Current Topics in Medicinal Chemistry, 2020, 20, 1291-1299 REVIEW ARTICLE

Send Orders for Reprints to [email protected] 1291 Current Topics in Medicinal Chemistry, 2020, 20, 1291-1299 REVIEW ARTICLE ISSN: 1568-0266 eISSN: 1873-4294 Impact Factor: The Microbiome: A Reservoir to Discover New Antimicrobials Agents 3.442 The international journal for in-depth reviews on Current Topics in Medicinal Chemistry BENTHAM SCIENCE Sébastien Boutin1,2,* and Alexander H. Dalpke3 1Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital Heidelberg, 69120 Hei- delberg, Germany; ²German Center for Lung Research (DZL), TLRC Heidelberg, 69120 Heidelberg, Germany; 3Institute of Medical Microbiology and Hygiene, Medical Faculty, Technische Universität Dresden, 01307 Dresden, Gemany Abstract: Nature offered mankind the first golden era of discovery of novel antimicrobials based on A R T I C L E H I S T O R Y the ability of eukaryotes or micro-organisms to produce such compounds. The microbial world proved to be a huge reservoir of such antimicrobial compounds which play important functional roles in every Received: November 11, 2019 environment. However, most of those organisms are still uncultivable in a classical way, and therefore, Revised: February 10, 2020 the use of extended culture or DNA based methods (metagenomics) to discover novel compounds Accepted: February 17, 2020 promises usefulness. In the past decades, the advances in next-generation sequencing and bioinformat- DOI: 10.2174/1568026620666200320112731 ics revealed the enormous diversity of the microbial worlds and the functional repertoire available for studies. Thus, data-mining becomes of particular interest in the context of the increased need for new antibiotics due to antimicrobial resistance and the rush in antimicrobial discovery. In this review, an overview of principles will be presented to discover new natural compounds from the microbiome. We describe culture-based and culture-independent (metagenomic) approaches that have been developed to identify new antimicrobials and the input of those methods in the field as well as their limitations. Keywords: Antimicrobial peptides, Metagenomics, Data mining, Bioinformatics, Microbiome, Antimicrobials agents. 1. INTRODUCTION The classical procedure to find antimicrobial compounds is to look for them in nature (Fig. 1). The first accessible From the 1930s to the 1960’s, the golden age of antimi- organisms producing antimicrobials were plants and those crobial discoveries took place. Those discoveries lead to the compounds were used for millennia. However, access to introduction in the clinics of the major classes of drugs cur- fungi and bacteria with microbial culture revolutionized the rently used. After the first discoveries, the research field fo- field [5]. The majority of antibiotics currently implemented cused more on the optimization of those drugs in the follow- in clinic originate from cultivable microorganisms [6]. In the ing 50 years [1]. The phase of optimization was always fol- environment, microbes, however, are never living in a lowed by the emergence of antimicrobial resistance leading monoculture environment and therefore, most of them have to an “arm race” to counteract those resistances [2]. Since the developed ways to communicate and/or defend themselves 2000’s, only a few drugs of entirely new classes were ap- against other bacteria/fungi. The billions of years of evolu- proved and discovered, leading to a threat in health due to tion involve in this arms race created a huge arsenal of those the steady increase of emerging pathogens and multi-drug compounds [7]. Most of them are products of “secondary resistant (MDR) bacteria [3]. The currently used drugs focus metabolism,” which are not required for survival under labo- on two major targets in 5 different modes of action. Some ratory conditions but are most likely increasing the fitness of drugs alter the structure of the cell by inhibiting the cell wall their producer in the native complex biome [5]. Those prod- synthesis or disrupting the cell membrane integrity while ucts are often encoded by a biosynthetic gene cluster (BGC), others target the molecular machinery inhibiting either the which is a physically clustered group of two or more genes DNA or RNA synthesis or the protein synthesis via the bac- as an operon within the genome. Together the BGCs will terial ribosome or metabolic pathways [4]. The emergence of encode cooperatively a complete biosynthetic pathway lead- MDR bacteria guided the field of antibiotic research into ing to the production of a specialized metabolite and possible actually two ways; understanding the molecular mechanisms chemical variants. Furthermore, genes involved in the regu- leading to the resistance to manage our use of the affected lation, such as transcription factors and transporters, are usu- drugs and developing new antibiotics targeting new path- ally also present in these clusters [8, 9]. ways or identifying new modes of action in known pathways. The field of microbiology observed in the last decades a shift in the paradigm of environmental and host-related mi- *Address correspondence to this author at the Department of Infectious crobes. The advances in culture-independent methods Diseases, Medical Microbiology and Hygiene, University Hospital Heidel- showed that microbes are part of a complex ecosystem and berg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany; the diversity of this microbiome was by far underestimated Current Topics in Medicinal Chemistry E-mail: [email protected] 1873-4294/20 $65.00+.00 © 2020 Bentham Science Publishers 1292 Current Topics in Medicinal Chemistry, 2020, Vol. 20, No. 14 Boutin and Dalpke Fig. (1). Summary of the pipelines used to discover microbiome derived antibiotic. The classical way consists to culture the organisms di- rectly from the environment and test them for antimicrobial production. An evolution of this way leads to the development of in situ culture where the organisms is grown in isolation in a permeable chamber put in the original environment (displayed as a iChip in the figure). The environmental DNA (eDNA) can be used to construct metagenomic library either via direct fragmentation and ligation to a vector or via a PCR targeted approach using conserved region of the biosynthetic gene clusters (BGCs) to amplify the diversity of BGCs within the environment. Those PCR products can also be integrated in a vector to construct a metagenomic library. The metagenomic data can also be ex- plore (“data-mining”) via bio-informatic to search for new BGCs and design new primers to isolate them from the metagenome. The pre- pared library can then be transferred into a heterologous host (i.e. Escherischia coli) to perform an antimicrobial test assay. Each part of the different path is explained and discuss in details in the separate sections of the manuscript. by classical culture. It is estimated that classical culture re- capacities as well as annotation of the sequences themselves, trieves around 1% of the microbiome of environmental sam- such approaches appear to be very promising. ples, yet reaches slightly better recovery in human-associated 2. THE WORLD’S MICROBIOME AS A POTENTIAL microbiomes, due to the optimization of growth media for SOURCE FOR NEW ANTIBIOTICS detection of human pathogenic bacteria [10]. Many research- ers are currently working on extending culture methods to The environmental microbiome is a source of new anti- improve culturability with impressive advances, especially in biotics, but unfortunately, culture-based approaches barely humans [11–13]. However, symbiotic microbes will still be scratch the surface of the diversity of those microbiomes difficult to culture and the production of antibiotic com- [14]. The advances in the metagenomic field pave the way pounds will be influenced by the growth conditions as well for the exploration of highly diverse microbiomes such as as other organisms growing in nature together with antimi- the soil and aquatic microbiome. The exploration of those crobial producers. Therefore, the field of drug discovery now two major environments showed that the BGCs involved in focuses on metagenomic approaches: This approach aims at the production of antimicrobial compounds by those mi- using the DNA from the environmental microbiome (eDNA) crobes are geographically heterogeneous and show a strong to discover antimicrobial functions by prediction instead of environmental specificity [15-17]. This work was the basis or prior to phenotypical identification of such compounds. of the exploration of diverse environments and geographi- Given the enormous technical developments in sequencing cally distinct biomes to mine for new antibiotics from a highly diverse environment, allowing a low re-discovery rate The Microbiome: A Reservoir to Discover New Antimicrobials Agents Current Topics in Medicinal Chemistry, 2020, Vol. 20, No. 14 1293 [18]. Naturally, the metagenomic approach can be used with cro-organisms. Those chambers improved the culture of mi- any source material, but the main focus of the research is crobes, which are dependent on environmental/host nutrients currently soil, water and host-associated microbiomes as opening the scope of cultured microbes for antibiotic discov- supported by the major consortium performing metagenom- ery. ics and offering a huge database for data-mining [19, 20]. An updated version of the diffusion chamber is the iChip. Soil is of high interest because it is one of the most diverse The principle

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