**4.3 New cultivation techniques**

38 Antimicrobial Agents

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

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

**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

recent methodology of antibiotic discovery.

**4.1 Improvements in screening platforms** 

tested due to extremely low hit rates.

represents a bottleneck of screening for antibiotics.

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 discovering novel antimicrobials.

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

Future Antibiotic Agents: Turning to Nature for Inspiration 41

Fig. 4. Strategies for identifying metabolic products of cryptic gene clusters (compiled and adapted from (Challis, 2008)). Based on homology searches, novel biosynthetic gene clusters are predicted from genome sequences. **A**) The modular structure of synthetases allows assumptions on putative substrates, which together define structural and physicochemical

**B**) Alternatively, the organism can be grown on a medium containing putative precursors labeled with stable isotopes to facilitate subsequent identification of final products by 2D NMR. **C**) The predicted synthetase can be expressed using recombinant DNA techniques and used in isolated form to reconstitute the product *in vitro*. **D**) The putative biosynthetic gene cluster can be knocked out and metabolites in culture supernatants analyzed by LC-MS

biosynthetic gene cluster-containing locus can be transferred to a heterologous host. The metabolome of the transgenic strain is compared to the untransformed host. **F**) Attempts to force expression of cryptic biosynthetic genes using induction of various endogenous

The term metagenomics refers to "the application of modern genomic techniques to the study of microbial organisms directly in their natural environments, by-passing the need for

features of secondary metabolites that guide the design of isolation procedures.

in comparison to the metabolome of the wild-type strain. **E**) Similarly, the entire

activators have also been made.

**4.6 Metagenomics** 

hand, environmental concentrations of numerous antibiotics are too low to be readily detected by conventional analytical methods or activity screening. Modern liquid chromatography-mass spectrometry (LC-MS) instruments combine high resolution and high sensitivity with the power of structure determination, and as such hold great potential for analysis of secondary metabolites in organic extracts of various, complex environmental samples (Davies, 2011).

An exciting new field in natural product research is imaging mass spectroscopy (IMS) (Esquenazi et al., 2009). Application of IMS enables analysis of spatial distribution of compounds in a substrate, such as a plant organ or a marine sponge. This method led to identification of various (endo)symbiotic microorganisms as true producers of secondary metabolites which were initially erroneously attributed to the host organism (Esquenazi et al., 2009; Simmons et al., 2008). Another promising application of IMS is the so-called thin layer agar natural product MALDI-TOF imaging. Here, microorganisms are grown on a thin agar film deposited on a MALDI plate, after which the sample is covered with a matrix and analyzed by MALDI. Thus, a complete set of metabolites produced under different culturing conditions (even in cocultures to trigger interspecies interactions) can be examined (Yang et al., 2009).

Finally, the soaking of potential ligands from environmental extracts into crystals of recombinant proteins was proposed as another method to enrich for and analyze the structures of compounds with desired affinity to bacterial targets (Davies, 2011). Ideally, structural information gathered on the isolated secondary metabolite should assist in identification of the biosynthetic pathway from (meta)genomic library sequences (see sections 4.5 and 4.6).
