Molecular Characterization of Bacillus-Genus Bacteria with Fibrinolytic Potential Isolated from Squashes «NTETE» in Brazzaville in the Republic of Congo

Abstract

Thromboses appearing in the blood and causing cardiovascular disease by the presence of fibrin remain a problem of concern worldwide. The fibrinolytic enzymes developed by bacteria of the Bacillus-genus are today an interesting and promising alternative to chemicals with multiple consequences. It is in this perspective that three (3) samples of cooked squash wrapped in sheets and consumed in Brazzaville and collected and in three Brazzaville markets were explored. The estimated count of the total flora of each sample for each of the three markets, namely Total, Moukondo and Tsiémé was carried out on PCA and represents respectively (5.2 ± 0.12) 107 CFU / g, (1.73 ± 0 , 16) 107 CFU / g, and (9.43 ± 1.06) 107 CFU / g while bacteria of the genus Bacillus are estimated in Mossel respectively at (3.5 ± 1.16) 106 CFU / g, (4, 01 ± 0.85) 106 CFU / g (8.96 ± 0.60) 106 CFU / g. Sixty-six isolates of bacteria of the genus Bacillus isolated from squash by conventional microbiology techniques have been phenotypically characterized. The morphological types characterized are essentially the bacillary form and the spherical form. The ability to produce fibrinolytic enzymes correlated with growth was assessed. The growth in terms of optical density varies from 0.800 to 0.97 and the enzymatic production in all the isolates tested varies from 12 to 21 mm. After DNA extraction from 36 isolates, PCR amplification of the rR16S gene revealed fragments of approximately 1500bp by electrophoresis on Agarose Gel. The sequencing of thirty-four (34) fragments made it possible to obtain fifteen (15) sequences having a strong similarity between them and also with the homologs of the databases (97% to 100%) and therefore the molecular identification of: Bacillus sp 40%, Bacillus amyloliquefaciens 6.66%, Bacillus subtilis 33.33%, Bacillus Pumilus 6.66%, Bacillus megaterum 6.66%, Bacillus velezensis 6.66%. Five (5) of these sequences have been submitted to GenBank and the accession numbers are successively: MK193815.1 (Bacillus subtilis strain ASM1), MK207434.1 (Bacillus subtilis strain ASM3), MK207435.1 (Bacillus pumilus strain ASM5) , MK207436.1 (Bacillus subtilis strain ASM4), MK207437.1 (Bacillus megaterium strain ASM2).The multiple alignment of sequences obtained shows a high conservation of this gene in bacteria of the genus Bacillus. The phylogenetic classification clearly shows this monophyletic class of bacteria of the genus Bacillus with very short distances(less than 3%).