Characterization and evaluation of the antibacterial potential of bacterial microbiota of cultivated soils of Cassava ( Manihot esculenta ) and Black pepper ( Piper nigrum ) in the city of Igarapé Açu – Pará, Brazil.

— Actinobacteria form a heterogeneous group of Gram-positive bacteria that have a great morphological and metabolic variety, are microorganisms with great biotechnological potential for the production of several substances of industrial and pharmaceutical interest, being considered the main source of antibiotics. Due to the increase in the occurrence of bacteria that are multiresistant to antibiotics used in clinical and hospital routine, the need to discover new substances with antibiotic capacity becomes evident. Thus, studies that assess the diversity and variety, composition and properties of metabolites produced by bacterial species present in cultivated soils are important and necessary. This work aimed to investigate the occurrence of actinobacteria in rhizosphere soils of areas cultivated with cassava (Manihot esculenta) and black pepper (Piper nigrum) in the city of Igarapé Açu - Pará, Brazil, to assess their potential as producers of antimicrobial substances. This is an analytical descriptive, experimental study that performs a microbiological analysis of soil samples. With great morphological diversity, 21 different bacteria were isolated, all with characteristics of actinobacteria. As a result, strains of actinobacteria with variable colonial characteristics were isolated, microscopically characterized as isolated and chained Gram-positive bacilli. Among the isolates, 8 strains were able to inhibit the growth of Escherichia coli and Klebsiella pneumoniae compared to in vitro. It emphasizes the need for more research aimed at the microbiology of Amazonian soils, due to the diversity of bacteria of biotechnological importance that can significantly contribute to the production of new antibiotics, thus generating a positive expectation in the fight against infections.


INTRODUCTION
Soil is considered an important source of chemically diverse and biologically active substances; these compounds have been of great importance for industry and especially for pharmaceuticals. The microbiota in this ecosystem is extremely rich and the bioprospecting of several microorganisms and the analysis of their metabolic activities allow the discovery of potential antibacterial agents and other substances of industrial interest 1 12 107 The Actinobacteria class is divided into 4 subclasses, 5 orders, 14 suborders, 50 families, 197 genera and 1936 species 4 , they are mostly Gram-positive, aerobic bacteria 5 , with high content of G+C in their DNA 6 , similar to some fungi, these bacteria reproduce mainly by sporulation, in which each spore produced can give rise to a new organism 7 .
Soils used for agriculture have different characteristics from uncultivated areas, due to anthropic action, type of fertilization and soil management can change the composition of the bacterial community in quantity and quality of microorganisms found in their rhizospheres 8,9 . The microorganisms present in the rhizosphere community of cultivated soils are of great importance for plants, as they influence their development and protect the roots against pathogens 10 .
The search for new species of actinobacteria in these places is an essential component for the discovery of new substances and, based on the properties presented by actinobacteria, the study of microorganisms present in rhizosphere environments is of great importance, which can provide the discovery of new compounds and products of biological origin, with biotechnological potential for the production of metabolites with antibacterial action or other pharmaceutical and industrial applications.
The Amazon population still practices on a large-scale subsistence culture in small areas of land. Cassava (Manihot esculenta Crantz) and black pepper (Piper nigrum), are plants adapted to the tropical region, resistant to heat, demanding moisture and rainfall 11 , both highly cultivated in regions of the state of Pará. The objective of this work is to characterize and evaluate the antibacterial potential of bacterial microbiota of cultivated soils of cassava (Manihot esculenta) and black pepper (Piper nigrum) in the city of Igarapé-ace -Pará.

-Sample collection:
To carry out this work, 24 soil samples were used, collected in groups of 4 samples at each point. The collection will take place through the use of sterile spatulas and the samples will be packed in disposable and sterile bags. Each sample will have 100 g of soil from each collection point, which will be georeferenced to meet the need for subsequent collections if necessary.

-Isolation of microorganisms
For bacterial isolation, samples from the rhizosphere, before seeding for bacterial isolation, underwent a pretreatment consisting of a 10g dilution of the sample in 90mL of sterile saline solution, followed by vertexing for 10 to 20 minutes. After shaking, each sample was heated in a 50°C water bath for 10 minutes, aiming to eliminate contaminating bacteria that spread quickly and are not the target of isolation. The resulting supernatant was seeded using the surface streak method, using 0.05 mL sterile and disposable loops, in 3 Petri dishes containing the culture media: modified Czapeck Agar, modified Hickey-Tresner Agar and the culture medium Vitamin Arginine. In all culture media, Amphotericin B (100μg/mL) was added to inhibit fungal growth. Petri dishes were incubated in a humid chamber bacteriological incubator for 24 hours up to 30 days at a temperature of 37°C to 45°C with daily visual observation of bacterial colony growth. The plates with bacterial growth were transferred to other plates and after purification they were kept in test tubes with slanted enrichment culture medium for their maintenance.

-Bacteria characterization
The isolated strains were characterized morphologically and physiologically through morphological characteristics of the colonies, microscopic characteristics of microorganisms and their metabolism through tests 8 biochemical tests. The macroscopic characterization was carried out by visual observation of color, shape and moisture characteristics, the evaluation of the microscopic aspect was carried out after making smears made of each characterized colony that were stained by the Gram stain method. The methodologies used in the biochemical tests were adapted from the manual of the National Health Surveillance Agency 12

-Antimicrobial Sensitivity Test (TSA)
The TSA was performed using the diffusion test on plates containing Muller Hinton Agar, and the potential for inhibiting the growth of Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli strains was evaluated, bacterial suspensions were prepared with an approximate concentration of 0.5 cfu. /mL and the suspensions were used to seed plates containing Muller Hinton agar medium, using sterile disposable swabs, after 30 minutes at room temperature, the isolated and characterized strains from the soil were inoculated and pricked at specific points in the previously seeded plates, the formation of bacterial growth inhibition halos around the bite was considered a potential inhibiting power.

III. RESULTS
Despite the simplified methodology, this work clearly demonstrates the possibility of the existence of bacteria with biotechnological potential in the Amazon region. The antibacterial action of the isolates against Gram-positive bacteria such as S. aureus was not found in this research, but on Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae there was inhibition of bacterial growth in vitro, this action was also described by Silva 7 , in which only Gram-negative bacteria were affected.
The Amazonian soil microbiota is characterized by abundance and diversity, our findings were characterized by the isolation of bacteria with various macro and microscopic morphologies, similar to the results 13,10 . The organisms found in this research showed a wide morphological variety, but the presence of isolated Grampositive bacilli or in chains predominated, similar findings are described in the literature, reinforcing the assertion of the vast amount of these microorganisms in the soil 3 .
Ecological studies of actinomycetes populations are extremely important, as these microorganisms form spores and conidia, allowing their survival in adverse conditions 14 . In addition, actinomycetes stand out for the possibility of synthesizing vitamins, substances that inhibit enzymatic activity 15 , antibiotics and other biologically active compounds 16 .
There was bacterial growth at all collection points, with no growth restriction in relation to the soil depth in the places. Isolated colonies were macroscopically characterized based on their color and appearance presented in the media used. (Table 1).
The microscopic analysis of bacterial colonies, performed using the Gram stain technique, showed a great diversity of bacterial morphologies (Figure 1 The biochemical characterization of the isolated colonies was performed using biochemical tests according to table 3. In rhizosphere soils of black pepper, 13 plates with bacterial growth were analyzed, which presented colony characteristics similar to those of cassava. (Table 4).
Considering the morphology of the bacteria found through the Gram staining technique, they were identified in isolated and paired Gram-positive bacilli (BGPi), Gram positive coccobacillus (CBGP), Gram positive bacilli in chain (BGPc) and Gram-positive cocci grouped and isolated (CGP), distributed according to table 5.
The biochemical characteristics of colonies isolated from rhizosphere soil samples of black pepper did not show any difference from the characteristics isolated from rhizosphere soils of cassava. For the evaluation of the sensitivity test and antimicrobial activity of the strains, all 33 bacterial strains isolated were submitted to the methodology (Figure 2), of which only 8 strains showed inhibitory activity on the tested bacteria (Table 6).

V. CONCLUSION
The Amazonian soil is still little explored in its biotechnological potential, the findings in this research demonstrate the possibility of discovering new bacterial strains with high potential for the production of new antibacterial drugs. Bacteria isolated from these soils proved to be organisms with relevant pharmaceutical potential. These organisms act as growth inhibitors of other microorganisms and are key agents in the biocontrol of diseases of human pathogens, and can significantly contribute to the production of antibiotics, thus generating a positive expectation in the fight against infections. The expected scientific benefits of greater knowledge about microbial diversity include, among others, better understanding of the roles played by microbial communities in terrestrial environments and knowledge of their interactions with other components of biodiversity.
Despite being relevant in several aspects, soil bacteria are still poorly studied and disseminated. It is proposed to carry out more in-depth research on the applications of actinobacteria in these processes, using more accurate tests and techniques to achieve these findings in order to highlight the economic importance of soil biota, and support the review and synthesis of the direct value and indirect soil biological species.