**3.1.4 Higher fungi**

Among the eukaryotes, fungal genomes are rich in biosynthetic gene clusters for encoding small molecule production (Miao & Davies, 2010). Fungi are the second largest group of eukaryotes next to insects and exceed not only the bacteria and actinomycetes, but also the higher plants in terms of the number of potential existing species. It looks like the world of fungi is one of the largest reservoirs for isolating further bioactive metabolites (Berdy, 2005).

Besides the discovery of new compounds, the re-evaluation of "old" substances, including microbial metabolites formerly believed to be inactive, have proven to be just as important. On numerous occasions such compounds have been shown to be active in later investigations, or were rediscovered by screening a different stock of microbes, or with specific screening methods. It is unpredictable how many "new" bioactive metabolites will be discovered in this way (Berdy, 2005).

An excellent example of this is pleuromutilin (Fig. 2). It was initially discovered in 1951 in a study of the culture broth of the edible basidiomycete mushroom *Pleurotus multilus* (Kavanagh et al., 1951). After more than 50 years, a derivative of pleuromutilin, named retapamulin, was approved in 2007 by the FDA for the treatment of bacterial skin infections. The low oral bioavailability of retapamulin seems to have been improved in the new derivative named BC-3205, which is being investigated in phase-I clinical trials by Nabriva (Butler & Cooper, 2011). Another pleuromutilin derivative, BC-7013 ([14-O-[(3 hydroxymethyl-phenylsulfanyl)-acetyl]-mutilin]), is in phase-I clinical trials as a topical

Sponges alone produce more than 3300 antibiotics and other bioactive compounds. It is noteworthy to mention that these isolated "animal" compounds very frequently show surprising analogy to microbial or algal products. As with the secondary metabolites produced by plants and their endophytes, it is not surprising that in numerous occasions the active compounds isolated from sponges proved to be derived from the microorganisms living in symbiosis with their host (Berdy, 2005). Marine microbes are particularly attractive because of the high potency required for bioactive compounds to be effective in the marine

Psammaplin A (Fig. 2) is a symmetrical bromotyrosine-derived disulfide natural product isolated from the *Psammaplysilla* sponge (Arabshahi & Schmitz, 1987), with *in vitro* antibacterial activity against MRSA. Based on the structure of psammaplins, Nicolaou et al. produced a library of 3,828 compounds. Six of these optimized antibacterial agents possessed more than 50-fold higher activities than the natural product, demonstrating MIC levels in methicillin-resistant/intermediate vancomycin-resistant strains of *S. aureus* at less than 1µg/ml. In order to construct these heterodimeric disulfide analogues they used a novel combinatorial disulfide exchange strategy, thus demonstrating the power of modern combinatorial techniques when applied to a base active structure from nature (Newman & Cragg, 2004; Nicolaou et al., 2001a; Nicolaou et al., 2001b). Most significantly, a number of these agents exhibited increased selectivity against bacterial cells over fibroblasts and

In similar efforts of the marine natural products community, many antibacterial agents have been identified from sponges (Laport et al., 2009). Despite their high number, none of them

Among the eukaryotes, fungal genomes are rich in biosynthetic gene clusters for encoding small molecule production (Miao & Davies, 2010). Fungi are the second largest group of eukaryotes next to insects and exceed not only the bacteria and actinomycetes, but also the higher plants in terms of the number of potential existing species. It looks like the world of fungi is one of the largest reservoirs for isolating further bioactive metabolites (Berdy, 2005). Besides the discovery of new compounds, the re-evaluation of "old" substances, including microbial metabolites formerly believed to be inactive, have proven to be just as important. On numerous occasions such compounds have been shown to be active in later investigations, or were rediscovered by screening a different stock of microbes, or with specific screening methods. It is unpredictable how many "new" bioactive metabolites will

An excellent example of this is pleuromutilin (Fig. 2). It was initially discovered in 1951 in a study of the culture broth of the edible basidiomycete mushroom *Pleurotus multilus* (Kavanagh et al., 1951). After more than 50 years, a derivative of pleuromutilin, named retapamulin, was approved in 2007 by the FDA for the treatment of bacterial skin infections. The low oral bioavailability of retapamulin seems to have been improved in the new derivative named BC-3205, which is being investigated in phase-I clinical trials by Nabriva (Butler & Cooper, 2011). Another pleuromutilin derivative, BC-7013 ([14-O-[(3 hydroxymethyl-phenylsulfanyl)-acetyl]-mutilin]), is in phase-I clinical trials as a topical

environment, due to the diluting effect of seawater (Zhang et al., 2005).

lymphocytes as compared to the natural product.

be discovered in this way (Berdy, 2005).

**3.1.4 Higher fungi** 

has yet been involved in clinical trial as an antibacterial agent.

antibiotic, while BC-3781 successfully completed a phase-II clinical trial for the treatment of acute bacterial skin and skin structure infections (ABSSSI) (US National Institutes of Health, 2011). Nabriva's lead product BC-3781 is the first of a new class of systemically available pleuromutilin antibiotics for the treatment of serious skin infections and pneumonia. BC-3781 is being developed for both oral and intravenous formulations.

#### **4. How do we search for natural antibiotics?**

Although the number of antibiotics present in nature may truly be huge, many of them are already known or will not be usable (i.e., will not display selective toxicity to bacteria, will be too weak, or will lack the desired pharmacokinetic properties) (Pelaez, 2006). Yet historically, the development of antibiotics from natural templates has seen an unprecedented gain compared to the *de novo* synthesis. The conventional discovery process of antibiotics from the pool of microbial natural products requires having a given microorganism grown in conditions appropriate to induce the production of (the desired) metabolite, which is then extracted and tested in a screen able to detect it as a hit. Finally, the compound has to be isolated from the original mixture and identified.

Identification of novel antibiotic types that occur in relatively low frequency in nature clearly requires innovative detection and characterization techniques. Numerous promising microbiological approaches supplemented with bioinformatic, genetic, and structural methods have been developed over the last decade to address the issue (Fig. 3).

Fig. 3. Postgenomic approaches in antibiotic discovery (adapted from (Davies, 2011)).

Future Antibiotic Agents: Turning to Nature for Inspiration 39

An obvious solution to increasing the throughput of fermentation is to miniaturize initial batches (i.e., perform microfermentations), allowing accommodation of larger numbers of strains and/or growth conditions simultaneously. At Cubist Pharmaceuticals they faced the challenge by encapsulating individual environmental microbes in ~2 mm alginate macrodroplets and growing them in media favouring actinomycete growth supplemented with antibiotics against single-cell eubacteria and fungi. This technology supports the fermentation and screening of up to 10 million actinomycetes per year (Baltz, 2006; Gullo et al., 2006). Similarly, a method that couples bacterial encapsulation in gel microdroplets with flow cytometry to detect those beads that contain microcolonies was reported (Zengler et al., 2002). This enables rapid isolation of bacterial strains from environmental samples in order

Since the vast majority of prokaryotes are not amenable to simple cultivation (indeed, only ~0.1% of existing prokaryotes have been cultured so far (Alain & Querellou, 2009)), numerous efforts to develop strategies for efficient bacterial growth *in vitro* have been made. Undoubtedly, expanding the accessible pool of antibiotic producers will raise the odds of

Attempts to recover diverse microorganisms from environmental samples by manipulating growth conditions (e.g. media formulation, light, temperature, agitation) have shown some success (Köpke et al., 2005; Uphoff et al., 2001; Zengler et al., 2002). However, the approach is strictly empirical and the yield is rather unpredictable. Moreover, the projects are often endangered by overgrowth of (common) opportunistic fast-growing microorganisms, especially when using nutrient-rich artificial media (Alain & Querellou, 2009). Furthermore, *in vitro* culturing attempts typically disregard the importance of chemical components or physical conditions of natural growth environments. Culturing *in situ* or under simulated natural conditions was demonstrated to be successful in some instances. For example, new bacteria were isolated from intertidal marine sediments using diffusion chambers and growth in seawater aquarium (Kaeberlein et al., 2002). The membranes of diffusion chambers allow for exchange of chemicals between the chamber and the environment, while restricting cell movement. Interestingly, two isolates easily grown in diffusion chambers could only be maintained in petri dishes in coculture, indicating the requirement for specific signaling between the two species as a marking of a favorable environment. Other studies found specific physical requirements for culturing different strains, such as high hydrostatic pressure (Alain et al., 2002) or carriers for adhesion (Yasumoto-Hirose et al., 2006). Previously uncultured bacteria were successfully recovered from soil, marine sediments or activated sludge by these innovative methods. Unfortunately, they are rather specialized

and as a result were not adopted by a wider scientific community.

**4.4 Direct isolation of metabolites from environmental samples** 

Direct sampling of natural products from the environment represents an alternative to microbial strain isolation and fermentation for production of secondary metabolites. In theory, this grants access to the complete metabolome, which cannot be retrieved by classical means because most microbes defy cultivation (see section 4.3). On the other

to prepare pure cultures for subsequent studies.

**4.3 New cultivation techniques** 

discovering novel antimicrobials.

These allow for laboratory culturing of previously inaccessible microorganisms as potential antibiotic producers, extracting genomes of uncultivable species from environmental samples or mining for and inducing expression of cryptic biosynthetic clusters to yield yet untapped secondary metabolites, direct solvent extraction and subsequent characterization of low molecular weight compounds from natural samples, and high-throughput fermentation of underexplored bacterial strains. In addition, intelligent strategies to avoid antibiotic rediscovery have been devised. In the following sections, we critically review the recent methodology of antibiotic discovery.

#### **4.1 Improvements in screening platforms**

Parallel fermentation coupled with whole-cell assays for antibiotic activity remains the cornerstone of antibiotic discovery. Yet, introduction of certain implementations are vital to detect antibiotic compounds that occur at low concentrations or to prevent rediscovery of old antibiotic types. For example, researchers at Merck developed a highly sensitive assay for detection of inhibitors of β-ketoacyl-[acyl-carrier-protein] synthase II (FabF), a component of FASII pathway, by introducing a plasmid that encodes antisense RNA against the *fabF* transcript in *S. aureus* (S.B. Singh et al., 2007). Thereby, FabF expression is knocked down to growth-limiting levels, resulting in a strain that is hypersensitive to FASII pathway inhibitors. The mutant is assayed in parallel with the control wild-type strain to monitor for differential sensitivity. This combination of target-based and wholecell screening had a high hit rate of 0.3% and led to discovery of platensimycin, a broad spectrum Gram-positive antibiotic, from a screen of ~250.000 natural product extracts (Wang et al., 2006). Another interesting approach was reported by a team at Cubist Pharmaceuticals. They constructed a model target organism (CM400) by using *E. coli* engineered to harbor multiple resistance markers (conferring resistance to 16 most frequent antibiotics) (Baltz, 2006; Gullo et al., 2006). In this way, the hits are preselected to belong to new antibiotic classes. Additionally, a derivative of CM400 (termed CM435) with increased permeability was created to achieve enhanced sensitivity to antibacterial compounds. However, this strategy requires enormous input of natural products to be tested due to extremely low hit rates.

#### **4.2 High-throughput fermentation focusing on relevant microorganisms**

It is absolutely essential to screen extracts of only those organisms that have the capacity to produce complex secondary metabolites. The size of the genome provides a good indication of metabolism complexity; actinomycetes, the most important group of antibiotic producers, have large genomes relative to other bacteria with up to 10% of all genes devoted to production of secondary metabolites, such as nonribosomal peptides and polyketides (Baltz, 2008; Donadio et al., 2007). Using relatively selective antibiotics, bacterial populations can empirically be enriched for rare species (Baltz, 2006 and references cited therein) after which microbial diversity in the remaining population is conveniently assessed by 16S rRNA gene sequencing (Amann et al., 1995; Rajendhran & Gunasekaran, 2011). If the population is considered interesting in terms of secondary metabolite-producing potential, thousands of strains are typically screened for antibiotic activity. This, however, is no trivial task and represents a bottleneck of screening for antibiotics.

An obvious solution to increasing the throughput of fermentation is to miniaturize initial batches (i.e., perform microfermentations), allowing accommodation of larger numbers of strains and/or growth conditions simultaneously. At Cubist Pharmaceuticals they faced the challenge by encapsulating individual environmental microbes in ~2 mm alginate macrodroplets and growing them in media favouring actinomycete growth supplemented with antibiotics against single-cell eubacteria and fungi. This technology supports the fermentation and screening of up to 10 million actinomycetes per year (Baltz, 2006; Gullo et al., 2006). Similarly, a method that couples bacterial encapsulation in gel microdroplets with flow cytometry to detect those beads that contain microcolonies was reported (Zengler et al., 2002). This enables rapid isolation of bacterial strains from environmental samples in order to prepare pure cultures for subsequent studies.
