Morphological and biochemical characterization of bacterial species of Bacillus, Lysinibacillus and Brevibacillus

ABSTRACT The objective of this work was to characterize reference bacteria strains, belonging to the genus Bacillus and species of correlated genera, by simplified morphological and biochemical methods. The morphological characterization is based on the aspects of the colonies, as well as cytomorphology of the species, by optical and scanning microscopy. For biochemical characterization, the sensitivity test to antimicrobials by disk-diffusion is performed. Moreover, the strains were characterized by extracting intracellular proteins. Characteristics such as shape, color, and consistency of the colonies, in addition to the type of spore and production of protein crystals were determinants for the morphological characterization of these species. The antibiogram revealed high resistance to β-lactam group antibiotics, in species of Bacillus cereus s.l group. In Bacillus subtilis s.l. group there was high susceptibility to antibiograms, mainly for species of B. subtilis. The protein profile provided specific protein patterns for some species, mainly bands of 130 e 65 kDa for B. thuringiensis, 140 e 130 kDa for Lysinibacillus sphaericus, and 115 kDa for Brevibacillus laterosporus. Our results showed that the morphological and biochemical characterizations, provided a simple identification, with easy interpretation, and low cost.

Many of these species have high biotechnological potential.Their biodiversity is used for many purposes, especially in the agricultural sector, such as biological control of pests and diseases vectors; solubilization of phosphorus; promotion of plant growth; and production of chemical substances (Lanna-Filho et al., 2010;Ramírez et al., 2014).
Due to the functional multiplicity of these bacteria, in recent years, interest in growth and home production (fermentation) of bacterial species of this genus has increased, mainly for direct application in crops (Monnerat et al., 2018;Lana et al., 2019).However, this fermentation process is often done inappropriately, generating undesirable contaminants at the end of production.Thus, it is necessary to know the material to be grown, and correctly identify it, to avoid possible pathogens and/or losses in organic and agricultural production (Valicente et al., 2020;ABCbio, Gabriela Teodoro Rocha et al. 2019).
Tough most of the correct and precise identification of these bacteria is carried out by molecular techniques, such as gene amplification by Polymerase Chain Reaction -PCR (Erlich, 1989), DNA sequencing and protein profile by spectrometry of mass by MALDI-TOF, these techniques are considered sophisticated tools, which require qualified professionals and a higher investment cost, making them ultimately inaccessible to rural producers (Assis et al., 2011).
Identification methods considered dependent on cultivation, which include the morphological analyzes of the colonies; Gram stain; and microscopic observations, to verify the production of endospores and protein inclusions, mainly in Bacillus thuringiensis species.As well as the use of biochemical and physiological tests, became simple and low-cost tools, which can be performed quickly and easily interpreted and accessed by farmers, which can help them establish an efficient quality control plan (Assis et al., 2011;Madigan et al., 2016).
The main objective of this work was to characterize bacteria strains of reference belonging to the genus Bacillus and species of related genera, by simple morphological and biochemical methods.

Bacterial strains
Among the 20 strains analyzed, all belong to Inverte-

Morphological characterization
The strains were inoculated in Petri dishes containing EMBRAPA-agar culture medium at 30 ºC for 72 h (Monnerat et al., 2007).After this period, the colonies were visualized by microscope transmission, model JEM-2100-Jeol, and classified according to the principles of Bergy's Manual of Systematic Bacteriology (De Vos et al., 2009).
For cytomorphological characterization, the strains were inoculated in EMBRAPA-liquid medium for 48 h at 30 °C and subsequently visualized by an electronic scanning microscope (SEM), model Zeiss DSM 962.
Strains were cultured in Embrapa-liquid culture medium, for 72 hours, at 30 °C, by incubation on a rotary shaker at 200 revolutions per minute (rpm).The turbidity of the bacterial solution was adjusted according to the manufacturer to 0.5 on the Mac Farland scale, which corresponds to an amount of 10 8 CFU/mL. 2 mL of the bacterial inoculum was streaked, on each Petri dish (15 cm) with Embrapa-agar culture medium.Sterilized forceps were used to deposited diffusion discs, containing the antibiotics.
The experimental design was entirely random, and three replications were performed per strain.To determine the positive and negative control, 20 strips of autoclaved filter paper were deposited in autoclaved dH 2 O Milli Q.
The plates were incubated at 30 °C for 24 h and, the formation of inhibition halos was evaluated.The measurement of halos was performed using a caliper in millimeters.
The determinations of the inhibition zone were based and adapted according to Bauer et al. (1966)

Protein profile -SDS-PAGE
To obtain cultures in their vegetative state, selected strains were grown in Embrapa-liquid culture medium, for 24 hours at 30 °C, in a rotary shaker at 200 rpm.The bacterial culture was centrifuged for 5 minutes at 12,000 rpm, at 4 °C.The pellets (bacterial mass) were resuspended in 1.0 mL of 10 mM tris-HCl solution, pH 8.0, containing 20% sucrose (w/v) and chilled 0.125% SDS.Immediately after the complete resuspension of the pellet, 20 µl of EDTA 0.5 M, pH 8.0 was added, and the samples were placed on ice for 10 minutes with moderate agitation.The cell solutions were centrifuged at 12,000 rpm for 5 minutes at 4 °C.The pellet was resuspended in 500 µl of 0.5 mM MgCl 2 solution, maintained at room temperature for 10 minutes, and then centrifuged for 5 minutes.
The supernatant, coming from the bacterial mass extraction process, was reserved and used for the analysis of the protein profile, since the objective was to analyze the total intracellular proteins of the bacterial isolates.
Samples were boiled together with the protein buffer at 90 °C for 5 minutes, in a 1:1 ratio.The analysis of the protein profile was performed by electrophoresis of proteins in polyacrylamide gel (SDS-PAGE 12%) and, fixed in a silver nitrate solution, and the process started with 40% methanol and 5% acetic acid for 12 h, followed by washing with 50% ethanol.Then, the gels were sensitized with a 0.2% sodium thiosulfate solution, followed by the impregnation solution of 0.2% silver nitrate, 0.028% formaldehyde.
Finally, gels were fixed with the protein banding solution, which contained a solution of 6% sodium carbonate, 0.4% sodium thiosulfate, and 0.018% formaldehyde.To stabilize the appearance of protein patterns, a solution containing 45% methanol and 10% acetic acid in 100 mL of dH 2 O was used.

RESULTS AND DISCUSSION
The morphological identification of Bacillus strains and related genera found that the 20 strains exhibited wide morphological and cytomorphological variety (Figures 1 and 2), between and within species.Nevertheless, the morphological structures observed are typical of these species grown in an Embrapa culture medium.Thus, the composition of the culture medium and the incubation conditions can strongly influence the morphological variety of bacteria and define details of diameter, shape, elevation, surface texture, color, and consistency of bacterial colonies (Rabinovitch et al., 2015).
The most common characteristics among the species were the form and elevation of the colonies, being 85% circular and 95% flat, except for strain S2785, B. amyloliquefaciens, which had a convex elevation.Furthermore, strain S2786, B. mycoides, had a rhizoid colony format, differing from the other strains.B. licheniformis also showed important morphological variations, such as irregular and small colonies, with a filamentous appearance and cream color (Figure 1).
The analysis of the surface of the colonies showed that 75% of the bacterial strains had a smooth and opaque surface, mainly for the species of the Bacillus cereus s.l.Vos et al., 2009;Logan & Halket, 2011).
The cytomorphological evaluation of the species by, the vegetative cells of the Bacillus and related strains showed sizes between 0.5 and 3 mm.Diverse spore shapes were found especially within species (Table 1 and Figure 2).
The ability to produce protein crystals with entomopathogenic action was also verified, mainly in the species B. thuringiensis (Figure 2) and Lysinibacillus sphaericus.et al., 2004).Spherical crystals have insecticidal activity to dipterans (Melatti et al., 2005).
It was found that in the same strain there was the formation of two protein inclusions, this being present in the S2566 isolate, which presented bipyramidal and cuboid crystals.The same result was observed by Praça et al. (2004), on the S997 (B.thuringiensis) strain, to which he found the three types of conformations.
Morphological and biochemical characterization of bacterial species of   Morphological and biochemical characterization of bacterial species of

Bacillus, Lysinibacillus and Brevibacillus
The production of crystals proteins by Brevibacillus laterosporus was not observed; however, Ruiu (2013) states that some strains of the same species form a lamellar parasporal inclusion with oval shape, firm and adjacent to the side of the spore.
However, it is important to note that due to the high phenotypic similarity between some species and subspecies, which can influence their correct characterization, and/or due to the dependence on the metabolic processes of the microorganisms, the result obtained by these techniques often become inconclusive because they may require long periods of evaluation (Assis et al., 2011;Celandroni et al., 2016).Thus, it is necessary to combine different identification techniques to provide a conclusive and accurate identification of these species.
In addition to the morphological characterization, it was observed that Bacillus species and related genera were resistant and susceptible to antimicrobials.Thus, it was verified that seven of the 20 antibiotics tested inhibited 100% the bacterial growth of the strains, being amikacin, ciprofloxacin, gentamicin, levofloxacin, erythromycin, tetracycline and vancomycin (Figure 3).The use of these antibiotics can help separate these Bacillus and related species from other strains of divergent genera that exhibit resistance to these  Antimicrobial resistance was also verified, and it was found that 70% of the strains were resistant to aztreonam, followed by the antimicrobial's oxacillin (65%), penicillin G (55%) and ampicillin (35%), all belonging to the chemical group of β -lactam.Furthermore, 45% of the strains It is important to emphasize that, even if there is a high level of resistance, on the part of species of the B. cereus Morphological and biochemical characterization of bacterial species of

Bacillus, Lysinibacillus and Brevibacillus
Strains belonging to the Bacillus subtilis s.l. group were highly susceptible to antimicrobials, especially the isolates of Bacillus subtilis, for example, strain S2794 was found to be resistant to chloramphenicol, ampicillin and penicillin, in contrast to isolates S2776 and S2790, which were resistant to only aztreonam.
The species of B. amyloliquefaciens S2785 was susceptible to all tested antibiotics, while isolates S2784 and S2788 were commonly resistant to penicillin, and S2791 to oxacillin.Within the same phylogenetic group, the species B. atrophaeus showed resistance to rifampicin (5 μg), and it is proposed that a better parameter for identification and separation of this specie in relation to others of the same phylogenetic group maybe it's unique orange color colony.
B. licheniformis strain was susceptible to chloramphenicol, ampicillin, and penicillin, nevertheless, it was shown to be resistant to aztreonam and oxacillin and to clindamycin.Unlike these results, Adimpong et al. (2012) reported that the 38 strains of B. licheniformis tested were susceptible to gentamicin, tetracycline and vancomycin; however, 50% of these strains were resistant to chloramphenicol and clindamycin, thus causing a variation in the resistance profile among several isolates of the same species.
It was found that species belonging to the related genera Lysinibacillus spp.and Brevibacillus sp. also showed a high profile of susceptibility to the antibiotics tested, mainly under the action of antibiotics from the β-lactam group, such as penicillin and ampicillin.A similar result was observed by Abdel-Salam et al. (2018), in which strains of L. sphaericus were highly susceptible to ampicillin.However, L. sphaericus strains showed small variations in resistance; strain S0002 was resistant to cotrimoxazole, while strain S0127 was resistant to oxacillin.
The Bacillus sp. was resistant to amoxicillin, aztreonam, cefepime, oxacillin and penicillin, a common resistance profile among almost all strains.
Antibiogram is a method for rapid characterization since, through the level of resistance, different strains showed different resistances between, the bacterial groups observed, and the specificity of resistance of some strains were verified for certain antibiotics.
In addition to the characterization of species by the mechanism of resistance and susceptibility to antimicrobials, the protein profile was also analyzed, and patterns were defined for each strain.Thus, the electrophoretic profile of proteins extracted from whole cells produced heterogeneous patterns of Bacillus strains proteins of related genera.Figure 4 shows the distribution of protein bands across the gel.Gabriela Teodoro Rocha et al.
The electrophoretic profile of the protein fractions of Bacillus species and related ones had a wide range of molecular weights that varied from 140 kDa to 10 kDa.
However, the strains shared a similar pattern with protein masses of 25 and 45 kDa, which was determined as a stan-  In addition, it was verified that the S2789 strain, considered a Bacillus sp., without official species identification, presented characteristics very similar to the species of the genus Lysinibacillus, since this species presented colonies with circular, smooth and mucous characteristics; spores with spherical shape and protein bands of 140 kDa.However, a molecular characterization is necessary to confirm the hypothesis that this species belongs to the genus Lysinibacillus and not to the genus Bacillus.
Hence, the morphological characterization becomes a fast and simplified identification parameter, mainly in species with high heterogeneity.However, this technique becomes useless, when there is a high homogeneity of phenotypic similarities between a large number of species of the same genus.Thus, combining morphological and biochemical characterization methods appear to produce an identification pattern with a higher degree of confidence (Figure 5).
antimicrobials.A similar pattern observed by Celandroni et al. (2016), in which strains of Bacillus spp.and Paenibacillus spp.were susceptible to ciprofloxacin and tetracycline, in addition to tigecycline and vancomycin.

Figure 3 :
Figure 3: Resistance and susceptibility profile of strains belonging to the genera Bacillus, Lysinibacillus and Brevibacillus.
were resistant to cefepime and ceftazidime.Most of the species in this study were resistant to antibiotics belonging to the β-lactam group, especially strains in Bacillus cereus s.l group.(B.thuringiensis, B. cereus, B. mycoides) (Figure 3).Resistance to these antimicrobials is conditioned to the production of nucleophilic enzymes (β-lactamase) that can promote β-lactam ring opening, present in the bacterial cell wall (Guimarães et al., 2010; Bautista & Teves, 2013; Madigan et al., 2016).Luna et al. (2007) verified the sensitivity to 24 antimicrobials in species B. cereus s. l. group, finding that strains of B. cereus and B. thuringiensis were resistant to amoxicillin, ampicillin, ceftriaxone, penicillin, and oxacillin, thus presenting a similar resistant profile; however, some strains of B. mycoides and B. pseudomycoides were susceptible to β-lactams.
laterosporus) showed proteins with a wide range of molecular weights, mainly with high molecular weight, ranging from 140 kDa to 20 kDa.However, Bre. laterosporus exhibited two different protein bands, at 115, 100 and 80 kDa, and the other strains analyzed did not obtain protein bands that could differentiate them or determine their exact identification.Protein profiles obtained by the SDS-PAGE polyacrylamide gel offered an effective and rapid approach to identify bacterial species in the Bacillus group.However, this electrophoretic technique has its own level of discrimination and combined with another methodology, can provide a more accurate identification of these species.The aim of this study was to characterize reference species of Bacillus and correlates by simplified morphological and biochemical methods and, because of the results presented, validations of the techniques discussed here will be carried out later.
Morphological and biochemical characterization of bacterial species of Bacillus, Lysinibacillus and BrevibacillusCONCLUSIONMorphological and biochemical characterizations, due to morphological homogeneity between some species and subspecies, provided a simple identification, of easy interpretation and low cost, considering mainly the microscopic observations and the analysis the protein profile.

Figure 5 :
Figure 5: Summary of essential characterizations found in Bacillus strains and correlated genus.
thuringiensis, since this protein is encoded by the cryI and cryIV genes, as observed in six Bt isolates.The protein profile of Bacillus cereus and Bacillus mycoides species were similar to B. thuringiensis species, all belonging to the same phylogenetic group (Bacillus cereus sensu lato).In B. cereus there was the presence of