Development of Novel Drugs from Marine Surface Associated Microorganisms

While the oceans cover more than 70% of the Earth’s surface, marine derived microbial natural products have been largely unexplored. The marine environment is a habitat for many unique microorganisms, which produce biologically active compounds (“bioactives”) to adapt to particular environmental conditions. For example, marine surface associated microorganisms have proven to be a rich source for novel bioactives because of the necessity to evolve allelochemicals capable of protecting the producer from the fierce competition that exists between microorganisms on the surfaces of marine eukaryotes. Chemically driven interactions are also important for the establishment of cross-relationships between microbes and their eukaryotic hosts, in which organisms producing antimicrobial compounds (“antimicrobials”), may protect the host surface against over colonisation in return for a nutrient rich environment. As is the case for bioactive discovery in general, progress in the detection and characterization of marine microbial bioactives has been limited by a number of obstacles, such as unsuitable culture conditions, laborious purification processes, and a lack of de-replication. However many of these limitations are now being overcome due to improved microbial cultivation techniques, microbial (meta-) genomic analysis and novel sensitive analytical tools for structural elucidation. Here we discuss how these technical advances, together with a better understanding of microbial and chemical ecology, will inevitably translate into an increase in the discovery and development of novel drugs from marine microbial sources in the future

Catabolism of Nucleic Acids by a Cystic Fibrosis Pseudomonas aeruginosa Isolate: An Adaptive Pathway to Cystic Fibrosis Sputum Environment

Pseudomonas aeruginosa is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). We undertook Biolog Phenotype Microarray testing of P. aeruginosa CF isolates to investigate their catabolic capabilities compared to P. aeruginosa laboratory strains PAO1 and PA14. One strain, PASS4, displayed an unusual phenotype, only showing strong respiration on adenosine and inosine. Further testing indicated that PASS4 could grow on DNA as a sole carbon source, with a higher biomass production than PAO1. This suggested that PASS4 was specifically adapted to metabolize extracellular DNA, a substrate present at high concentrations in the CF lung. Transcriptomic and proteomic profiling of PASS4 and PAO1 when grown with DNA as a sole carbon source identified a set of upregulated genes, including virulence and host-adaptation genes. PASS4 was unable to utilize N-Acetyl-D-glucosamine, and when we selected PASS4 mutants able to grow on this carbon source, they also displayed a gain in ability to catabolize a broad range of other carbon sources. Genome sequencing of the mutants revealed they all contained mutations within the purK gene, encoding a key protein in the de novo purine biosynthesis pathway. This suggested that PASS4 was a purine auxotroph. Growth assays in the presence of 2 mM adenosine and the complementation of PASS4 with an intact purK gene confirmed this conclusion. Purine auxotrophy may represent a viable microbial strategy for adaptation to DNA-rich environments such as the CF lung

Identification of the Antibacterial Compound Produced by the Marine Epiphytic Bacterium Pseudovibrio sp. D323 and Related Sponge-Associated Bacteria

Surface-associated marine bacteria often produce secondary metabolites with antagonistic activities. In this study, tropodithietic acid (TDA) was identified to be responsible for the antibacterial activity of the marine epiphytic bacterium Pseudovibrio sp. D323 and related strains. Phenol was also produced by these bacteria but was not directly related to the antibacterial activity. TDA was shown to effectively inhibit a range of marine bacteria from various phylogenetic groups. However TDA-producers themselves were resistant and are likely to possess resistance mechanism preventing autoinhibition. We propose that TDA in isolate D323 and related eukaryote-associated bacteria plays a role in defending the host organism against unwanted microbial colonisation and, possibly, bacterial pathogens

Assessing the Effectiveness of Functional Genetic Screens for the Identification of Bioactive Metabolites

A common limitation for the identification of novel activities from functional (meta) genomic screens is the low number of active clones detected relative to the number of clones screened. Here we demonstrate that constructing libraries with strains known to produce bioactives can greatly enhance the screening efficiency, by increasing the “hit-rate” and unmasking multiple activities from the same bacterial source

Development of Novel Drugs from Marine Surface Associated Microorganisms

While the oceans cover more than 70% of the Earth’s surface, marine derived microbial natural products have been largely unexplored. The marine environment is a habitat for many unique microorganisms, which produce biologically active compounds (“bioactives”) to adapt to particular environmental conditions. For example, marine surface associated microorganisms have proven to be a rich source for novel bioactives because of the necessity to evolve allelochemicals capable of protecting the producer from the fierce competition that exists between microorganisms on the surfaces of marine eukaryotes. Chemically driven interactions are also important for the establishment of cross-relationships between microbes and their eukaryotic hosts, in which organisms producing antimicrobial compounds (“antimicrobials”), may protect the host surface against over colonisation in return for a nutrient rich environment. As is the case for bioactive discovery in general, progress in the detection and characterization of marine microbial bioactives has been limited by a number of obstacles, such as unsuitable culture conditions, laborious purification processes, and a lack of de-replication. However many of these limitations are now being overcome due to improved microbial cultivation techniques, microbial (meta-) genomic analysis and novel sensitive analytical tools for structural elucidation. Here we discuss how these technical advances, together with a better understanding of microbial and chemical ecology, will inevitably translate into an increase in the discovery and development of novel drugs from marine microbial sources in the future

Marine bacteria as a source of new antibiotics

The pre-antibiotic era was characterised by the lack of antibiotics and hence, the lack of adequate means for the treatment of numerous infectious diseases. The ground-breaking discovery of penicillin and other antibiotics, which came to be widely used in clinical practice, brought great benefit and contributed to the increase of the average human life expectancy from 47 years in 1900 to 74 years (in men) and 80 years (in women) in 2000 in the USA (Lederberg 2000). Furthermore, in 1941 Skinner and Keefer reported a shocking 82 % mortality among 122 consecutive patients who had been treated for Staphylococcus aureus bacteremia before antibiotics were available (Skinner and Keefer 1941), as contrasted to 20-40 % mortality rates being reported in the post-antibiotic era (Mylotte, McDermott et al.1987; Shurland, Zhan et al. 2007). The use of antimicrobials is not limited to clinical applications. Currently antimicrobials, such as, for example, chitosan based products, triclosan and various bacteriocins, are widely used in hand washing products, toothpastes and in the food industry to increase the shelf life of various products (Stephen, Saxton et al. 1990; Waaler, Rolla et al. 1993; Barkvoll and Rolla 1994; Bhargava and Leonard 1996; Gould 1996; Jones, Jampani et al. 2000; Haas, Marie et al. 2005; Galvez 2007; Dutta, Tripathi et al. 2009). By comparison to chemical disinfectants, these natural products are generally less toxic and also provide great advantages, such as biodegradability, biocompatibility as well as chemical and physical versatility (Gould 1996; Dutta, Tripathi et al. 2009). Nevertheless, the use of antibiotics for the treatment of diseases historically remains the main focus of antimicrobial research. Ever since the discovery of penicillin there have been many attempts to find novel antimicrobials due to the inevitability of development of bacterial resistance towards the widely used antibiotics. In the past years much of the effort in that direction was focused on terrestrial sources. Nowadays, more than ever before, the exploration of new and under-explored sources becomes extremely important in the process of finding biologically active compounds (“bioactives”) with novel chemical structures. The majority of antibiotics currently used in clinical practice are of natural product origin (Newman, Cragg et al. 2003; Singh and Barrett 2006; Von Nussbaum, Brands et al. 2006). For example, 70 out of the 90 antibiotics marketed in the years 1982–2002 originated from natural products (Newman, Cragg et al. 2003). Notably, the quinolones or fluoroqinones, one of the most successful classes of synthetic antibiotics, are also based on the structure of the natural product quinine (Demain and Sanchez 2009) (Figure 1). In fact, chemical modifications based on a natural product scaffold is a widely used approach in modifying the chemical and physical properties of the molecule, thus making it useful for a particular pharmacological application (Walters, Murcko et al. 1999; Leeson, Davis et al. 2004). It has been suggested that the success of natural compounds is due to the fact that they have undergone natural selection and, therefore, are best suited to perform their activities (Nisbet and Moore 1997; Muller-Kuhrt 2003; Koehn and Carter 2005). Thus, further research on bioactive natural products may provide a source of new chemical structures that can guide the design of novel chemical compounds (Breinbauer, Vetter et al. 2003; Nicolaou, Chen et al. 2009), as well as reveal yet unknown modes of action (Urizar, Liverman et al. 2002).32 page(s

Antibiotic Discovery: Combatting Bacterial Resistance in Cells and in Biofilm Communities

Bacterial resistance is a rapidly escalating threat to public health as our arsenal of effective antibiotics dwindles. Therefore, there is an urgent need for new antibiotics. Drug discovery has historically focused on bacteria growing in planktonic cultures. Many antibiotics were originally developed to target individual bacterial cells, being assessed in vitro against microorganisms in a planktonic mode of life. However, towards the end of the 20th century it became clear that many bacteria live as complex communities called biofilms in their natural habitat, and this includes habitats within a human host. The biofilm mode of life provides advantages to microorganisms, such as enhanced resistance towards environmental stresses, including antibiotic challenge. The community level resistance provided by biofilms is distinct from resistance mechanisms that operate at a cellular level, and cannot be overlooked in the development of novel strategies to combat infectious diseases. The review compares mechanisms of antibiotic resistance at cellular and community levels in the light of past and present antibiotic discovery efforts. Future perspectives on novel strategies for treatment of biofilm-related infectious diseases are explored

Marine biofilm bacteria evade eukaryotic predation by targeted chemical defense

Many plants and animals are defended from predation or herbivory by inhibitory secondary metabolites, which in the marine environment are very common among sessile organisms. Among bacteria, where there is the greatest metabolic potential, little is known about chemical defenses against bacterivorous consumers. An emerging hypothesis is that sessile bacterial communities organized as biofilms serve as bacterial refuge from predation. By testing growth and survival of two common bacterivorous nanoflagellates, we find evidence that chemically mediated resistance against protozoan predators is common among biofilm populations in a diverse set of marine bacteria. Using bioassay-guided chemical and genetic analysis, we identified one of the most effective antiprotozoal compounds as violacein, an alkaloid that we demonstrate is produced predominately within biofilm cells.Nanomolar concentrations of violacein inhibit protozoan feeding by inducing a conserved eukaryotic cell death program. Such biofilm-specific chemical defenses could contribute to the successful persistence of biofilm bacteria in various environments and provide the ecological and evolutionary context for a number of eukaryote-targeting bacterial metabolites

Production of antimicrobial compounds by marine epibiotic bacteria

The aim of this thesis was to obtain antimicrobial compounds from marine eukaryote-associated bacteria. The unique environment, present on the surfaces of marine eukaryotes, creates conditions that promote and favour the production of bioactive compounds, such as antimicrobials, by giving the producers a clear advantage in the competition for nutrients. This study has demonstrated the abundance of antimicrobial producing bacteria on two marine algae, Delisea pulchra and Ulva australis. Two antimicrobial compounds, violacein and tropodithietic acid (TDA), have been successfully purified and chemically identified from two different bacterial isolates. Moreover, the production of multiple bioactive compounds was observed for both these bacteria.This study also made an attempt to understand the role of the antimicrobial compounds for the producer organisms. Consequently, the effect of violacein on biofilm formation, as well as the possible role of TDA in the defence of both the producer bacterium and the host, have been proposed. The importance of environmental conditions for the expression of bioactive compounds has also been demonstrated, for example, by showing the necessity of high iron concentrations for the production of bioactives in isolates Ul56 and 0245. Notably, despite the absence of a close phylogenetic relationship between these two isolates, they have shown similar trends in terms of production of bioactive compounds. For the first time, this work also described the construction and analysis of a large insert DNA library in E. coli using genomic DNA from a collection of cultured isolates with demonstrated antimicrobial activity. This approach proved to be successful and led to a substantial increase in the positive hit rates in the functional screening of the library, and provided invaluable information concerning the genes, potentially involved not only in the biosynthesis, but also in the processes associated with the production of bioactive compounds, such as transport and resistance