Boottanun et al. AMB Expr (2017) 7:16 DOI 10.1186/s13568-016-0302-0 ORIGINAL ARTICLE Open Access Secondary metabolites from Bacillus amyloliquefaciens isolated from soil can kill Burkholderia pseudomallei Patcharaporn Boottanun1,2, Chotima Potisap1,2, Julian G. Hurdle3 and Rasana W. Sermswan1,2* Abstract Bacillus species are Gram-positive bacteria found in abundance in nature and their secondary metabolites were found to possess various potential activities, notably antimicrobial. In this study, Bacillus amyloliquefaciens N2-4 and N3-8 were isolated from soil and their metabolites could kill Burkholderia pseudomallei, a Gram-negative pathogenic bac- terium also found in soil in its endemic areas. Moreover, the metabolites were able to kill drug resistant isolates of B. pseudomallei and also inhibit other pathogenic bacteria such as Staphylococcus aureus, Escherichia coli and Acinetobac- ter baumannii but not the non-pathogenic Burkholderia thailandensis, which is closely related to B. pseudomallei. Since the antimicrobial activity of N3-8 was not partially decreased or abolished when treated with proteolytic enzymes or autoclaved, but N2-4 was, these two strains should have produced different compounds. The N3-8 metabolites with antimicrobial activity consisted of both protein and non-protein compounds. The inhibition spectrum of the precipi- tated proteins compared to the culture supernatant indicated a possible synergistic effect of the non-protein and peptide compounds of N3-8 isolates against other pathogens. When either N2-4 or N3-8 isolates was co-cultured with B. pseudomallei the numbers of the bacteria decreased by 5 log10 within 72 h. Further purification and characterization of the metabolites is required for future use of the bacteria or their metabolites as biological controls of B. pseudomal- lei in the environment or for development as new drugs for problematic pathogenic bacteria. Keywords: Bio-control, Antimicrobial peptides, Pathogenic bacteria, Secondary metabolites Introduction substance from Bacillus licheniformis could inhibit food Bacillus spp. are Gram-positive bacteria found diversely spoilage bacteria (Guo et al. 2012). Moreover, the culture in nature especially in soil. In unsuitable conditions such supernatant from Bacillus spp. isolated from soil named as high temperature, radiation and harsh chemical rea- KW and SA was reported to contain N-acyl homoserine gents, they can form endospores for survival (Errington lactone that significantly decreased biofilm formation of 2003). Besides spore forming, Bacillus spp. are also able Burkholderia pseudomallei (Ramli et al. 2012). In addi- to produce secondary metabolite products (Sansine- tion, the Bacillus strain TKS1-1 in endospore form, was nea and Ortiz 2011), which is an additional function to used to reduce the incidence of citrus bacterial canker compete against other organisms. Bioactive compounds (Huang et al. 2012). Some Bacillus spp. can produce sev- from Bacillus subtilis, bacteriocin-like substances, were eral types of active compounds such as B. amyloliquefa- reported to inhibit several clinical bacteria such as Lis- ciens FZB42 that has 8.5% of the genome dedicated for teria monocytogenes, Staphylococcus aureus, Bacil- the synthesis of secondary metabolites (Chen et al. 2007). lus cereus, Salmonella typhi (Xie et al. 2009) and also a It can produce lipopeptides; surfactin, fengycin, bacillo- mycin D, polyketide (difficidin) and dipeptide bacilysin that can suppress growth of Erwinia amylovora (Chen *Correspondence: [email protected] 1 Department of Biochemistry, Faculty of Medicine, Khon Kaen University, et al. 2009). 123 Mitraparb Rd, Muang District, Khon Kaen Province 40002, Thailand Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Boottanun et al. AMB Expr (2017) 7:16 Page 2 of 11 Melioidosis is one of infectious diseases that pose a agar (NA) plates and incubated at 37 °C for 18–24 h. Bac- significant public health problem in Southeast Asia and terial isolates with colony morphology of large, dry, white North Australia. The causative agent is B. pseudomal- color with wavy, lobed margins were selected to sub- lei, a Gram-negative bacteria which can be found in soil culture and confirmed by the Gram’s stain. These isolates and water in endemic areas (Cheng and Currie 2005). were tested for their ability to kill B. pseudomallei and Melioidosis is the third most cause of death among the other pathogens. infectious diseases in northeast Thailand and is respon- sible for 20% of community acquired septicemias with Agar well diffusion method for screening of antimicrobial a 40% mortality rate (Wiersinga et al. 2012). The bacte- activity rium is intrinsically resistant to several antibiotics and Antimicrobial activity against B. pseudomallei or other acquired resistant to ceftazidime the drug of choice after pathogens of culture supernatants and precipitated pro- lost a penicillin-binding protein 3 gene (Chantratita tein from culture supernatants of Bacillus spp. were et al. 2011). Burkholderia pseudomallei was found to be investigated by the agar well diffusion method (Umer unevenly distributed in soil and that the physicochemi- et al. 2013). In brief, overnight 1% cultures in Luria Ber- cal properties of soil may partially influence the presence tani (LB) medium of B. pseudomallei and other indica- and absence of the bacterium (Ngamsang et al. 2015; tor bacteria were inoculated into fresh LB medium and Palasatien et al. 2008). Some microbes in soil may also incubated at 37 °C, 200 rpm for 4 h until the log phase inhibit or compete by producing some active compounds and were then used at approximately 105–106 CFU/ml against the bacteria. to swab on Müller-Hinton agar (MHA) plates. The plates In this study, it was therefore of interest to screen for were punched to obtain 6.6 mm wells by sterile pipette Bacillus spp. in soils that were negative for B. pseudomal- tips and then 100 µl of sterile supernatant or precipitated lei and characterize the metabolites that could inhibit or proteins were added into each well. Ceftazidime (Sigma- kill B. pseudomallei. The Bacillus spp. themselves or their Aldrich, St. Loius, MO, USA) at 50 µg/ml concentration antimicrobial metabolites might then be used as bio-con- was used as the positive control and minimal medium trols to prevent and reduce the incidence of melioidosis was used as the negative control. The plates were left in endemic areas. at room temperature for 1 h before being incubated at 37 °C, for 18–24 h. Inhibition activity was evaluated by Materials and methods measuring the diameter of inhibition zone against B. Bacterial strains pseudomallei. Bacterial strains used to test for the inhibition spectrum of B. amyloliquefaciens metabolites are listed in Addi- Species identification tional file 1: Table S1. They were obtained from the Meli- N2-4 and N3-8 with inhibitory activity against B. pseu- oidosis Research Center, Faculty of Medicine Khon Kaen domallei were selected for gDNA extraction with an RBC University, Thailand and also kindly provided by Asso- kit (RBC ribosicen, Taiwan) and were then used for PCR ciate Professor Julian G. Hurdle’s laboratory, Center for amplification using universal primers against the con- Infectious and Inflammatory diseases, Institute of Bio- served region in 16s rDNA gene (Rd1; 5′AAGGAGGT sciences and Technology, Texas A&M University, USA. GATCCAGCC3′, Fd1; 5′AGTTTGATCCTGGCTCAG3′) Our bacterial strains were deposited in culture collec- (Ghribi et al. 2012). The master mix of PCR contained tion belonging to World Data Centre for Microorganism 2.5 µl of 10X PCR buffer, 0.16 mM dNTP, 2.0 mM MgCl2, (WDCM) as MRCKKU (registration number 1130). 0.1 µM of each primer, 0.04 unit/ml of Taq DNA poly- merase, 50 ng of DNA template and DNase-free water Isolation of Bacillus spp. from soil to a final volume of 25 µl. The PCR products were ana- Twenty-five soil samples that were confirmed as nega- lyzed with 1.2% agarose gel electrophoresis and stained tive for B. pseudomallei by culture and semi-nested PCR with SYRB Gold. The expected PCR products of 1500 bps (Ngamsang et al. 2015) were used for isolation of Bacillus were sequenced (First Base laboratories Sdn Bhd, Malay- spp. sia) and the BLASTn program was used (Altschul et al. The protocol for isolation of Bacillus spp. from soil was 1997; National Center for Biotechnology Information according to Travers et al. (1987) described with some 2015) to identify their species. modifications. One gram of each soil sample was mixed with 10 ml sterile distilled water and boiled at 100 °C for Production kinetics of antimicrobial metabolites 5 min to kill other vegetative cells. Thereafter, superna- N2-4 and N3-8 were cultured in duplicate in 200 ml tants were diluted by 10-fold serial dilution and 100 µl of of minimal medium composed of 5.0 g l-glutamic −2 −3 −4 the 10 , 10 , 10 dilutions were spread onto nutrient acid, 0.5 g KH2PO4, 0.5 g K2HPO4, 0.2 g MgSO4·7H2O, Boottanun et al. AMB Expr (2017) 7:16 Page 3 of 11 Partial purification of proteins 0.01 g MnSO4·H2O, NaCl 0.01 g, FeSO4·7H2O 0.01 g, CuSO4·7H2O 0.01 g and CaCl2·2H2O 0.015 g/L. This was Culture supernatants from N2-4 and N3-8 were pre- supplemented with 1% w/v sterile glucose as described by cipitated using 20, 40, 60 and 80% saturated ammonium Jamil et al. (2007) at 37 °C with 200 rpm shaking for 102 h.