Broad-spectrum antimicrobial activity by Burkholderia ...

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Jun 18, 2018 - Sawana A, Adeolu M, Gupta RS. Molecular signatures and phylo- genomic analysis of the genus Burkholderia: proposal for division.
RESEARCH ARTICLE Rojas-Rojas et al., Microbiology DOI 10.1099/mic.0.000675

Broad-spectrum antimicrobial activity by Burkholderia cenocepacia TAtl-371, a strain isolated from the tomato rhizosphere squez-Murrieta,1 Fernando Uriel Rojas-Rojas,1 Anuar Salazar-Gómez,1 María Elena Vargas-Díaz,1 María Soledad Va Ann M. Hirsch,2,3 Ren e De Mot,4 Maarten G. K. Ghequire,4 J. Antonio Ibarra1,* and Paulina Estrada-de los Santos1,*

Abstract The Burkholderia cepacia complex (Bcc) comprises a group of 24 species, many of which are opportunistic pathogens of immunocompromised patients and also are widely distributed in agricultural soils. Several Bcc strains synthesize strainspecific antagonistic compounds. In this study, the broad killing activity of B. cenocepacia TAtl-371, a Bcc strain isolated from the tomato rhizosphere, was characterized. This strain exhibits a remarkable antagonism against bacteria, yeast and fungi including other Bcc strains, multidrug-resistant human pathogens and plant pathogens. Genome analysis of strain TAtl-371 revealed several genes involved in the production of antagonistic compounds: siderophores, bacteriocins and hydrolytic enzymes. In pursuit of these activities, we observed growth inhibition of Candida glabrata and Paraburkholderia phenazinium that was dependent on the iron concentration in the medium, suggesting the involvement of siderophores. This strain also produces a previously described lectin-like bacteriocin (LlpA88) and here this was shown to inhibit only Bcc strains but no other bacteria. Moreover, a compound with an m/z 391.2845 with antagonistic activity against Tatumella terrea SHS 2008T was isolated from the TAtl-371 culture supernatant. This strain also contains a phage-tail-like bacteriocin (tailocin) and two chitinases, but the activity of these compounds was not detected. Nevertheless, the previous activities are not responsible for the whole antimicrobial spectrum of TAtl-371 seen on agar plates, suggesting the presence of other compounds yet to be found. In summary, we observed a diversified antimicrobial activity for strain TAtl-371 and believe it supports the biotechnological potential of this Bcc strain as a source of new antimicrobials.

INTRODUCTION Burkholderia sensu lato includes Burkholderia, Paraburkholderia, Caballeronia species and Robbsia andropogonis, and it has been suggested that Burkholderia rhizoxinica may represent another genus [1–4]. In Burkholderia sensu stricto, a group of 24 species are included in the Burkholderia cepacia complex (Bcc) [5]. This group comprises known opportunistic pathogens for immunocompromised patients and is responsible for nosocomial infections. However, the Bcc is also part of the soil microbial community [6]. Together, the different Bcc species produce a wide range of antimicrobial compounds that inhibit fungal and bacterial (phyto)

pathogens [7]. Peptidic molecules with activity against filamentous fungi and yeasts include the cepacidines produced by B. cepacia AF 2001 [8], xylocandins by B. pyrrocinia ATCC 39277 [9], glidobactins by some B. cepacia strains [10], occidiofungin by Burkholderia contaminans MS14 [11, 12], burkholdins by Burkholderia ambifaria 2.2 N [13, 14], and altericidins synthesized by B. cepacia KB-1 that have limited activity on the phytopathogenic fungus Alternaria kikuchiana [15]. In addition to these peptides, several other secondary metabolites have been characterized as antifungal in the Bcc, including hydroxamate siderophores [16], uncharacterized volatile compounds [17] and phenazine-

Received 23 March 2018; Accepted 15 May 2018 Author affiliations: 1Instituto Politecnico Nacional, Escuela Nacional de Ciencias Biológicas, Prol. Carpio y Plan de Ayala s/n, Col. Santo Tomas, Del. Miguel Hidalgo. Mexico, Cd. de, Mexico; 2Dept. of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA, USA; 3Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA; 4Centre of Microbial and Plant Genetics, University of Leuven, Kasteelpark Arenberg 20 box 2460, 3001, Heverlee-Leuven, Belgium. *Correspondence: J. Antonio Ibarra, [email protected]; Paulina Estrada-de los Santos, [email protected] Keywords: Burkholderia cepacia complex; antagonism; inhibition; antimicrobial compounds. Abbreviations: Bcc, Burkholderia cepacia complex; BLIS, bacteriocin-like inhibitory substance; CAS, Chrome Azurol S; DCM, dichloromethane; DOE-JGI, Department of Energy-Joint Genome Institute; ESI-TOF-MS, electrospray ionization time-of-flight mass spectrometry; EtOAc, ethylacetate; HEX, hexane; LB, Luria–Bertani; NCBI, National Center of Biotechnology Information; NRP, non-ribosomal peptide; PacBio, Pacific Biosciences; PDA, potato dextrose agar; PDB, potato dextrose broth; PGPR, plant growth promoting rhizobacterium; RP-HPLC, reversed-phase high-performance liquid chromatography. Four supplementary figures are available with the online version of this article. 000675 ã 2018 The Authors

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like pigments [18]. Unlike antifungal compounds, only some antibacterial compounds have been described in Bcc strains. Two types of bacteriocins encoded in B. cepacia complex genomes have been produced recombinantly and showed antagonistic activity against other Bcc strains: the lectin-like bacteriocin from Burkholderia cenocepacia AU1054, which has activity against B. ambifaria, Burkholderia anthina, B. cenocepacia, B. contaminans and Burkholderia metallica [19], and the colicin M-like bacteriocins from B. ambifaria AMMD and MEX-5 [20]. Other examples of proteinaceous compounds produced by B. cepacia complex strains are the bacteriocin-like inhibitory substance (BLIS) produced by Burkholderia ubonensis A21 that acts against Burkholderia pseudomallei, the pathogen causing melioidosis [21], and a phage-tail-like bacteriocin, also known as tailocin [22], produced by B. cenocepacia BC0425, which inhibits several Bcc and non-Bcc species [23]. Also, secondary metabolites such as hydroxamate and catecholate siderophores [24, 25], the acetylenic compounds cepacins [26], a polyene compound produced by B. cenocepacia P525 [27], and the enacyloxins produced by Bcc species [28] have been reported to have antibacterial activity. Broad antimicrobial activity against fungi, yeast and bacteria has been reported for pyrrolnitrin produced by Burkholderia pyrrocinia [29, 30], B. cepacia [18, 31–36] and B. cenocepacia [37]. The variety of antimicrobial compounds produced by the Bcc, underlines the biotechnological potential of this group of species. Most of the antagonism studies in Burkholderia have focused on the activity of a single compound in a single strain. Indeed, by genome mining multiple genes and gene clusters have been found in several genomes of Bcc strains [38]. Our research group carried out a study exploring the ability of different environmental Burkholderia species to produce antagonistic compounds. From this search we found that B. cenocepacia TAtl-371 [39] was able to antagonize not only bacteria but also multiple fungi and yeasts, some of them with clinical and agricultural relevance. The aim of this work was to characterize the antimicrobial activity spectrum of B. cenocepacia TAtl-371 and the compounds responsible for such activity.

METHODS Micro-organisms and culture media B. cenocepacia TAtl-371, originally identified as Burkholderia unamae and in the current study reclassified as B. cenocepacia, was isolated from the tomato rhizosphere by Caballero-Mellado et al. [39] in Atlatlahuacan, Morelos, Mexico. The type strain B. cenocepacia J2315T was used as the reference [40]. The antimicrobial activity from TAtl-371 and J2315T was tested against the bacterial and fungal strains listed in Table 1. All strains were stored in 35 % (v/v) glycerol at 70  C and propagated in the appropriate culture medium and temperature before the experiments. The fungal strains were stored and cultured on potato dextrose

agar (PDA) plates at room temperature. The culture media employed included both potato dextrose broth (PDB) and Luria–Bertani (LB) broth. For solid and soft media preparation, agar was added to a final concentration of 1.5 and 0.5 %, respectively.

Multilocus sequence analysis The housekeeping genes gltB, lepA and recA and 16S rRNA fragment were PCR-amplified, sequenced and analysed according to Estrada-de los Santos et al. [41]. The atpD gene was also PCR-amplified as described by Baldwin et al. [42], and included in this analysis. Plant beneficial activities The B. cenocepacia strains were tested for siderophore production, phosphate solubilization, and growth on phenol, as previously described [39]. Paraburkholderia xenovorans LB400T was used as a positive control. Transmissibility marker gene detection The presence of cblA and esmR genes, two transmissibility marker genes associated with B. cenocepacia epidemic strains, was investigated using the specific primers CBL1/ CBL2 and BCESM1/BCESM2 following the PCR conditions previously described [43, 44]. Antimicrobial activity assays The ability to produce antimicrobial compounds was detected using the double-layer agar technique for bacteria and yeast as described previously [45]. Briefly, B. cenocepacia TAtl-371 and J2315T were spotted (2 µl volume) on PDA at 30  C for 72 h. Next, plates were exposed to chloroform vapours and overlaid with soft agar medium seeded with the respective bacteria or yeast strains to be tested. Antimicrobial activity was reported as an inhibition zone around the B. cenocepacia TAtl-371 and J2315T colonies. For fungi, cylinders of 5 mm carrying mycelial growth were cut from 10-day-old cultures on PDA and placed in the centre of a fresh PDA plate. At 8 equidistant points to the fungal cylinder, 5 µl volumes of B. cenocepacia cells grown to OD600 0.1 were inoculated. The plates were incubated for 2 to 3 days at 29  C and measured inhibition zones [46]. Antimicrobial production in batch cultures TAtl-371 and J2315T strains were grown in aerated 400 ml PDB batch cultures for 72 h at 30  C. The supernatant fraction was recovered by centrifugation (2722 g for 30 min at 4  C) and 200 ml were frozen for 72 h, at 20  C. Lyophilization was performed for 3 days or until the supernatant was completely dry. The resulting powder was rehydrated with 5 ml of distilled water thus concentrating 40 times. Drops of 10 µl were placed on PDA plates overlaid with soft PDA inoculated with the strains of interest. Effect of temperature, pH, chemicals and enzymes on antimicrobial activity Various temperatures, pH values, chemicals and enzymes were tested for their effects on the antimicrobial activity of the culture concentrated supernatant of strain TAtl-371. For

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Table 1. Antimicrobial activity spectrum of Burkholderia cenocepacia TAtl-371 and J2315T on agar plates and TAtl-371 supernatant Susceptibility tested strain

Burkholderia cenocepacia J2315T

Inhibition by TAtl-371 lyophilized batch culture supernatant

TAtl-371

Clinically important bacteria Acinetobacter baumannii ATCC BAA-747*

+

Pseudomonas aeruginosa PAO1

+

Staphylococcus aureus 55C1

+

+

+

Burkholderia cepacia complex B. ambifaria LMG 17828

+

LMG 17829

+

LMG 19182T

+

LMG 19466

+

LMG 19467

+ ND

MEX-5

+

B. anthina LMG 20980T

+

ND

LMG 20983

+

ND

B. arboris LMG 24066T

+

+

+

R-132

+

+

+

B. cenocepacia LMG 19230

+

LMG 18863

+

LMG 18824

+

LMG 16659

+

LMG 21462

+

+

+

+

ND

LMG 6986 LMG 18829 LMG 18828 LMG 18827

+

LMG 24506 LMG 18826

ND

+

+

+

+

+

+

+

ND

+

ND

B. cepacia LMG 1222T*

ND

LMG 18821 B. contaminans LMG 16227

+

+

R-12710

+

ND

+

B. dolosa LMG 18943T*

+

+

LMG 18941

+

+

B. diffusa LMG 24065T

+

+

LMG 24266

+

+

B. lata LMG 6992

+

ND

R-9940

+

ND

B. latens LMG 24064T

+

LMG 24264 R-11768

+

+

ND

+

+

B. metallica LMG 24068T

+

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Table 1. cont. Susceptibility tested strain

Burkholderia cenocepacia J2315

R-2712

T

Inhibition by TAtl-371 lyophilized batch culture supernatant

TAtl-371 +

B. multivorans LMG 13010T*

+

+

LMG 18825

ND

B. pyrrocinia LMG 14191T

+

+

LMG 21824

+ ND

B. seminalis LMG 24067T

+

+

LMG 24272

ND ND

B. stabilis LMG 14294T

+

+

LMG 14086

+

+

B. vietnamiensis LMG 10929T

ND

LMG 18835

ND

B. ubonensis LMG 20358T

+

LMG 24263

+

ND

Non-Burkholderia cepacia complex B. glumae LMG 2196T

+

+

+

1277

+

+

+

B. gladioli LMG 2216T

+

+

+

P. phenazinium LMG 2247T

+

+

P. phymatum LMG 21445T

+

P. tuberum LGM 21444T

+

Paraburkholderia

P. unamae MTl-641T

+ ND

P. xenovorans LB 400T

+

Cupriavidus C. gilardii LMG 5886T

+

+

C. metallidurans LMG 1195T

+

+

C. numazuensis LMG 26411T

+

C. plantarum LMG 26296T

+

+

C. taiwanensis LMG 19424T

+

+

Enterobacteriaceae Escherichia coli DH5a

ND

GM33

ND

JM110

+

Raoultella planticola ATCC 33531T

+

R. terrigena ATCC 33257T

+

+

Salmonella Typhimurium LT2

+

+

ATCC 13311*

+

+

ATCC 14028*

+

+

S. Typhi ATCC 6539

+

+

Tatumella citrea LMG 22049T

+

+

T. ptyseos H36T

+

+

T. punctata LMG 22050T

+

+

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+

+ +

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Table 1. cont. Susceptibility tested strain

Burkholderia cenocepacia J2315

T

T

TAtl-371 +

+

Agrobacterium tumefaciens AGJI-04

+

+

P. savastanoi pv. phaseolicola PSRF-01

+

+

T. terrae SHS 2008

+

Inhibition by TAtl-371 lyophilized batch culture supernatant

Phytopathogenic bacteria

Ralstonia solanacearum LMG 2299T LMG 2300

+ +

+

+

Robbsia andropogonis

+

+

Xanthomonas axonopodis pv. phaseoli XHFR-02

+

+

ATCC 10231

+

+

30

+

+

Clinically important yeast Candida albicans

C. glabrata CBS 138

+

43

+

+

Phytophthora sp. PTCA-14

+

+

Pythium sp. PYFR-14

+

+

Rhizoctonia sp. RHCH-14

+

+

Phytopathogen fungi and oomycetes

*Multidrug-resistant micro-organism. ND, not determined. The antimicrobial activity of both strains was tested on PDA after the growth of the producer strain for 72 h using the double-layer technique. ATCC, American Type Culture Collection. LMG, BCCM/LMG Belgian Co-ordinated Collections of Micro-organisms.

analysis of thermal stability, the sample was exposed to temperatures ranging from 30 to 100  C for 1 and 2 h. The effect of pH values between 1.0 to 13.0 was investigated by adjusting the supernatant with different 1 buffers: a glycine-HCl buffer for ranges of pH 1.0–3.0; an acetate-based buffer for pH 4.0–5.0; a citric acid-phosphate buffer for pH 6.0–7.0; a Tris-HCl buffer for pH 8–9; a glycine-NaOH buffer for pH 10.0–12.0; and a KCl-NaOH buffer for pH 13.0 [47]. The pH stability was determined by an overnight incubation of the fraction in the appropriate 1 buffer at different pH values at 4  C. After, the pH of the fraction was restored by adding the appropriate 2 or 3 buffer (glycine-HCl or citric acid phosphate). The given pH in this later step was adjusted to the one observed for the supernatant at the end of the incubation period for strains TAtl-371 and J2315T cultures, pH 3.0 and 6.0, respectively. The effect of chemicals was determined by incubating the supernatant at 25  C for 1 h with the following: water, dimethyl sulfoxide, acetonitrile, benzyl alcohol, methanol, ethanol, acetone, propanol, butanol, ethyl acetate, chloroform, benzene, ethyl benzene, xylene and trichloroethylene (10 % v/v). The proteolytic enzymes tested were pepsin, pancreatin, lysozyme, proteinase K, commercial Streptomyces griseus protease, peptidase and lipase (Sigma-Aldrich). These enzymes were tested at working concentrations of 10 and 20 mg ml 1 at 37  C for 1 and 2 h. Media served as a negative control for antimicrobial activity, lyophilized non-inoculated PDB was

used instead of the culture supernatant. After all the treatments, the antibacterial activity of the culture supernatant was tested by placing 100 µl drops on PDA and overlaying it with soft medium inoculated with T. terrea SHS 2008T.

Reversed-phase high-performance liquid chromatography The culture supernatant, fractions and purified compounds were analysed by RP-HPLC using a C18 column (4.6 by 250 mm) fitted to a Perkin Elmer HPLC system with a Perkin Elmer Flexar PDA Plus LC detector. Analysis of the chromatogram was performed using CHROMERA software. The mobile phase was composed of 0.1 % trifluoroacetic acid (A) and methanol (B). A 30 min gradient from 90 : 10 to 20 : 80 (A:B) was used. Absorbance of the effluent was monitored at 280 nm. B. cenocepacia TAtl-371 genome analysis The draft genome of B. cenocepacia TAtl-371 was generated at the Department of Energy-Joint Genome Institute (DOEJGI) using the Pacific Bioscienc (PacBio) sequencing technology [48] and deposited in The European Nucleotide Archive database (SAMN05443026) and the National Center of Biotechnology Information (NCBI) database (BioProject PRNJA332745) (Rojas-Rojas et al. in revision). Genes or proteins related to (the biosynthesis of) antimicrobial compounds were identified using the antiSMASH

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platform for secondary metabolite biosynthesis [49] and probabilistic profile hidden Markov models using HMMER (http://hmmer.org) to detect remote homologues to proteinaceous antimicrobial compounds using different profile databases of bacteriocin and enzyme sequences. Comparative genomics analysis using TAtl-371 and J2315T genomes was performed with progressiveMAUVE (http://darlinglab. org/mauve/mauve.html) [50] to identify loss or gain of genes between both strains.

Antibacterial activity in iron-deficient and ironsufficient conditions Antibacterial activity of B. cenocepacia TAtl-371 was tested under iron-deficient and iron-sufficient (50 and 100 µM Fe3+) conditions against all strains sensitive to TAtl-371 (Table 1). Iron-deficient and iron-sufficient PDA plates were prepared as previously reported [51] using a method adapted from the CAS [52] with a CAS solution without Fe3+ to prepare CAS plates. Antagonism was tested following the protocol described above. Cloning, expression, purification and activity of bacteriocin LlpA B. cenocepacia TAtl-371 llpA genes were amplified by PCR Q5 High-Fidelity DNA Polymerase (New England BioLabs), using a C100 Thermal Cycler (Bio-Rad). The amplification of the llpA88 bacteriocin gene was performed using primers FURR0001 (5¢TTGCTAcatatgAAGATCCTTGGCTCAAAT GAAACA3¢) with NdeI restriction site (marked with small letters) and FURR0002 (5¢TTGCTActcgagTCAGAAG GCGGCACCC3¢) with XhoI. PCR products were purified using the GeneElute PCR Clean-Up Kit (Sigma), digested with NdeI and XhoI (New England BioLabs), ligated in pET15b with T4 DNA ligase (Invitrogen) and transformed into E. coli DH5a. Restriction and ligase enzymes were used according to the supplier’s specifications. Preparation of competent E. coli cells and heat shock transformation were performed according to standard methods [53]. Transformants were verified for the presence of an insert by colony PCR using the appropriate primers. The confirmed plasmid (pET15_llpA88) was purified and sequenced using primers PGPRB-5104 and PGPRB-5105 [19] at GATC Biotech (Constance, Germany). Plasmids with inserts were used to transform E. coli BL21 (DE3) chemically competent cells. Induction of expression, lysis of harvested cells by mechanic breakdown from E. coli BL21 (DE3) carrying expression constructs pET15_llpA88 have been described previously [54]. The protein concentration of the free cell protein extract was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Recombinant protein LlpA88 was purified by nickel affinity chromatography, using an Äkta Purifier (GE Healthcare Life Sciences) with a 5 ml HisTrap column as described previously [19]. The Bcc strains sensitive to B. cenocepacia TAtl-371 were inoculated on 10 ml of soft LB agar and a 10 µl spot of purified recombinant LlpA88 (1 mg ml 1) was applied. Dialysis buffer (NaCl 200 mM, TRIS 20 mM; pH 7.5) was used as a negative

control. Plates were incubated at 37  C and scored for the presence of halo the following day.

Sequential solvent fractionation and purification of secondary metabolites with antibacterial activity Purification of secondary metabolites with antibacterial activity was performed by sequential solvent fractionation as follows. B. cenocepacia TAtl-371 culture supernatant was lyophilized and rehydrated with 5 % of its initial volume and then extracted with hexane (HEX), dichloromethane (DCM) and ethyl acetate (EtOAc). Each step of the extraction was performed three times with individual batches and combined into one fraction to be concentrated on a rotary evaporator. Concentrated fractions were dissolved in methanol, spotted on agar plates and overlaid with T. terrea SHS 2008T for evaluation of antibacterial activity. The active fraction was analysed by TLC on silica 60 plates, using HEX:EtOAc as a mobile phase (50 : 50 v/v), followed by UV detection at 254 and 365 nm. Compounds were separated and purified on a silica gel (70–230 mesh) column using TLC conditions. Active extract and purified compounds were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) as described above. The active peak, designated as compound 1, was tested with all sensitive strains spotting 10–20 µg and using the double-layer technique. Mass spectrometry The accurate molecular mass of compound 1 was obtained using an electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) high accuracy method at the SCRIPPS Center of Metabolomics (https://masspec.scripps. edu/).

RESULTS Bacterial identification and plant-beneficial activities Strain TAtl-371 was originally identified as B. unamae [39], but the newly analysed 16S rRNA sequence was highly similar (>97 %) to that of the Bcc (data not shown). To ascertain the identity of this strain, a MLSA was carried out. The maximum likelihood tree positioned strain TAtl-371 together with B. cenocepacia (Fig. S1, available in the online version of this article). The strain was originally characterized as a plant growth promoting rhizobacterium (PGPR) [39], and these traits were re-tested. Strong siderophore production and weak phosphate solubilization by strain TAtl-371 were observed (Fig. S2). These traits were less pronounced for B. cenocepacia J2315T, the type strain of the species. The TAtl-371 phenotypes are in agreement with the previous report. However, the ability to fix nitrogen could not be confirmed; strain TAtl-371 grew on semisolid N-free medium but it did not reduce acetylene to ethylene in nitrogen fixation experiments (data not shown). Strain TAtl-371 was also able to grow on phenol as the sole carbon source, as reported previously.

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Antimicrobial spectrum of B. cenocepacia TAtl-371 The antibacterial and antifungal activities of B. cenocepacia TAtl-371 and J2315T were tested on PDA plates after 72 h with several bacterial and fungal strains (Fig. 1, Table 1). Both strains inhibited the growth of several Gram-negative bacteria, including Bcc species from cystic fibrosis patients such as Burkholderia arboris, B. cenocepacia, Burkholderia dolosa, Burkholderia latens, Burkholderia multivorans, B. pyrrocinia, Burkholderia seminalis and Burkholderia stabilis. Also, Staphylococcus aureus, Salmonella Typhimurium, Salmonella Typhi, the rice pathogen Burkholderia glumae, the soil species Tatumella terrea, the clinical species Tatumella ptyseos, and also the phytopathogen Ralstonia solanacearum were inhibited. Strain TAtl-371 showed a significantly wider inhibitory spectrum than strain J2315T. For example, beside the strains inhibited by both strains, TAtl-371 also inhibited the multidrug-resistant pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (Gammaproteobacteria), the Bcc species B. ambifaria, B. anthina, B. cepacia, B. contaminans, Burkholderia diffusa, Burkholderia lata, B. metallica and B. ubonensis, as well as other members of the Betaproteobacteria (Paraburkholderia, Cupriavidus) and Gammaproteobacteria (Raoultella planticola, E. coli). The TAtl-371 antagonistic spectrum also comprised the phytopathogenic species Agrobacterium tumefaciens (Alphaproteobacteria), R. andropogonis (Betaproteobacteria), Xanthomonas axonopodis pv. phaseoli and Pseudomonas savastanoi pv. phaseolicola (Gammaproteobacteria) were also negatively affected. Moreover, B. cenocepacia TAtl-371 inhibited the growth of fluconazole-resistant yeasts Candida albicans 30 and Candida glabrata 43, as well as the phytopathogenic oomycetes Phytophthora sp. PTCA14 and Pythium sp. PYFR-14and the basidiomycete Rhizoctonia sp. RHCH-14. These results demonstrate the remarkable ability of B. cenocepacia TAtl-371 to inhibit several clinically and agriculturally important micro-organisms. To characterize the antimicrobial activities of B. cenocepacia TAtl-371 grown in liquid medium, lyophilized supernatant was obtained and drops from 10 to 100 µl were used to test antimicrobial activity against the sensitive strains. Interestingly, several bacteria were no longer sensitive to concentrated TAtl-371 supernatant: A. baumannii, P. aeruginosa, some strains of different Bcc species such as B. ambifaria, B. cenocepacia, B. dolosa, B. latens, B. metallica, B. multivorans B. stabilis and B. ubonensis, non-Bcc species Paraburkholderia phymatum, P. xenovorans, and other genera like Cupriavidus numazuensis, E. coli, R. planticola, S. Thyphi and S. Typhimurium, T. ptyseos, R. solanacearum and C. glabrata (Table 1). These data suggested that the antimicrobial activity of B. cenocepacia TAtl-371 might result from different compounds of which some could only be produced in solid media.

Physicochemical properties of the antimicrobial activity of TAtl-371 concentrated supernatant T. terrea SHS 2008T was the strain most sensitive to TAtl371 and selected as an indicator strain to further

characterize the corresponding antimicrobial activity present in the concentrated supernatant. The activity in the supernatant was capable of inhibiting T. terrea SHS 2008T after boiling for 2 h (Fig. 2A). Interestingly, the antibacterial activity was even only slightly reduced when the TAtl-371 supernatant was autoclaved (121  C, 15 psi for 15 min) (data not shown). The B. cenocepacia TAtl-371 culture supernatant was active over a wide pH range, from 1 to 11 (Fig. 2B). The activity was preserved after an overnight exposure to pH values between 1.0 and 13.0, and then being readjusted to pH 3.0, corresponding to the pH of the TAtl-371 strain culture at the end of the incubation period. Moreover, the antibacterial activity was stable even after mixing the supernatant with solvents of different polarities or with 2-mercaptoethanol (data not shown). A diminished antimicrobial activity was observed in the B. cenocepacia TAtl-371 supernatant after treatment with 20 mg ml 1 peptidase for 1 h, suggesting the presence of some proteinaceous compounds. Taken together, the data strongly suggest that the anti-Tatumella activity of the TAtl-371 culture supernatant is linked to one or more thermally and chemically stable compound (s). Additionally, it is likely that some of the inhibitory activity is of a proteinaceous nature.

Genes encoding antimicrobial compounds in B. cenocepacia TAtl-371 genome While this project was in progress, the final draft genome of the TAtl-371 strain was obtained (Rojas-Rojas et al. in revision) and screened for genes coding for potential antimicrobial compounds. Through homology searches genes linked to the biosynthesis of a non-ribosomal peptide (NRP), siderophores, chitinases and bacteriocins were identified. In this regard, a cluster of 15 genes likely responsible for the NRP siderophore ornibactin was found in chromosome 1. Additionally, a cluster of eight genes related to the hydroxamate siderophore pyochelin was also found in the same genetic element. These clusters have been reported in more than 40 genomes of different Burkholderia strains including B. cenocepacia J2315T [38]. Moreover, two genes encoding lectinlike bacteriocins (LlpA) were also found (llpA87 and llpA88). Also, a gene cluster encodes a tailocin present in the TAtl-371 genome (scaffold 1). Finally, two genes encoding chitinases were found: one on the larger scaffold and one on the medium-sized scaffold. The genes coding for the siderophores, chitinases and phage-tail-like bacteriocin were found in the genomes of both TAtl-371 and J2315T. The genes coding for LlpA bacteriocins on TAtl-371 genome, however, are located in a gene block that is not present on J2315T. The block contains also four hypothetical proteins, a methyl transferase and an integrase in a fragment of 7.1 Kbp. The block is only present in other B. cenocepacia strains, namely MC0-3, CR318, HI2424, AU1054, FL-5-3-30-S1-D7 and VC12802. Taken together these searches showed that TAtl-371 encodes multiple genes with potential antimicrobial activity and some of these are not common within the species.

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Fig. 1. Growth inhibition of bacteria, fungi, and oomycetes by Burkholderia cenocepacia TAtl-371 and J2315T. (A) Antagonism of B. cenocepacia J2315T and TAtl-371 against bacteria. A colony of B. cenocepacia TAtl-371 (on the left of each plate) and J2315T (on the right of each plate) was overlayed with soft agar seeded with the indicator strains: (a) Acinetobacter baumanni ATCC BAA-007; (b) A. baumanni ATCC BAA-747; (c) Burkholderia multivorans LMG 13010T; (d) Burkholderia dolosa LMG 18943T; (e) Staphylococcus aureus 55C1; (f) Salmonella Typhimurium LT2; (g) Agrobacterium tumefaciens, AGJI-04; (h) Robbsia andropogonis LMG 2129T; (i) Burkholderia glumae LMG 2196T; (j) Ralstonia solanacearum LMG 2299T; (k) Tatumella terrea SHS 2008T; (l) Tatumella ptyseous H36T; (m) Pseudomonas aeruginosa PAO1; (n) Candida albicans 30; (o) Candida glabrata 43. (B) Antagonism of B. cenocepacia J2315T and TAtl-371 against phytopathogenic basidiomycete Rhizoctonia sp. and the oomycetes Pythium sp., Phytophthora sp.

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Fig. 2. Effects of temperature and pH on the antibacterial activity of the culture supernatant from Burkholderia cenocepacia TAtl-371 against Tatumella terrea SHS 2008T. (A) Temperature effect on the B. cenocepacia TAtl-371 culture supernatant. Samples were exposed to the indicated temperatures for 1 h (black bars) or 2 h (hatched bars). The white bar indicates activity for the control samples. (B) Activity on different pH ( ) and stability to pH changes ( ).

Antimicrobial activity in iron-deficient and ironsufficient condition The siderophores’ ornibactin and pyochelin production was identified in the TAtl-371 genome. Given that antimicrobial activity has been reported for several siderophores [55], we decided to study the involvement of these compounds in the antimicrobial activity of TAtl-371. PDA plates with different Fe3+ concentrations were used for these experiments. First, we confirmed that the production of siderophores is modified by variation in the concentration of Fe3+ using a method adapted from the Chrome Azurol S (CAS) assay [52]. Siderophore production-associated halos on PDA and PDA without Fe3+ (hydroxyquinoline-treated) were not significantly different (Fig. S3) demonstrated that some Fe3+ remained on the PDA plates. However, after hydroxyquinoline treatment and the addition of 50 and 100 µM Fe3+ the siderophore production was clearly reduced (Fig. S3). A significant difference in antagonism was identified for only two micro-organisms. Inhibition of P. phenazinium LMG 2247T and C. glabrata 43 was decreased at both 50 and 100 µM Fe3+ (Fig. 3). This result suggests that siderophore production of TAtl-371 contributes to the inhibition of both species. Antagonism by bacteriocin LlpA To enable testing the potential antibacterial activity of TAtl371 LlpA bacteriocins, heterologous expression in E. coli was performed. The B. cenocepacia TAtl-371 gene encoding llpA88 was cloned into pET15b and strain E. coli BL21 pET15_llpA88 was obtained. Expression of the 30 kDa protein was detected for E. coli BL21 pET15_llpA88 after IPTG induction. The same methodology was followed for llpA87 but its expression was not observed (data not shown). After recombinant protein purification, bacteriocin activity of LlpA88 was assayed applying spots of purified protein on indicator-seeded agar plates to visualize growth inhibition as a halo formed in a confluent lawn of bacterial target cells.

Among the tested strains of Bcc species, only nine were sensitive to LlpA88 (Table 2). Also, other Burkholderia, Paraburkholderia, Cupriavidus, Rasltonia, Pseudomonas and Xanthomonas species were negative (data not shown).

Identification of an antimicrobial compound purified from the TAtl-371 culture supernatant In order to identify additional antimicrobial compounds, present in the culture supernatant of TAtl-371, the supernatant concentrated by lyophilization was analysed by RPHPLC. The resulting chromatographic pattern showed the presence of a predominant peak signal with a retention time of 3.01 min (Fig. 4A). This peak was collected, lyophilized and tested for antagonism showing antimicrobial activity against T. terrea SHS2008T. After a sequential solvent fractionation of the lyophilized culture supernatant, only the DCM extract had antimicrobial activity against T. terrea SHS2008T, indicating that the antimicrobial compound(s) had been fractionated from the original culture supernatant. This solvent extract was analysed by RP-HPLC and the predominant peak signal found was to be the same as that observed on the original lyophilized culture supernatant (Fig. 4A). Further purification of the DCM extract led to the isolation of a compound, named compound 1, which was only active against T. terrea SHS2008T (Fig. 4B). ESI-TOFMS analysis in the positive ion mode exhibited an intense [M+H]+ ion at m/z 391.2845 for compound 1 (Fig. S4).

DISCUSSION Bacteria from the genus Burkholderia are ubiquitous, and some species act as human, animal or plant pathogens, whereas others are opportunistic pathogens, e.g. many members of the Bcc. As a result of their metabolic versatility, Burkholderia species secrete numerous metabolites, including antimicrobial compounds [7]. A significant amount of information related to the production of antimicrobial compounds synthesized by Bcc species and also by

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Fig. 3. Inhibition of Burkholderia cenocepacia TAtl-371 on PDA plates with different concentrations of Fe3+. (A) antagonism against Paraburkholderia phenazinium LMG 2247T. (B) antagonism against Candida glabrata 43. The experiments were performed in triplicate and halo sizes were measured (mm).

the B. pseudomallei group exists. Bioinformatics analysis have demonstrated the presence of different gene clusters for antimicrobials in Burkholderia genomes. However, the majority of the studies have focused on the activity of a specific type of compound for individual strains. Hence, the aim of this study was to describe the antagonistic performance of an environmental B. cenocepacia strain, and to characterize multiple compounds involved in antimicrobial activity. Bacteria in the Bcc are reported to synthesize antimicrobial compounds [38], but most studies have focused on clinical strains and, to the best of our knowledge, not on environmental isolates. We tested several environmental isolates from our in-house Burkholderia collection, first selecting B. cenocepacia TAtl-371. Strain TAtl-371 was originally identified as B. unamae and characterized as PGPR [39]. However, the MLSA revealed that this strain belongs to the species B. cenocepacia. Despite being identified as a member of the Bcc, strain TAtl-371 does not contain the cblA and esmR genes, two transmissibility factors associated with the highly transmissible epidemic strains of B. cenocepacia (data not shown), suggesting that strain TAtl-371 is unlikely to be pathogenic. This strain exhibited a remarkable broad range of antimicrobial activity against clinically and agronomical important micro-organisms, including opportunistic pathogens, plant pathogens, environmental isolates and even some human pathogenic yeasts and phytopathogenic fungi and oomycetes. We observed that B. cenocepacia antimicrobial activity

was strain-dependent, e.g. the antagonistic spectra of the TAtl-371 and J2315T strains differed. More bacterial strains and fungi were sensitive to TAtl-371. Recently, a lectin-like bacteriocin was described in B. cenocepacia AU1054 [19]. The authors reported that the expressed LlpA has a genusspecific, narrow-spectrum activity, contrary to what is shown with strain TAtl-371. A previous study performed with several Bcc strains showed activity especially from the B. cenocepacia isolates, against a strain of S. aureus and one of C. albicans, but showed no activity against P. aeruginosa [28], which contrasts with our results. Although B. cepacia, B. contaminans and B. ambifaria inhibit phytopathogenic fungi and oomycetes [56], B. cenocepacia TAtl-371 inhibits a different group of phytopathogenic fungi, namely Rhizoctonia sp. and the oomycete Pythium sp. and Phytophthora sp. We did not find any reports on the inhibition of A. tumefaciens, R. andropogonis, B. glumae, R. solanacearum and X. axonopodis by other B. cenocepacia strains, susceptible bacteria belonging to different phylogenetic clades. Thus, B. cenocepacia TAtl-371 most likely synthesizes a different array of antimicrobial compounds than those described for other Burkholderia species. With regard to the activity on pathogenic yeasts, our results showed that the TAtl-371 strain inhibits the growth of both C. albicans and C. glabrata. These species are important in nosocomial infections worldwide [57, 58]. C. albicans growth inhibition has been documented for a B. cenocepacia strain [28], but an effect on C. glabrata has not been reported. Not many antifungal compounds are available for therapeutic use because several have negative effects on

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Table 2. Antagonism of recombinant LlpA88 bacteriocin against the Burkholderia cepacia complex Strain

Recombinant LlpA88*

Strain

B. cenocepacia LMG 19230

B. latens +

LMG 20358

LMG 18863

LMG 24263

LMG 18824

R-11768 LMG 24064T

LMG 16659 LMG 16656

Recombinant LlpA88*

T

B. ambifaria

LMG 21462

LMG 19467

LMG 6986

LMG 26702

LMG 18829

LMG 17828

LMG 18828

LMG 17829

+

LMG 18827

LMG 19182T

+

LMG 24506

LMG 19466

+

LMG 18826

B. diffusa

B. cepacia

LMG 24266

LMG 1222T

LMG 24065T

B. multivorans

B. arboris

LMG 13010T

LMG 24066T

LMG 18825

R-132

B. vietnamiensis

B. seminalis

LMG 18835

LMG 24272

LMG 10929T

LMG 24067T

B. stabilis

B. metallica LMG 24068T

LMG 14086 LMG 14294

T

+

R-2712

B. dolosa

B. lata

LMG 18941

LMG 6992

LMG 18943T

R-9940

B. anthina

B. contaminans

LMG 20983 LMG 20980T

+

+

R-12710

+

LMG 16227

+

B. pyrrocinia LMG 21824 LMG 14191T

An amount of 10 µl of purified recombinant LlpA at 1 mg ml complex strains.

1

was spotted and overlaid with soft agar previously inoculated with the B. cepacia

mammalian cells. With increasing resistance of C. albicans and C. glabrata strains to various antifungal drugs, it is critical to continue investigating compounds that could limit the growth of these pathogens. On the other hand, we found that peptidase treatment reduced the antimicrobial activity, strongly suggesting that some compound(s) in the supernatant fraction are of proteinaceous nature. Prerequisites for potential biomedical and biotechnological applications of antibacterial proteinaceous compounds include high stability to physical and chemical factors [59]. High thermal stability has been shown for such proteinaceous antibacterial compounds as bacteriocins [60, 61], peptides [62] and lipopeptides [63]. From a biotechnological perspective, heat stability is a useful

characteristic during food preparation and preservation as many food-processing methods require a heating step. Additionally, the stability at different pH values suggests the potential of B. cenocepacia TAtl-371 compounds to resist the harsh intestinal environment, which could make them useful as antibiotics. Similar features have been reported for non-proteinaceous antibacterial compounds such as CF66I, which is produced by B. cepacia CF-66 [64]. From our results, we believe that B. cenocepacia TAtl-371 antibacterial compounds have potential value. To determine their potential feasibility, the compounds should be purified and tested for endurance at high temperature and extreme pH, in addition to their toxicity in cells and animals. In this context, we tested haemolytic activity of strain TAtl-371 and its

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Fig. 4. Analysis by RP-HPLC and antimicrobial activity of B. cenocepacia TAtl-371 batch culture supernatant, DCM (dichloromethane) extract and purified compound 1. (A) RP-HPLC chromatogram. (B) activity of (i) dichloromethane fraction; (ii) batch culture supernatant and (iii) purified compound 1 on Tatumella terrea SHS2008T

supernatant on blood agar plates. The output was negative for both (data not shown), and this encourages us to continue the study of strain TAt-371, its antimicrobial activity and its potential use in biotechnology. The TAtl-371 genome analysis identified two gene clusters encoding for the siderophores ornibactin and pyochelin. Both siderophores have been reported to be involved in antibacterial activity on B. paludis MSh1T [24] and B. contaminans MS14 [25], members of the Bcc. Siderophores are secondary metabolites produced by micro-organisms to survive under iron-deficient conditions [65]. Antimicrobial activity mediated by siderophores has been reported to change in response to different concentrations of Fe3+ in culture media [51]. Using different concentrations of Fe3+, the inhibition decreased only with P. phenazinium LMG 2247T and C. glabrata 43. This shows that the broad antimicrobial activity of B. cenocepacia TAtl-371 is not solely due to siderophore production, but siderophores contribute to this activity. The heterologous expression of LlpA88 encoded by the TAtl-371 genome resulted in the inhibition of nine Bcc strains. By contrast, these strains were not sensitive to J2315T. Homologues of these genes have been reported in strains of Pseudomonas [66–68], Burkholderia [19] and Xanthomonas [67]. Heterologous expression and a similar antagonistic spectrum was reported by Ghequire et al. in 2013 for a lectin-like bacteriocin identical to LlpA88

encoded by the B. cenocepacia AU1054 genome [19]. Interestingly, most of LlpA88-sensitive strains belong to the species B. ambifaria, and thus further experiments are in progress in order to identify the mode of action of this bacteriocin on B. ambifaria. In Pseudomonas this bacteriocin was shown to use the essential outer-membrane protein BamA as a target (Ghequire et al. 2018, in press), though it remains unclear whether this is the case for Burkholderia LlpA(s) as well. A tailocin was found to be encoded on a gene cluster in scaffold 1 of the TAtl-371 genome. This gene cluster is widely distributed in Burkholderia and other genera. This compound was purified from the culture supernatant of B. cenocepacia BC0425 and showed activity against Bcc, non-Bcc strains and P. aeruginosa PAO1 and derivatives [69]. Also, the tailocin showed activity when exposed at 50  C for 1 h and lost activity at 80  C for 1 h and at 100  C for 3 min. A similar thermal treatment was applied on the TAtl-371 lyophilized culture supernatant. However, activity persisted against all sensitive strains indicated in Table 1, suggesting that the antagonism observed on the TAtl-371 culture supernatant is not completely given by the tailocin, if this compound is expressed since by SDS-PAGE the tailocin is not detected in the supernatant of the TAtl-371 strain as it was reported previously for B. cenocepacia BC0425 [69]. In B. gladioli, one of the proteins of this gene cluster was reported to act as a T3SS effector with a broad-

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spectrum antifungal activity [70]. However, no similar proteins where found in the TAtl-371 culture supernatant.

A. Humm and Maskit Maymon in the Hirsch lab for preparing the genomic DNA for sequencing.

Chitinases have been reported and characterized mainly in strains of B. gladioli [71, 72], but only one chitinase has been characterized in the Bcc [73]. Although two chitinaseencoding genes were identified in the TAtl-371 genome, no chitinolytic activity was detected in culture supernants of PDB, or other media designed to induced chitinases expression (data not shown), suggesting that TAtl-371 chitinase genes are not involved in the antifungal activity observed against R. solani.

Conflicts of interest The authors declare that there are no conflicts of interest.

Attempts to isolate the antagonistic compound derived in the purification of compound 1, which only has anti-T. terrea activity. The determination of the molecular mass of compound 1 showed that the same molecular mass has not been verified to be produced by any Burkholderia species, suggesting a new molecule produced by Bcc. Nuclear magnetic resonance spectroscopy experiments and knock out mutagenesis are underway to reveal the chemical structure of compound 1 and the genes involved in the production of this compound. Elucidation of the chemical nature of this potentially novel compound and the knock out mutants will be instrumental to identify the corresponding biosynthetic gene(s).

Conclusions B. cenocepacia TAtl-371 displays the broadest antimicrobial spectrum reported so far amongst the Bcc species. The characterization of the antibacterial compounds suggests that the compound(s) preserve its/their structure and function even under extreme conditions, making them useful for biomedical and biotechnological applications. Siderophores are involved in the inhibition of some bacteria, and the lectinlike bacteriocin is related to the inhibition of some Bcc strains. However, the spectrum goes beyond those compounds suggesting that more substances are involved in the inhibition activity. Funding information This project was partially supported by grants SIP 2014–0353, SIP 2015–0358, SIP 2016–0077, SIP 2016–1245, SIP 2017–0492 and SIP 2017–1714. The sequencing of the B. cenocepacia TAtl-371 genome was funded by the Joint Genome Institute of the U.S. Department of Energy Office of Science User Facility, which is supported under contract number DE-AC02-05CH11231. The purification of lectin-like bacteriocins was supported by grant 1529718N (MGKG, FWO Vlaanderen). Acknowledgements We thank Nestor Delgado Perez for technical assistance on chitinase experiments. FURR received a fellowship from CONACyT. MGKG was supported by a postdoctoral fellowship from FWO Vlaanderen (12M4618N). Acinetobacter baumannii ATCC BAA-007 and ATCC BAA7474 were kindly donated by Dr Maria Guadalupe Aguilera Arreola (ENCB-IPN). Agrobacterium tumefaciens AGJI-04, Pseudomonas savastanoi pv. phaseolicola PSRF-01, Xanthomonas anhonopodis pv. phaseoli XHFR-02, Phytophthora sp. PTCA-14, Pythium sp. PYFR-14 and Rhizoctonia sp. RHCH-14 were provided by MSc Belen Chavez-Ramírez (ENCB-IPN). Pseudomonas aeruginosa PAO1 was generously donated by Dr Carlos Cervantes Vega (IIQB-UMSNH). Candida strains were provided by Dr Lourdes Villa-Tanaca (ENCB-IPN). We acknowledge Ethan

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