobial activity were fractionated ultrafiltration system, with a 50 kDa retention membrane (Amicon Ultra).The degree of purification of each step was assessed via denaturing polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970) stained with silver nitrate (Heukeshoven & Dernick 1985).The protein concentration was determined through the Folin-Ciocalteu reagent method (Lowry et al. 1951).

Characterization of the partially purified substance
T h e f ra c t i o n s s h o w i n g a n t i m i c ro b i al activity were set apart and assessed for maintenance of antimicrobial activity under different conditions.Thermal stability was determined by exposing aliquots in an Eppendorf Microtube with a volume of 1000 μL at 100°C, in a dry bath, for 3, 5 and 10 min.Aliquots were also exposed to 121 °C/105 kPa for 15 min.To evaluate stability at low temperatures, aliquots were refrigerated at 4 °C for 10, 20 and 30 days and frozen at -20 °C for 30 days.To determine sensitivity to the proteolytic enzymes, aliquots were treated at 37 °C for 60 min with trypsin and papain (2 mg/mL), with 10 mM sodium phosphate buffer pH 6.0.
The chemical stability of the antimicrobial activity against organic solvents and detergents was also tested.To this test, 25 µL of the chemicals were added to 50 µL of partially purified substance, and incubated for 60 min at 37 °C.The solvents employed were: acetone (NEON), methanol (DINAMICA), ethanol (NEON), chloroform (ACROS) xylene (Synth), dimethyl sulfoxide (DMSO) (Synth), butanol (NEON) and ethyl ether (ALPHA) at a final concentration of 50% (v/v).The detergents used were Tween 20 and Tween 80 at a final concentration of 10% (v/v) (Motta et al. 2007a).The potential synergistic effects of organic solvents were also investigated in this study.At the end of each treatment, the aliquots were examined to verify antimicrobial activity against the indicator bacteria Listeria monocytogenes ATCC 7644 (Motta & Brandelli 2002).

Inhibitory spectrum
The antimicrobial activity was tested against cultures of clinical and food isolates.Suspensions of these indicator cultures were prepared in 0.85% NaCl, corresponding to 0.5 turbidity on the MacFarland scale (approximately 1.5 x 10 8 CFU/mL), and spread with a swab on Mueller Hinton agar plates.Twenty µL of the partially purified fractions were applied in duplicate.The plates were incubated for 24 h at the optimum temperature for each indicator microorganism.

Identification of the selected isolate
The bacterial isolate sed 1.4 was identified at genus level as Bacillus sp.through the MALDI-TOF/MS analysis, considering the score result (score 2.048).The data obtained were used to construct a dendrogram, which is arranged according to the levels of similarity among the phenotypic expression of proteins present in the strains (Figure 1).The comparison was carried out with Bacillus sp.sed 1.4 and Bacillus strains present in the MALDI-TOF database.Based on Figure 1, it could be suggested that the isolate Bacillus sp.sed 1.4 is more closely related to Bacillus altitudinis, with a distance level near 100, whereas its relationship to Bacillus pumilus is more distant, slightly greater than 100.However, both bacteria share an ancestor with common phenotypic characteristics.

Growth curve and production of the antimicrobial substance
The antimicrobial activity production by Bacillus sp.sed 1.4 was observed during the exponential growth phase at 9 h cultivation.Maximum antimicrobial activity of 200 AU/mL was reached in the stationary phase, between 12 and 30 h (Figure 3), followed by a decline to 100 AU/mL after 33 h incubation.The indicator bacterium was L. monocytogenes ATCC 7644.After obtaining this result, crude supernatant obtained at 24 h cultivation was sampled and used for purification steps.The pH of the culture remained constant (7.0) throughout the incubation period.

Partial purification of the antimicrobial substance
The precipitation with ammonium sulfate was carried out with the crude supernatant obtained after 24 h culture.It was found that the grouped saturation bands (0 to 60%) reached an activity of 3200 AU/mL when tested against the indicator strain L. monocytogenes ATCC 7644.
The pellet resulting from precipitation with ammonium sulfate was resuspended with 10 mM sodium phosphate buffer pH 6.0, and applied to a gel filtration chromatography.Of the 30 fractions collected, fractions 8 and 9 showed antimicrobial activity corresponding to 100 AU/ mL each.Three repetitions were performed and the elution profile was the same.
The fractions exhibiting antimicrobial activity were grouped and subjected to ultrafiltration using membranes with a cut-off molecular weight of 50 kDa.In this purification step, activity was observed in the retained fraction, with inhibition zones of 12 mm on average; while the antimicrobial activity of 100 AU/mL was maintained, the filtered fraction did not show antimicrobial activity against the indicator strain L. monocytogenes ATCC 7644.
As seen in Table I, it was noted that the antimicrobial substance showed 22.37 AU/ mg specific activity in the crude supernatant, while the fractions obtained after gel filtration chromatography presented 769.23 AU/mg, with a final yield of 2%.The protein profiles of the crude supernatant, ammonium sulfate precipitate and the antimicrobial substance resulting from gel filtration chromatography, was evaluated by means of polyacrylamide gel electrophoresis (Figure 4).In the polyacrylamide gel, a main band of the active fractions eluted from the Sephadex G-200 column was observed, with a molecular weight of approximately 148 kDa.

Characterization of the antimicrobial substance
In assessing the stability of the studied antimicrobial substance, it was seen that antimicrobial activity was maintained at 100% when treated at 100 °C for up to 5 min, but complete inactivation occurred after exposure for 10 min at the same temperature, and also at 121 °C/15 min.When refrigerated at 4 °C, it maintained 100% antimicrobial activity for 10 days, and for 30 days when frozen at -20 °C.The antimicrobial substance was completely inactivated when treated with the proteolytic enzymes papain and trypsin.
As for the effects of chemicals and detergents, it can be seen that there was complete inactivation when treated with Tween 80.When antimicrobial stability was tested against acetone, 66% residual activity was observed and above 80% when tested with methanol, ethanol, chloroform, xylene, diethyl ether, dimethyl sulfoxide, butanol and Tween 20, as shown in Table II.

Determination of the antimicrobial activity spectrum
In this evaluation, the antimicrobial substance demonstrated activity against the tested Listeria species, with inhibition zones between 10 and 13 mm (Table III).However, there was no inhibition of gram-negative bacteria, yeasts and other gram-positive species.

DISCUSSION
The identification of Bacillus sp.sed 1.4 was performed using MALDI-TOF and by sequencing the 16S rRNA gene.However, the methodologies were not sufficient for identification at the species level.Although the 16S rRNA gene is widely used for the identification of bacteria, it is limited for the genus Bacillus as it contains several groups of closely related species.This can make identification challenging.Bacillus altitudinis, Bacillus stratosphaericus e Bacillus aerophilus and Bacillus pumilus, for example, have very similar sequences of the 16S rRNA gene, making it difficult to identify them based on this type of analysis (Starostin et al. 2015).Elbanna et al. (2014), report that B. altitudinis and B. pumilus showed 99% similarity to each other after having their 16S rRNA gene sequences analyzed.Thus, techniques such as differential PCR of the genes rpoB, gyrB, nifD, recA and atpD, have been studied as alternatives in distinguishing among of Bacillus species (Ki et al. 2009, Bhandari et al. 2013, Quintero et al. 2018).For instance, the rpoB gene, which is homogeneous within cells because it is a single-copy gene, has relatively long sequences (approximately 3.5 kb in Bacillus), and many of these sequences are available in public databases (Ki et al. 2009).
An Acad Bras Cienc (2021) 93(Suppl.4) e20201820 8 | 13 finding confirms the close relationship between species.Data from MALDI-TOF/MS demonstrate that Bacillus sp.sed 1.4 is closely related to Bacillus altitudinis, with a level of similarity near to 100.The correlation with the species Bacillus pumilus showed a similarity level to the clusters of 400.Based on the reference database obtained in a comparison of MALDI-TOF data, Starostin et al. (2015) identified bacterial isolates such as Bacillus pumilus and Bacillus altitudinis.The dendrograms also showed that both groups have a similarity level of 400.
Bacillus sp.sed 1.4 was evaluated for its antimicrobial potential.The antimicrobial substance produced by this isolate demonstrated maximum activity in the stationary phase between 12 and 30 h, which is characteristic of a secondary metabolite.In a previous study, Boottanun et al. (2017), investigated the production of metabolites with antimicrobial activity secreted by Bacillus amyloliquefaciens.These metabolites were produced early in the incubation period until reaching a steady state between 12 and 72 h.Chalasani et al. (2015) also detected the beginning of antimicrobial substance production at 9 h cultivation, reaching a maximum production at 40 h.The activity of some secondary metabolites decreases when the producing bacteria enter the stationary phase, due to the adsorption of molecules on the surface of the producing cells and proteolytic degradation (Guo et al. 2012, Chikindas et al. 2018).
The antimicrobial substance produced by Bacillus sp.sed 1.4 was partially purified using precipitation with ammonium sulfate at 0 -60% saturation, gel filtration chromatography and an ultrafiltration membrane.The antimicrobial substance has an apparent molecular mass greater than 50 kDa, as it was retained in the ultrafiltration membrane and showed approximately 148 kDa during electrophoresis, which is included in the fractionation range of the Sephadex G-200 resin (5 to 600 kDa).According to the literature, the antimicrobial substances produced by Bacillus are generally small, showing molecular weight below 30 kDa (Zhao et al. 2018).Antimicrobial peptides produced by Bacillus spp.evaluated by denaturing polyacrylamide gel electrophoresis often shown a molecular weight of approximately 3 kDa or less (Chalasani et al. 2015, Regmi et al. 2017).However, the large molecular weight observed in this study may suggest that the substance is secreted in the form of aggregates.This corroborates findings described by Cladera-Olivera et al. (2004), in which Bacillus licheniformis P40 produced an antimicrobial substance of approximately 150 kDa when the sample was eluted using Sephadex G-100.These results are also similar to those reported for the bacteriocin Linocin M18 (Valdés-Stauber & Scherer 1994).It has been suggested that the association of molecules in large aggregates is possibly due to the highly hydrophobic nature of the peptides.Motta et al. (2007b) demonstrate the effects of a peptide produced by Bacillus P34 on the growth of Listeria monocytogenes and Bacillus cereus with antimicrobial activity of 160 AU/mL over a period of 24 h.At the end of the partial purification process, the fraction was purified 32.9 times with a yield of 2% and specific activity of 769.23 AU /mg.A protease produced by Bacillus cereus was purified by chromatographic precipitation techniques with ammonium sulfate (50%), ion exchange (DEAE-Cellulose) and filtration gel (Sephadex G-100) by Lakshmi et al. (2018), resulting in a protein with specific activity of 300 AU/mg and recovery of 34.6%.Mothe & Sultanpuram (2016) purified an enzyme produced by a new species Bacillus caseinilyticus and it was found that purification using DEAE cellulose column chromatography increased purity by 20.74 times, with specific activity of 89.2 AU/mg.
The effects of proteolytic enzymes, thermal treatments and organic solvents and detergents on the antimicrobial substance produced by Bacillus sp.sed 1.4 suggest that the substance has a protein nature (Sharma et al. 2018, Aunpad & Nabangchang 2007) The trypsin cleavage site is highly specific, cleaving the peptide bond after residues with long positively charged side chains, such as arginine and lysine, while papain presents broad specificity, cleaving peptide bonds of basic amino acids, leucine, or glycine (Sangeetha & Abraham 2006, Berg et al. 2014).Antimicrobial peptides that are not sensitive to trypsin probably do not have a lytic site for this protease; nevertheless, inactivation by pancreatic proteases such as trypsin is The stability of the antimicrobial substance to thermal treatments, organic solvents and detergents, resembled those observed for some antimicrobial compounds of peptide nature produced by Bacillus spp.(Motta et al. 2007b, Ebrahiumipour 2014, Chalasani et al. 2015, Lee & Chang 2018).
While the studied substance inhibited the growth of different species of Listeria, it was not able to inhibit Gram-negative bacteria, yeasts and the other Gram-positive species tested, demonstrating a more restricted spectrum of activity.According to Liu (2015), antimicrobial substances produced by Grampositive bacteria have less potential to inhibit the growth of gram-negative bacteria.This is due to the fact that Gram-negative bacteria have an outer membrane, which functions as an impermeable barrier for the cell, making it difficult for antimicrobial peptides to reach the cytoplasmic membrane (Garcia-Gutierrez et al.

2019).
The antimicrobial peptide studied by Lee & Chang (2018) was tested against a range of Gram-positive and Gram-negative bacteria, and the results obtained showed that the substance was able to inhibit the growth of Gram-positive bacteria such as Bacillus cereus and Listeria monocytogenes.Torres et al. (2015) also found antimicrobial activity against strains of Listeria monocytogenes.In view of the results presented, it is suggested that the substance may be a class IIa bacteriocin, an anti-Listeria type bacteriocin.
Although foodborne disease associated with L. monocytogenes is not as common as those of Salmonella, Campylobacter or Escherichia coli, the mortality rate can be considered the highest.Approximately 30% of invasive listeriosis cases end in death, and therefore L. monocytogenes can be considered a pathogen Table III.Spectrum of action of the partially purified antimicrobial substance according to the inhibition zone (mm).

Indicator microorganism Inhibition zone (mm)
Listeria monocytogenes ATCC 7644 12 Listeria monocytogenes 4B (carcass) 11 Listeria innocua L 10 (buffalo milk) 11 Listeria monocytogenes QF Oxford (sliced cheese) 10 Listeria seeligeri BQ Oxford (countertop) 11 Listeria seeligeri BP Palcam -2 (countertop) 13 Listeria seeligeri PF Oxford (sliced ham) 10 Listeria seeligeri MP Oxford (hands) 11 Listeria seeligeri BP Oxford (countertop) of food and public health importance (Haggerty et al. 2018, Gray et al. 2018).Often introduced into the processing environment through raw foods, Listeria species can adhere to a variety of surfaces, with some strains persisting for several years and acting as a source of continuous crosscontamination (Colagiorgi et al. 2017, Gray et al. 2018).L. monocytogenes is a difficult organism to eradicate and its presence still occurs despite the best management practices, such as cleaning and sanitizing after contact with food and sterilizing non-food contact surfaces, in addition to equipment maintenance (Drew & Clydesdale 2015).Thus, the search for alternative antimicrobial substances to control strains of L. monocytogenes is a point of great interest (Leite et al. 2016).Based on the results of this work, the substance produced by Bacillus sp.sed 1.4 presented the potential for application in food preservation, as it inhibited all Listeria species tested.

Figure 1 .
Figure 1.Dendrogram of Bacillus species showing the levels of similarity in the phenotypic expression of proteins present in the isolates.

Figure 2 .
Figure 2. Phylogenetic tree obtained from the 16S rRNA gene of the sequences with closer similarities suggested by BLAST and identified isolates from other studied sediments.

Figure 3 .
Figure 3. Growth curve of the Bacillus sp.sed 1.4 isolate.Growth (•) and antibacterial activity (■) were monitored during growth in TSB at 30 °C.Each point represents the average of three independent experiments.The indicator strain was Listeria monocytogenes ATCC 7644.The error bars represent the standard deviation (n=3).

Figure 4 .
Figure 4. Electrophoresis in polyacrylamide gel stained with silver nitrate showing the protein bands of the stages of partial purification of the antimicrobial substance produced by Bacillus sp.sed 1.4.A: G-200, B: precipitated in the 0-60% range, C: crude supernatant, MW: molecular weight marker.

Table I .
Concentration of soluble proteins and antimicrobial activity from the fractions resulting from the purification steps, produced by Bacillus sp.sed 1.4.

Table II .
Effect of different chemicals and organic solvents on the antimicrobial activity of the partially purified crude supernatant produced by Bacillus sp.sed 1.4.Tests were performed by incubating the partially purified crude supernatant and treating it at 37ºC for 1 h.Residual activity was estimated against Listeria monocytogenes ATCC 7644.