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

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 blaTEM-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.


Introduction
Plant tissues represent a significant source of natural substances for pharmaceutical and biotechnological interest. Drug discovery has been based on medicinal plants for centuries [1][2][3]. However, endophytes are capable to biosynthesize a plethora of natural products and compounds which are originally believed to be produced only by their host plants [4,5], and therefore, they are considered alternative suppliers of characteristic phytochemical compounds and represent a vast unexplored reservoir of unique chemical structures [6].
ese plant-symbiont microorganisms that live in intimate interaction establish a mutualistic interaction with the host plant by exchanging nutrients and protection; they produce antibiotics and other substances that can protect the plant against stress conditions such as attack by herbivores, pests, and plant pathogens without causing apparent disease symptoms [7][8][9].
Likewise, phytopathogenic fungi represent a severe threat to several crops, thus affecting production and quality. Modern agriculture is entirely dependent on agrochemicals; although they can improve crop yield, quality, and shelf-life, they negatively affect the environment and human health. In this regard, issues related to sustainability and practices in defense of the environment have drawn considerable attention [35][36][37].
Approximately, 13 million people suffer from parasitic diseases caused by Leishmania protozoa infection. Parasite resistance and host toxicity of currently available drugs are a reality and a concern mainly in subtropical countries [38,39]. On the other hand, microbial resistance to antibiotics has been rapidly spread, is responsible for 33,000 deaths in Europe, and became a concern to public health [40].
We aimed to explore the endophytes of Polygala paniculata and isolate antibiotic-and biotechnology-related enzymes-producing microorganisms. Herein, we present the Burkholderia cepacia COPS, a sequence-type (ST) 1870 strain isolated from P. paniculata roots collected in the Brazilian Atlantic Forest. In addition to its draft-genome, we presented the COPS enzymes and antimicrobial activities.
Burkholderia spp. consist of emerging sources of a plethora and diverse natural products potentially relevant for therapeutic/medicine, biotechnological, and agriculture applications [41]. is genus is a versatile producer of antimicrobial compounds and enzymes and exhibits plant growth-promoting properties. We can find such Gramnegative bacteria in several habitats, ranging from humans (as pathogens) to plants (as endophytes) [42][43][44][45][46][47][48].

Screening of Antimicrobial and Enzymatic Activities.
Fourteen bacterial isolates were randomly selected (four from rhizosphere and ten endophytes) and qualitatively tested by the overlay test [44,67,68] for the antimicrobial activity screening. A total of 100 μL of precultured isolates in International Streptomyces Project medium 2 (ISP2) (malt extract: 10 g/L; yeast extract: 4 g/L; and glucose: 4 g/L) [69] was adjusted to OD 600 between 0.3 and 0.6 and inoculated in the center of the Petri dishes containing ISP2 agar and incubated at 28°C for 72 hours. en, the isolates were exposed to chloroform for inactivation, followed by a 30minute evaporation step. Subsequently, semisolid brainheart infusion agar (BHI), previously inoculated with test microorganisms (Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 11775, and Candida albicans ATCC 10231), was poured onto the inactivated isolate. e antagonism test for phytopathogenic fungi was performed as described by Quiroga et al. [70]. e isolated strains were streaked in potato dextrose agar (PDA) at the edges of the plates and incubated at 28°C until their complete growth. Next, a 0.6 cm plug of the phytopathogenic fungus mycelium was placed at the top of the plate containing the grown isolate. e plates were incubated again at 28°C. As a negative control, each phytopathogen was cultured in PDA for indicating the time of the inhibition evaluation [71]. All tests were performed in triplicates. All pathogenic strains used in this study are listed in Table 1.
e assessment of enzymatic potential consisted in a preculture of Polygala paniculata-derived bacteria in 3 mL of tryptic soy broth (TSB-KASVI) and incubated for 48-72 hours at 28°C. en, 2 μL of the culture was transferred to a M9 enzymatical solid medium (200 mL/L of stock solution (64 g/L Na 2 HPO 4 .7H 2 O; 15 g/L KH 2 PO 4 ; 2.5 g/L NaCl; 5 g/L NH 4 Cl)); 2 mL/L 1 M MgSO 4 ; 10 g/L; 0.1 mL/L CaCl 2 1 M; 15 g/L agar, pH 7.2, with different supplements, depending on the activity to be studied: (1) 0.5% yeast extract and 1% soluble starch for amylase activity; (2) 0.5% yeast extract and 1% carboxymethyl cellulose for cellulase activity; (3) 0.5% yeast extract and 1% pectin, pH 8.0 for pectin-pectate lyase; and (4) 0.5% yeast extract and 1% pectin, pH 5.0 for pectinpolygalacturonase. e lipase/esterase media consisted of peptone, 10 g/L; NaCl, 5 g/L; CaCl 2 .H 2 O, 0.1 g/L; agar, 15 g/ L; pH 7.4, supplemented with 1% (v/v) of Tween 20 and Tween 80 for lipolytic and esterastic activities, respectively. e following components were used for protease medium: 5 g/L of tryptone; 2.5 g/L of yeast extract; 1.0 g/L of glucose; 2.5 g/L of NaCl; 15 g/L of agar; and the pH adjusted to 7.0. All components were sterilized at 121°C for 15 minutes, and 100 mL skimmed milk was added for completing one liter. e experiment was performed in triplicate, and the isolates were incubated for 48 h at 28°C. Congo red dye was used as a revealer (15 minutes) followed by a washing step with 5 M NaCl for the cellulase activity visualization. Iodine tincture was used for amylase and pectinases tests. e enzymatic production of protease, lipase, and esterase activities was visualized as a bright halo around the colonies [72].

Natural Products Extraction.
e isolate GLB 2 was selected for the next experiments due to its broad-spectrum, high bioactivity rates in antimicrobial screening, and capacity to biosynthesize multiple enzymes. e natural products extract (NPE) of the isolate GLB 2 was obtained through the inoculation of 10 μL of a preculture in roundbottom tubes (12 mL capacity) containing 3 mL of ISP2 and incubated at 220 rpm/28°C for three days. Subsequently, the culture was inoculated in 100 mL of ISP2 in a 250 mL flask and maintained under the same conditions for seven days. e culture was then centrifuged at 4,500 rpm for 10 min and extracted by solid-phase using polypropylene mesh packages containing 1.5 g of Amberlite ® XAD16 resin (Sigma-Aldrich), which were added to the fermented broths and overnight incubated on a rotary shaker under the same conditions described. e resin bags were then removed and packed in glass tubes containing 20 mL of MeOH : EtOAc (1 : 1). e extracts were dried and concentrated by Vacufuge plus (Eppendorf ), resuspended at a 50 mg/mL concentration in 100% dimethyl sulfoxide (DMSO) and maintained at − 80°C [73].

Antagonism Index in Phytopathogens.
e isolate GLB 2 NPE antagonism index (AI) was determined by the inoculation of 500 μg of its NPE in a sterile disk placed on top of the plate. A plug of phytopathogenic fungus was inoculated in the center. e assay positive control consisted of culturing each phytopathogen, as described above, in the presence of 500 μg of benomyl, whereas for the negative control, each phytopathogen was cultured to indicate the expected fungi growth. All measurements were performed in triplicate ( Figure 1).

Minimum Inhibitory Concentration.
e minimum inhibitory concentration (MIC) of the NPE was performed in triplicate, according to recommendations of the Clinical and Laboratory Standards Institute (CLSI) [74]  International Journal of Microbiology pneumoniae ATCC 700603, and Pseudomonas aeruginosa ATCC 27853. An overnight culture of each pathogen was diluted until reaching a 5 × 10 5 CFU.mL − 1 final concentration in Müeller-Hinton cation adjusted (MHCA) broth with several concentrations of COPS NPE (from 100 to 1.5 μg/mL at 1% DMSO) and incubated for 24 h, at 37°C. As a positive control, ciprofloxacin was used at the same concentration gradient, and bacterial suspensions in MHCA broth and 1% DMSO were used as negative controls. e bioactivity was analyzed by measuring each well's optical density 24 hours after administration of NPE in a microtiter plate reader.

In Vitro Activity Assay against Leishmania spp.
Cultures of promastigote forms of Leishmania infantum strain MHOM/BR/1972/LD and L. major, maintained at − 80°C in a freezing solution (DMSO/fetal bovine serum-1 : 10), were thawed and transferred to 9 mL of Schneider's medium (Sigma-Aldrich, USA) supplemented with 10% inactivated fetal bovine serum (Vitrocell Embriolife, BRA), 10% human urine from male volunteers aged between 25 and 35 years, and 1% of penicillin and streptomycin. e cultures were then centrifuged for 5 min at 5000 rpm, and the pellet was resuspended in 1 ml of the same medium, which was transferred to a 50 mL capacity cell culture bottle containing 9 mL of fresh medium and incubated at 26°C in 5% CO 2 .
e toxicity assay consisted of the inoculation of promastigotes in the stationary phase (10 7 cells/mL) in 96 well plates containing different concentrations of NPE (200, 100, 50, 25, 10, and 1 μg/mL). ey were tested in biological triplicates and experimental duplicates. e OD 550 was measured by a spectrophotometer ( ermo Scientific Multiskan GO spectrophotometer) 24 hours after the administration of NPE. Amphotericin B (Sigma-Aldrich, USA) at 100 μM was used as a positive control. e cell viability percentage was calculated from the absorbance of the negative control, which represents 100% of cell viability (% of living cells � test OD 550 × 100/negative control OD 550 ), and IC 50 was measured by nonlinear regressions of the values found for each concentration in, at least, three independent experiments.

Statistical Analysis.
e results were analyzed by GraphPad Prism 8.0.1 software (San Diego, California, USA), and the Shapiro-Wilk test was applied to all data obtained. Subsequently, one-way ANOVA (one-way analysis of variance), followed by Dunnett's multiple comparisons test were applied using a statistical significance at p < 0.05 (95%).  e statistical significance in Leishmania assays was calculated by Tukey's multiple comparison test after oneway ANOVA analysis.
2.8. Genomic DNA Isolation. Isolate GLB 2 was cultivated for three days/28°C in 20 mL of ISP2 broth (malt extract, 10 g/L; glucose, 4 g/L; yeast extract, 4 g/L; pH 7,3), and cells were harvested by centrifugation at 8000 rpm for 10 minutes. Genomic DNA was then extracted using DNeasy Kit (Qiagen) with a lysis process consisting of four cycles of incubation of cell pellets at 65°C for 15 minutes in 180 μL of ATL buffer followed by freezing at − 80°C for 15 minutes. DNA was eluted in 100 μL of H 2 O, and its quality was analyzed on 0.7% agarose gel stained with hydra green and quantified using a NanoDrop.

Antimicrobial and Enzymatic Potential Screening.
Fourteen bacterial isolates were randomly selected (endophytes and from rhizosphere) and tested by overlay assay against S. aureus ATCC 29213, E. coli ATCC 11775, and C. albicans ATCC 10231 and phytopathogenic fungi as a primary selection on solid media. e results of the antimicrobial and enzymatic screening assay data are summarized in Tables 2 and 3. We observed that protease was the most abundant enzymatic activity detected, followed by pectinase at pH 8.0 (pectate lyase). Based on such results, we selected the isolate GLB2, which showed the antagonism activity against all tested pathogens and presented the enzymatic activity. We coined the isolate Burkholderia cepacia COPS strain in this study.

Burkholderia cepacia COPS Genome.
Root endophyte GLB2 isolate, which revealed a broad-spectrum and potent activity against all pathogens tested, was identified as Burkholderia cepacia based on 16s rRNA BLAST (GenBank accession number MN939546) search. e phylogenetic analysis revealed the isolate belongs to the Burkholderia sensu stricto group and is closely related to Bcc genomovar.
Genome sequencing of Burkholderia cepacia COPS strain generated 1,970,487 reads with an average length of 35-251 bp. e assembled genome estimated in 8.3 Mbp distributed in 80 contigs with an N50 of 275,353 bp. e functional annotation predicted 1885 genes, of which 1,838 are protein-coding genes (CDSs), 21 tRNAs, 26 misc RNAs, and 66.87% of a GC content. e draft-genome sequence was deposited in GenBank under accession number WIXR00000000.
Phylogenetic inferences (neighbor-joining and maximum likelihood) of COPS strain and Bcc reference strains of individual 16S rRNA and housekeeping genes atpD and phaC showed low-resolution bootstrap values. In addition, COPS strain is clustered separately from B. cepacia UCB 717 forming a single branch, whereas gltB, gyrB, lepA, recA, and trpB were strongly supported, as well lineages clearly grouped ( Supplementary Material 1-8).
Pairwise digital DNA-DNA hybridization (dDDH) of COPS strain compared to 13 sequences of other Burkholderia cepacia strains revealed identity levels ranging from 87.90% to 80.70%, whereas ANIm, ANIb, and tetranucleotide frequency signature (TETRA) values of Burkholderia sp. LK4, B. reimsis BE51, and B. lata LK27 were interestingly slightly higher when compared to B. cepacia strains. Table 4 summarizes the genome level comparisons of COPS to other B. cepacia strains.

Activity against Leishmania spp.
We tested the activity of B. cepacia COPS NPE in vitro against Leishmania infantum and Leishmania major. A 24-hour treatment with NPE concentrations ranging from 200 to 1 μg/mL potentially inhibited L. infantum promastigotes with cell viability ranging from 27% to 78.25%, respectively, with IC 50 of 86.6 μg/mL. Moreover, the NPE in the highest concentration tested was more effective than amphotericin B at 100 μM (28.6% cell viability).
On the other hand, the effect of COPS NPE against Leishmania major was not so potent compared to L. infantum.

Discussion
Burkholderia spp. are nonfermenting bacteria spread worldwide and highly able to adapt to various ecological niches [43,46,[97][98][99]. Although Bcc is considered potentially life-threatening as an opportunistic pathogen in hospital environments, especially in patients with cystic fibrosis, Burkholderia spp. are versatile for biotechnological applications, and their potential has been exploited for bioremediation, plants growth promotion, biological control, and broad-spectrum agents in several members of this group [41,46,47,100].
Burkholderia genus (sensu lato) comprises more than 100 species that possess the ability to adapt to various ecological niches [99,101]. Members of this complex group exhibit genomes ranging from 7 to 9 Mbp, resulting in several taxonomic rearrangements [46,99], rapid mutation, and adaptation [102]. Bcc species exhibit high genetic   International Journal of Microbiology similarity and is phenotypically indistinguishable [102][103][104], leading to often misidentifications interfering the effective treatment and epidemiological studies [52,105].
Our analyses concerning COPS strain for pathogenicity determinants using PathogenFinder 1.1 revealed a prediction of pathogenic potential of 0.829, whereas the reference B. cepacia ATCC 25416 was 0.774. Interestingly, Z-scores of TETRA correlation were slightly higher (Burkholderia sp. LK4, B. reimsis BE51, and B. lata LK27) when compared to B. cepacia strains. e analysis of sequence variations of recA and hisA genes offers further discriminatory support at species-level identification [53,104,106]. However, multilocus sequence typing offers more sensitivity to identify species within Bcc [57,[107][108][109]. Our results showed a significant branch support (100%) and a strong association with B. cepacia UCB717 and revealed novel alleles of gyrB, lepA, and phaC, which led us to describe the novel ST 1870. As mentioned, Burkholderia spp. may be found in a wide range of niches, and genetic variations might occur in response to niche adaptation [41,109].
Regarding the individual alignments of atpD and phaC, our results showed low-resolution bootstrap values, which corroborate inconsistencies found in MLST, ANI, and DNA hybridization of the soil isolate Burkholderia catarinensis sp. nov., formerly reported as B. cepacia [110]. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) enabled the proper identification of novel species of Burkholderiales. Interestingly, B. catarinensis exhibits physiological characteristics that differ from most other Bcc species [52].
In summary, the present study confirms that COPS strain is closely related to B. cepacia based on the wholegenome ANI, individual housekeeping gene sequence analysis, and MLST. Moreover, we present for the first time the identification of a B. cepacia symbiotically/endophytically associated with Polygala paniculata suggesting that Polygala genus plays a role in harboring microorganisms for biotechnological applications. e detection of in vitro production of active compounds may be tricky. Usually, quorum sensing (QS) also controls the production of multiple antimicrobial substances. e cell density can promote a signaling system that transcribes certain genes for their interaction with their hosts and increases resistance to stresses [43,111]. However, the presence of nutrients in the fermentation medium might affect the biosynthesis of QS signaling molecules, such as N-acyl homoserine lactones. Keum et al. [112] demonstrated that glucose (present in ISP2 at 4 g/L in its composition) increases the biomass, but suppresses the production of pyrrolnitrin, which is effective against fungi, yeasts, and Gram-positive bacteria [113]. Nevertheless, Figure 4 shows that the COPS NPE produced by the cultivation in ISP2 still exhibited potent inhibition against phytopathogenic fungi (lanes D, E, and F). Interestingly, regarding cell proliferation, the cultivation of B. cepacia COPS in ISP2 showed higher cell density comparing to other media, such as Luria-Bertani medium [114], PDB [67], and 2S4G [115]. However, in disk diffusion assay, the NPEs obtained by cultivation in different media presented a nonsignificant activity (lane B). In contrast, the overlay assay using ISP2 showed a moderate activity against B. cereus and higher inhibition zones against E. coli and E. faecalis (lane C). us, these data corroborate related studies [112,116,117] concerning multiple secondary metabolites' production.
In agrobiology, the broad-spectrum activity of microorganisms that inhibit plant pathogens is critically important for biological control because the ability to antagonize phytopathogens can indirectly promote the host plant * * * * * * growth [99,118]. In this context, Orlandelli et al. [118] evaluated antagonism and competitive interactions of endophytic fungi isolated from Piper hispidum against Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa, and Moniliophthora perniciosa. Although fungus Lasiodiplodia theobromae showed activity against all tested phytopathogens in a dual culture assay, it exhibited antagonism indexes of 60.09% against M. perniciosa, 64.79% in A. alternata, and 54.16% in Colletotrichum sp. In our study, the NPE of Burkholderia cepacia COPS showed better inhibition toward M. perniciosa, thus exceeding the positive control (74.17%). On the other hand, the COPS crude extract showed mild effects toward A. alternata (22.53%) and Colletotrichum sp. (42.27%). e phytopathogenic fungus Moniliophthora perniciosa, which causes the witches' broom disease in cacao crops, is responsible for 90% losses in the cacao annual production [119,120]. erefore, it is of interest to have a microorganism, such as B. cepacia COPS, that could help in the cacao crops infection control.
Interestingly, de Almeida Lopes et al. [121] isolated three endophytic strains of Bcc from soybean plants. e cultivation in nutrient broth produced bioactive lipopeptides, extracted by different methods (methanol, ethyl acetate, and ammonium sulfate precipitation), and qualitatively tested regarding their capacity to inhibit fungal (S. sclerotiorum, P. sojae, and R. solani) and bacterial (X. axonopodis pv. glycines and P. savastanoi pv. glycinea) plant pathogens. e inhibition rates against the phytopathogenic fungi exceeded 70%; whereas, in our study, 200 μg of the COPS NPE promoted a potent inhibition of Sclerotinia sclerotiorum and showed a moderate activity against P. sojae.
Our analysis revealed that COPS NPE can potentially inhibit C. paradoxa but exhibits moderate and slight activities against Colletotrichum sp. and Fusarium verticillioides, respectively. Such phytopathogenic fungi and others are responsible for high losses in the production of several crops worldwide [122,123]. C. paradoxa causes the black rot postharvest disease in pineapple [124] and also infects sugarcane [125,126], palm trees, cacao plants, and several other crops [127]. F. verticillioides is a producer of fumonisin, a carcinogenic mycotoxin [128], and other species have been described as emergent and opportunistic pathogens in humans [129]. Fávaro et al. [125] monitored E. nigrum endophytically inoculated in sugarcane plants, and its extract significantly reduced the diameter of Fusarium verticillioides, Colletotrichum falcatum, Ceratocystis paradoxa, and Xanthomonas albilineans colonies at concentrations ranging from 0.1 to 2.0 mg/mL.
As discussed above, the COPS strain was capable of producing lipase in solid medium, and its NPE potentially inhibited L. infantum and L. major. ese observations corroborate the work of Alves et al. [130] that investigated the antileishmanial activity of crude extracts of lipase-producing endophytic fungi toward Leishmania amazonensis. e antileishmanial activity of lipases of Vermisporium sp. (78.88%), Emericella nidulans (39.65%), Dichotomophtora portulacae (63.17%), and Dichotomophtora boerhaaviae (98.13%) was detected at 5 mg/mL in amastigote forms, suggesting an enhancement of antileishmanial activity by lipases due to their thermal stability and resistance to several organic solvents, including alcohols [131]. erefore, a detailed analysis of compounds produced by B. cepacia COPS is fundamental for a complete understanding of its potent antileishmanial effect.
Considering that the leishmaniasis treatment with pentavalent antimonials is known to be ineffective and unsafe and therapies based on pentamidine and amphotericin B are considered toxic and exhibit recurrence rates, it is clear that new treatment alternatives are necessary [132][133][134][135][136]. Although widely described as a producer of antibacterial and antifungal compounds [41,42,[137][138][139], we emphasize that the Burkholderia species' antileishmanial activity was unknown so far. As we demonstrated in this study, Burkholderiales may offer promising candidates to treat neglected diseases of which resistance and toxicity of current treatments represent a global public health concern.

Conclusions
is research's novelty lies in the isolation of rhizospheric and endophytic bacteria associated with Polygala paniculata and isolation and biological activity determination of the root endophyte Burkholderia cepacia COPS strain. Our results demonstrated that Polygala paniculata is a promising source of microorganisms for the fight against Leishmania spp., bacteria of clinical importance, and phytopathogens.

International Journal of Microbiology
Data Availability e data used to support the findings of this study are found in laboratory notebooks of Laboratory of Microbiology and Biomolecules (LaMiB), Department of Morphology and Pathology, Federal University of São Carlos, Brazil.

Conflicts of Interest
e authors declare that they have no conflicts of interest.