Discovery of a Novel Lineage Burkholderia cepacia ST 1870 Endophytically Isolated from Medicinal Polygala paniculata Which Shows Potent In Vitro Antileishmanial and Antimicrobial Effects

Abstract

In this study, we report the isolation and identification of an endophytic strain of Burkholderia cepacia (COPS strain) associated with Polygala paniculata roots. Polygala plants are rich sources of promising microbiomes, of which the literature reports several pharmacological effects, such as trypanocidal, antinociceptive, anesthetic, anxiolytics, and anticonvulsant activities. B. cepacia COPS belongs to a new sequence type (ST 1870) and harbors a genome estimated in 8.3 Mbp which exhibits the aminoglycosides and beta-lactams resistance genes aph(3')-IIa and bla _TEM-116, respectively. Analysis performed using MLST, average nucleotide identity, and digital DNA-DNA hybridization support its species-level identification and reveals its novel housekeeping genes alleles gyrB, lepA, and phaC. The root endophyte B. cepacia COPS drew our attention from a group of 14 bacterial isolates during the primary screening for being potentially active against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 9341, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231 and exhibited the broad-spectrum activity against phytopathogenic fungi. In addition, COPS strain showed production of protease, lipase, and esterase in solid media, and its natural product extract showed potent inhibition against fungal plant pathogens, such as Moniliophthora perniciosa, whose antagonism index (89.32%) exceeded the positive control (74.17%), whereas Sclerotinia sclerotiorum and Ceratocystis paradoxa showed high percentages of inhibition (85.53% and 82.69%, respectively). COPS crude extract also significantly inhibited S. epidermidis ATCC 35984, E. faecium ATCC 700221 (MIC values of 32 μg/mL for both), E. faecalis ATCC 29212 (64 μg/mL), and S. aureus ATCC 25923 (128 μg/mL). We observed moderate antagonistic activity against A. baumannii ATCC 19606 and E. coli ATCC 25922 (both at 512 μg/mL), as well as potent cytotoxic effects on Leishmania infantum and Leishmania major promastigote forms with 78.25% and 57.30% inhibition. In conclusion, this study presents for the first time the isolation of an endophytic B. cepacia strain associated with P. paniculata and enough evidence that these plants may be considered a rich source of microbes for the fight against neglected diseases.

Figure 1

Schematic representation of the quantitative assay for the AI determination: (a) paper disk containing NPE of Burkholderia cepacia COPS; (b) phytopathogenic fungus mycelium. The inhibition index was calculated by the formula using the means of the mycelial growth measurements in centimeters.

Figure 2

Consensus tree obtained from a neighbor-joining phylogenetic analysis using the Jukes-Cantor method (bootstrap with 1000 replicates) based on the concatenated full length of seven housekeeping and 16S rDNA gene of B. cepacia COPS strain compared to reference sequences of Bcc members.

Figure 3

Antimicrobial activity of NPE (a) produced by B. cepacia COPS. Columns (b) and (c) correspond to different crude extracts' antibacterial activity compared to overlay assay (top to bottom: Bacillus cereus, Escherichia coli, and Enterococcus faecalis). Lanes (d) COPS NPE, (e) benomyl, as positive control, and (f) negative control represent the antifungal effect. From top to bottom: Moniliophthora perniciosa, Ceratocystis paradoxa, Sclerotinia sclerotiorum, Alternaria alternata, and Fusarium verticillioides.

Figure 4

Evaluation of enzymatic production of Burkholderia cepacia COPS in solid media: (a) protease; (b) esterase; (c) lipase. Activity of B. cepacia COPS NPE at different concentrations against promastigotes of L. infantum (d) and L. major (e) after 24 hours of exposure. Values represent two independent experiments performed in triplicate. ^∗∗∗Indicates statistical significance compared to the negative control, with values of p < 0.001.