Progress in Microbes and Molecular Biology Review Article 1 Microbes from Peat Swamp Forest — The Hidden Reservoir for Secondary Metabolites? Kuan-Shion Ong1,2*, Vengadesh Letchumanan3, Jodi Woan-Fei Law3, Catherine M. Yule2,4, Sui-Mae Lee1,2 1Tropical Medicine and Biology Multidisciplinary Platform, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, 47500, Subang Jaya, Selangor. 2School of Science, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, 47500, Subang Jaya, Selangor. 3Novel Bacteria and Drug Discovery (NBDD) Research Group, Microbiome and Bioresource Research Strength (MBRS), Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, 47500, Bandar Sunway, Selangor Darul Ehsan, Malaysia. 4School of Science and Engineering, University of the Sunshine Coast, Sippy Downs, Queensland, 4556, Australia. Abstract: Antimicrobial resistance is a significant threat to the healthcare sector. For the past century, there has been a decline in the discovery of new antibiotics. This has urged researchers to bio-prospect for new bioactive agents from microbes originating from untapped environments, as well as to explore the potential of other microbial genera apart from the well- known Streptomyces. Tropical peat swamp forests are an example of such an environment. Two novel antimicrobial-producing bacteria from the genera Burkholderia and Paenibacillus have been identified to produce potent antimicrobials. These two genera of bacteria have recently gained tremendous interest due to their genome complexity. They are known as multifaceted organisms not only because of their genetic content, but also due to their positive interactions with the environment along with a plethora of organisms including plants and animals. The interactions observed are attributed to their genomes and to their production of secondary metabolites including antimicrobials. Hence, this review provides an overview of the nature of tropical peat swamp forests, taxonomy and production of secondary metabolites of both Burkholderia and Paenibacillus, as well as discussing the future perspective of isolating antimicrobial-producing microbes from tropical peat swamp forests. Keywords: Antimicrobials; Burkholderia; Paenibacillus; resistance; secondary metabolite; tropical peat swamp forest Received: 18th March 2020 Accepted: 20th April 2020 Published Online: 25th April 2020 Citation: Ong K-S, Letchumanan V, Law JW-F, et al. Microbes from Peat Swamp Forest — The Hidden Reservoir for Secondary Metabolites?. Prog Mircobes Mol Bio1 2020; 3(1): a0000077. https://doi.org/10.3687/pmmb.a0000077 INTRODUCTION Antimicrobials have been used for generations as prophylaxis to prevent initial or recurrence of infection, and as agents to destroy, inhibit or prevent pathogenic action of microbes[1]. Over time, use of antimicrobials has created an inevitable selective pressure leading to the evolution of microbes to resist the action of antimicrobials. Microbes can gain resistance towards antimicrobials intrinsically through mutations or by acquiring the ability via conjugation[2]. This phenomenon is further exacerbated by the extensive use of antimicrobials to control infections which has unprecedentedly accelerated the process and emergence of resistant microbes[3]. This is an alarming issue as the rapid emergence of antimicrobial- resistant pathogens limits treatment options and increases mortality. According to the Director General of the World Health Organization (WHO), we are heading towards a post-antibiotic era in which common infections and injuries could once again kill[4]. Therefore, there is an urgent need for alternative measures to tackle the crisis of antimicrobial resistance. One of the methods is by bioprospecting for new antimicrobials from untapped resources such as the tropical peat swamp forests where extreme conditions and low nutrients promote competition among microbes. The review will discuss the nature of tropical peat swamp forests, taxonomy and production of secondary metabolites of both Burkholderia and Paenibacillus, as well as discuss the future prospects of isolating antimicrobial-producing microbes from tropical peat swamp forests. TROPICAL PEAT SWAMP FORESTS Tropical peat swamp forests (TPSFs) are unique wetland ecosystems periodically flooded by fresh water Copyright @ 2020 by Ong K-S and HH Publisher. This work under licensed under the Creative Commons Attribution-NonCommercial 4.0 International Lisence (CC-BY-NC4.0) *Correspondence: Kuan-Shion Ong, Tropical Medicine and Biology Multidisciplinary Platform, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, 47500, Subang Jaya, Selangor. kuanshion@gmail.com 2 from rainfall[5]. Out of 30–45 million hectares of global wetlands in the world, about 18.1 million hectares are TPSFs widely distributed around Southeast Asia[6]. TPSFs contribute to almost 20% of the total global terrestrial organic carbon and form one of the largest terrestrial organic carbon sinks. Carbon is stored in the forest biomass with trees up to 70m tall, but most of it is sequestered peat layers up to 25m deep. Disturbance of TPSFs due to drainage and fire causes the release of greenhouse gases such as methane and carbon dioxide to the atmosphere. Burning of TPSFs results in about 25% of the total global greenhouse gas emissions from deforestation and forest degradation which result in an estimated 3% of total global anthropogenic greenhouse gas emissions. Furthermore, TPSFs hold a key role in regional hydrology (movement and distribution of water). This is because peat can hold 5–10 times its weight in water. This is important as the water stored can act as a buffer in reducing water velocity thus minimizing the impact of downstream flooding[5]. TPSFs grow on a substrate formed by the accumulation of layers of peat (partially decomposed organic matter) up to 25m deep. They are usually dome-shaped due to the buildup of peat, and are permanently waterlogged with a pH range of 2.9 to 4.5[8]. The leaching of tannic acids (20.2 ± 2.3 mg/l) from the leaves of endemic plants causes the dark brown, acidic water in TPSFs[9]. The dark brown water reduces light penetration and impedes photosynthesis by algae, which together with the slow flow rates and high temperatures create an anoxic environment, while toxic secondary compounds leach from the leaf litter which then reduces the microbial decomposition of organic matter (decay rates of 0.0006–0.0016 k day-1 for endemic plants), hindering nutrient recycling thus creating a highly concentrated carbon reservoir[5]. In addition, TPSFs are known to be ombrotrophic, receiving nutrients and water solely from rainfall and dust. The lack of nutrient input and slow decomposition rate results in a low nutrient environment in TPSFs[8]. BACTERIAL COMMUNITY IN TPSFS It was previously thought that such extreme environmental conditions (acidic, waterlogged and low nutrient availability) meant low bacterial diversity. However, metagenomic studies have revealed the complexity of the genetic information of the bacterial community and high diversity[10]. Kanokratana et al. (2011)[10] deduced that bacteria constituted the most abundant microbial group in a Thailand TPSF. From the bacterial sequences identified, Proteobacteria was the largest species group (37.9% of total bacteria), which comprised mostly Alpha-proteobacteria, followed by Acidobacteria (35.0% of total bacteria). Other key minor bacterial phyla include Verrumicrobia (5.7%), Planctomycetes (9.6%), Actinobacteria (2.5%), Bacteroidetes (1.1%), Nitrospirae (1.8%), Firmicutes (0.4%) and others unclassified bacteria (6.0%). Moreover, the bacterial population (determined using next generation sequencing) in a Malaysian TPSF showed a similar pattern[11] as the Thailand TPSFs and was consistent with a previous study conducted by Jackson et al. (2009)[12] which showed that TPSFs are dominated by Proteobacteria and Acidobacteria (more than 50% of the total bacteria population). Antimicrobial-producing bacteria from TPSFs The slow degradation of organic matter in TPSF results in low levels of nutrients which creates a highly competitive environment[13]. Consequently, it is likely that bacteria would produce secondary metabolites such as antimicrobial compounds to secure their niche and resources. Such phenomena are consistent with the isolation of antimicrobial producing bacteria from two different TPSF: the southeast Pahang and Selangor TPSF. These antimicrobial producing isolates were identified via a polyphasic taxonomic approach to be novel species from the genus Burkholderia and Paenibacillus, namely Burkholderia paludis sp. nov. (from Pahang TPSF)[14] and Paenibacillus tyrfis sp. nov. (from Selangor TPSF)[15]. Burkholderia and their secondary metabolites The Burkholderia genus consists of a group of ubiquitous bacteria that occur in aquatic environments, soil, plant rhizospheres and animals. Burkholderia are mesophilic Gram-negative rods, oxidase positive, motile microorganisms. Burkholderia can be characterized phenotypically by their pigmentation, presence of hydroxyl fatty acids of 14, 16 and 18 carbon atoms, possession of distinct polar lipids, and by having Q8 cellular respiratory quinones[14]. The genus can be divided into three groups: Burkholderia sensu stricto, Paraburkholderia and Caballeronia[16]. Burkholderia sensu stricto is a group of closely related Burkholderia species that share a high degree of 16S rRNA (98–100%) and recA (94–95%) gene sequence similarity which makes them difficult to be differentiated using conventional molecular techniques. To differentiate different species of Burkholderia sensu stricto, multilocus sequence analysis (MLSA) is usually adopted as the technique provides the discriminatory power needed for both identification and differentiation[17]. Burkholderia sensu stricto species have diverse ecological roles and have been used in biocontrol and bioremediation. Several Burkholderia sensu stricto species can be used for biocontrol agents as they can produce secondary metabolites to repress soil borne pathogens. Some Burkholderia sensu stricto species can act as plant growth promoters. They can also be used for bioremediation of recalcitrant xenobiotics, for instance, Burkholderia xenovorans can degrade chlorinated toxic phenolic compounds commonly found in pesticides and herbicides[18]. In contrast, nearly all Paraburkholderia species (e.g. Paraburkholderia bryophila, Paraburkholderia tropica and Paraburkholderia nodosa) and Caballeronia (e.g. Caballeronia ginsengisoli, Caballeronia terrestris and Caballeronia humi) are plant growth promoters as they are able to fix nitrogen and supply nutrients to their plant hosts[16, 19]. Many secondary metabolites with antimicrobial activity are produced by the Burkholderia species have been identified. They usually possess antifungal and/ or antibacterial activity (Table 1). Microbes from Peat... 3 Ong K-S et al. Compounds Burkholderia species Bioactivity References 2-pyrrolidone-5-carboxylic acid Burkholderia sp. HD05 Antifungal Zhang et al. (2019)[20] Bis-(2-ethylhexyl) phthalate Burkholderia gladioli OR1 Antibacterial Bharti et al. (2015)[21] Burkholdines Burkholderia ambifaria 2.2N Antifungal Tawfik et al. (2010)[22] Cepacidin A Burkholderia cepacia Antifungal Lee et al. (1994)[23] Cepacins A and B Burkholderia cepacia SC 11 Antibacterial Parker et al. (1984)[24] Cepafungin Burkholderia sp. Antifungal Shoji et al. (1990)[25] Cepalycin Burkholderia cepacia Antifungal Abe and Nakazawa (1994)[26] Enacyloxins Burkholderia ambifaria AMMD Antibacterial Mahenthiralingam et al. (2011)[27] Gladiolin Burkholderia gladioli Anti-mycobacterium Song et al. (2017)[28] Icosalide Burkholderia gladioli Antibacterial Dose et al. (2018)[29] Iminopyrrolidines Burkholderia plantari #9424 ICMP Antibacterial Mitchell and Teh (2005)[30] Occidiofungin Burkholderia contaminans MS14 Antifungal Lu et al. (2009)[31] Phencomycin Burkholderia glumae 411gr-6 Antibacterial Han et al. (2014)[32] Pyochelin Burkholderia paludis Antibacterial Ong et al. (2017)[33] Pyrazoles derivatives Burkholderia glumae #3729 ICMP Antibacterial Mitchell et al. (2008)[34] Pyrrolnitrin Burkholderia cepacia Antifungal, antibacterial El-Banna and Winkelmann (1998)[35] Vietnamycin Burkholderia vietnamiensis Antibacterial Rowe et al. (2016)[36] Xylocandin Burkholderia cepacia Antifungal Meyers et al. (1987)[37] content which ranges from 39 to 59 mol%, and genome size ranges from 3.02 Mbp (eg. Paenibacillus darwinianus) to 8.82 Mbp (eg. Paenibacillus mucilaginosus)[38,39]. This group of bacteria can be isolated from a variety of environments, mainly from soil. They are often associated with humans, animals, and plants. The majority of the Pae- nibacillus spp. are producers of antimicrobial compounds and enzymes that are useful for bioremediation (Table 2). Furthermore, some of these compounds can be utilized as bio-fertilizers for plant growth promotion or bio-pesticides against root pathogens[39]. Paenibacillus and their secondary metabolites The genus Paenibacillus comprises aerobic/facultative anaerobic and endospore-forming bacteria, with a majority of them typically showing Gram-positive cell wall structures[38,39]. This genus was initially included in the genus Bacillus based on morphological characteristics prior to reclassification. The genus Paenibacillus — which means “almost a Bacillus” was then proposed by Ash et. al (1993)[40] using phylogenetic classification[39]. The Paenibacillus spp. have MK-7 as major quinone, anteiso-C15:0 as major cellular fatty acid, DNA G + C Compounds Paenibacillus species Bioactivity References Paenibacillin Paenibacillus polymyxa OSY-DF Antibacterial He et al. (2008)[41] Paenicidin A Paenibacillus polymyxa NRRL B-30509 Antibacterial Lohans et al. (2012)[42] Penisin Paenibacillus sp. A3 Antibacterial Baindara et al. (2016)[43] Polymyxin Paenibacillus polymyxa Antibacterial Nation and Li (2017)[44] Colistin Paenibacillus polymyxa Antibacterial Tambadou et al. (2015)[45] Octapeptin Paenibacillus tianmuensis Antibacterial Qian et al. (2012)[46] Paenibacterin Paenibacillus tiaminolyticus OSY-SE Antibacterial Huang et al. (2014)[47] Pelgipeptin Paenibacillus elgii B69 Antibacterial Ding et al. (2011)[48] Gavaserin Paenibacillus polymyxa Antibacterial Pichard et al. (1995)[49] Fusaricidins Paenibacillus polymyxa KT-8 Antibacterial Kajimura and Kaneda (1997)[50] Table 1. Antimicrobials produced by Burkholderia species. Table 2. Antimicrobials produced by Paenibacillus species. 4 FUTURE PERSPECTIVES IN TPSF MICROBIAL CULTIVATION Thus far, only two bacteria (Burkholderia paludis sp. nov and Paenibacillus tyrfis sp. nov) with antimicrobial- producing ability from TPSF have been successfully cultivated and identified. These bacteria such as Burkholderia from Proteobacteria and Paenibacillus from Firmicutes are common phyla of bacteria dominating the TPSFs. Hence, what needs to be improved in order to isolate the uncommon bacteria with antimicrobial- producing ability from TPSF? The possible reasons for isolating those common bacteria are the suitability of media used and the incubation period. The use of alternative culture media The failure in cultivating peat-inhabiting bacteria using culture dependent techniques is often due to the usage of conventional media such as nutrient and tryptone soy media. Such media contain near-neutral pH with high mineral salt content that do not simulate the acidic, low nutrient conditions of TPSFs[8]. Besides that, it favors fast growing bacteria and these fast-growing bacteria will outgrow the other slow growing bacteria[51]. Therefore, there is a need to use diluted acidic media with low salt content such as MM1, Medium M1, Medium M2, Medium M31, nitrate mineral salt media, peat extract medium and R2A in order to cultivate various peat-inhabiting bacteria at the same time suppressing the fast-growing bacteria (Table 3). Table 3. Examples of media with low salt content. Type of Media Media Compositions References MM1 100 mM NaCl, 10 mM (NH4)2SO4, 5 mM MgSO4, 1 mM CaCl2, 8mM KH2PO4, 16 mM K2HPO4 and micronutrient Mehta and Rosato (2005)[52]; Schulte and Bonas (1992)[53] Medium M1 0.25 g/L KNO3, 0.1 g/L KH2PO4, 0.1 g/L MgSO4, 0.02 g/L CaCl2.2H2O, 0.1 g/L yeast extract, 0.005 g/L Na2MoO4 and 0.05% (w/v) carbon source Dedysh et al. (2006)[54] Medium M2 0.1 g/L (NH4)2SO4, 0.1 g/L MgSO4, 0.02 g/L CaCl2.2H2O and 0.05% (w/v) carbon source Dedysh et al. (2006)[54] Medium M31 0.1 g/L KH2PO4, 20 mL Hutner’s basal salt, 1 g/L N-acetylglucosamine, 0.1 g/L peptone and 0.1 g/L yeast extract Kulichevskaya et al. (2012b)[55] Nitrate mineral salt media 1 g/L KNO3, 1 g/L MgSO4.7H2O, 0.717 g/L Na2HPO4.12H2O, 0.272 g/L KH2PO4, 0.2 g/L CaCl2.6H2O and 0.005 g/L ferric ammonium EDTA Dedysh and Dunfield (2011)[56] Peat extract medium 500 ml of supernatant (400 g of wet peat mixed with 200 ml of distilled water) and 500 ml of base Medium M2 Dedysh et al. (2006)[54] R2A 0.5 g/L yeast extract, 0.5 g/L proteose peptone, 0.5 g/L casamino acid, 0.5 g/L dextrose, 0.5 g/L soluble starch, 0.3 g/L sodium pyruvate, 0.3 g/L KH2PO4 and 0.05 g/L MgCl2 Dedysh et al. (2006)[54]; Edenborn and Sexstone (2007)[57]; Taylor et al. (2002)[58] Based on other studies on northern wetlands, several types of bacteria were isolated using the minimal media shown in Table 3. For example, an acidophilic methane- oxidizing bacterium was isolated using minimal mineral medium containing vitamin mixture with methane as sole carbon source. In another study conducted by Dedysh et al. (2006)[54], Alpha-proteobacteria, Beta-proteobacteria, Gamma-proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes were isolated using Medium M1, Medium M2 and diluted R2A media. However, acidobacteria and plactomycetes were not found in the same study which might due to the reason that some Acidobacteria such as Granulicella species are inhibited by the presence of phosphates found in most minimal media[59]. Therefore, agar selection is one of the main criteria leading to successful cultivation of peat-inhabiting bacteria. Prolonged incubation time Most peat inhabiting bacteria are slow growing even under optimal growth conditions. These bacteria are usually fastidious facultative anaerobes such as methanotrophs, acidobacteria and planctomycetes. In a study conducted by Kulichevskaya et al. (2012a)[60], colonies of Telmatocola sphagniphila (planctomycetes) were developed after 4 weeks of incubation using modified Medium M2 supplemented with trace element and vitamins under 5% CO2 (v/v) condition. Telmatobacter bradus which is a facultative anaerobe belonging to the phylum Acidobacteria requires 4 weeks to grow using Medium M2[59]. In another example, Telmatospirillum sibiriense which is an acidotolerant facultative anaerobic, only had observable colonies after 5 months of incubation on N-free minimal media supplemented with a reducing agent[61]. This also indicates that the presence of reducing agents might promote the growth of fastidious facultative anaerobes. However, there is no published result on the successful isolation of strict anaerobes from either the northern wetlands or TPSFs, which suggest the need for other reducing agents to be included in the culture media[51]. Furthermore, a more stringent method should be applied during sampling collection where peat samples are to be placed immediately in anaerobic conditions prior to sample transportation. This reduces the exposure of oxygen to the anaerobic bacteria at the same time simulating the actual anoxic conditions in the TPSF. To sum up, there is a need to incubate the peat culture for a prolonged duration with anoxic conditions, minimal nutrients, and with appropriate supplements in order to isolate anaerobes from TPSF. GENOMIC APPROACH TO DISCOVER BIOACTIVE SECONDARY METABOLITES PRODUCTION Whole genome sequencing of bacteria has become increasingly common for routine use in microbiological Microbes from Peat... 5 laboratories. Subsequently, a large quantity of DNA sequence data from different microorganisms is currently available in public databases. As a result, this creates a path for uncovering novel natural products from microbes by utilizing new bioinformatics tools[62]. For instance, many recent studies have been performing whole genome sequencing of drug-prolific producers such as the Streptomyces spp.[63–69] for further investigation of their secondary metabolite production ability This could also be useful for the prediction of novel products of non- ribosomal peptide synthetases (NRPSs) and polyketides synthases (PKSs) through application of various sequence analysis tools[62]. Similarly, this strategy has been applied for Burkholderia and Paenibacillus[70,71,72]. By taking the genus Burkholderia as an example, the determination of genome sequences of these bacteria has essentially created a route for in silico structural prediction, wet lab experimental design, and execution. Genome-guided approaches, which are made possible through accessibility of extensive genome sequence data coupled with genome- mining technologies, have warrant the discovery of structurally and functionally diverse natural products from numerous Burkholderia strains[71, 73,74]. CONCLUSION Tropical peat swamp forests are indeed a promising environment to source for secondary metabolites. However, culture-dependent methodologies should be scrutinized to ensure cultivation of rare bacterial species with important ecological and commercial roles which have never been captured before. Besides, whole genome sequencing of the bacteria in the near future may allow further understanding of the antimicrobial synthesizing capability of these bacteria. Nevertheless, efforts should be made to culture microbes from different genera with similar potential to discover new bioactive secondary metabolites. Conflict of Interest The authors declare that there is no conflict of interest in this work. Author Contributions K-SO performed the literature search, critical data analysis and performed the writing of this review. Technical support and proofreading were contributed by JW-FL and VL. K-SO, CMY and S-ML founded the review writing project. Acknowledgments The authors would like to thank School of Science and Tropical Medicine and Biology Multidisciplinary Platform, Monash University Malaysia for their support. References 1. Banin, E, Hughes, D, and Kuipers, OP. Bacterial pathogens, antibiotics and antibiotic resistance. FEMS Microbiol Rev 2017; 41(3): 450–452. 2. Munita, JM and Arias, CA. Mechanisms of antibiotic resistance. Microbiol Spect 2016; 4(2): 1–37. 3. Holmes, AH, Moore, LS, Sundsfjord, A, et al. Understanding the mechanisms and drivers of antimicrobial resistance. The Lancet 2016; 387(10014): 176–187. 4. WHO. Antimicrobial resistance in the European Union and the world. . 2012. 5. Yule, CM. 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