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Insight & Intelligence : Jun 6, 2013
Metagenomics May Hold Key to Novel Antibiotic Discovery
This relatively new science has the potential to fill a technological void.!--h2>
In the June 3rd issue of the New York Times, the push to speed up the approval of new antibiotics got front-page billing. “We are facing a huge crisis worldwide not having an antibiotics pipeline,” said Janet Woodcock, CDER’s director at the FDA. “It’s bad now,” she said, “and the infectious disease docs are frantic. But what is worse is the thought of where we will be five to 10 years from now.”
The number of FDA approvals of new antibiotics has dropped even as multi-drug-resistant strains of bacteria have proliferated. In late 2012, the FDA recommended approval of telavancin (Vibativ, a vancomycin derivative) for the treatment of hospital-acquired pneumonia when other drugs are unsuitable. The approval came nearly two years after the FDA had rejected the drug for a second time because clinical data did not meet the agency’s guidelines.
Due to the dearth of novel antibiotics in company pipelines, scientists have been investigating new ways of finding these drugs, including the relatively new enterprise known as metagenomics.
Tip of Microbial Iceberg
And as Jo Handelsman, Ph.D., currently professor of molecular, cellular, and developmental biology at Yale’s Howard Hughes Medical Institute, has pointed out, antibiotics in use today, including erythromycin and vancomyin, originated from laboratory-cultured soil bacteria. But she says, these organisms represent only the tip (0.1%) of a microbiological iceberg; the vast majority of soil denizens are unculturable by standard lab methods.
Dr. Handelsman—who, with colleagues at the University of Wisconsin, coined the term metagenomics—and other scientists say that this relatively new science, or the culture-independent genomic analysis of assemblages of uncultured microorganisms, has the potential to fill that “technological void.”
A useful recent definition of metagenomics, offered by UC Berkeley’s Kevin Chen, Ph.D., and Lior Pachter, Ph.D., defines it as “the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species.”
Dr. Handelsman defines it as “the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms.”
Two types of analysis have been used to obtain information from metagenomic libraries: a function-driven approach, in which metagenomic libraries are initially screened for an expressed trait, and a sequence-driven approach, in which libraries are initially screened for particular DNA sequences.
Some studies, she explains, have used random sequencing to generate massive databases of DNA sequences while other investigations have involved screened clones to find genes of a particular family or that share a particular motif. But with both of these approaches, explains Dr. Handelsman, genes of interest can be identified only if they share sequence similarity with genes that have already been discovered in cultivatable organisms. Novel genes and gene families, by definition, are overlooked by such approaches.
Functional metagenomics involves screening metagenomic libraries for the expression of a function, such as the appearance of a pigment, enzymatic activity, or for an antibiotic. Because, Dr. Handelsman says, clones in these screens are selected by phenotype (such as expression of an antibiotic activity) and not by homology to some known sequence, the strategy can identify new genes as well as novel activities of known gene families.
To date, scientists have used these technologies to both identify novel antibiotics derived from the environment, and to discover novel resistance genes in unexpected places.
In 2012, scientists at the Rockefeller University demonstrated that culture-independent antibiotic discovery methods “have the potential to provide access to novel metabolites with modes of action that differ from those of antibiotics currently in clinical use.”
Dimitris Kallifidas, Ph.D., Hahk-Soo Kang, Ph.D., and Sean F. Brady, Ph.D., working in the Rockefeller’s Laboratory of Genetically Encoded Small Molecules, reported last year in the Journal of the American Chemical Society the identification of a novel, soil-derived antibiotic. The investigators noted that propagation of DNA extracted directly from environmental samples in lab-grown bacteria provides a means to study natural products encoded in the genomes of uncultured bacteria, but that gene silencing can hamper the functional characterization of gene clusters captured on such environmental DNA clones.
The scientists showed that overexpression of transcription factors found in sequenced environmental DNA-derived biosynthetic gene clusters, along with traditional culture-broth extract screening, could identify novel bioactive secondary metabolites from otherwise-silent gene clusters.
The studies led to the successful isolation of Tetarimycin A, a tetracyclic methicillin-resistant Staphylococcus aureus (MRSA)-active antibiotic, from the culture-broth extract of Streptomyces albus cultures. The bacteria growing in the cultures had been cotransformed with an environmentally derived type-II polyketide biosynthetic gene cluster and its pathway-specific Streptomyces antibiotic regulatory protein (SARP) cloned under the control of the constitutive ermE* promoter.
And although overuse of antibiotics has been widely blamed for the evolution and acquisition of antibiotic-resistance genes, scientists say that little is known about the diversity, distribution, and origins of resistance genes, especially for the “unculturable majority” of environmental bacteria.
Metagenomic tools and phylogenetic analysis have revealed that the environment comprises a reservoir of antibiotic resistance gene determinants (ARGDs) and that the majority of ARGDs acquired by human pathogens may have an environmental origin.
While at the University of Wisconsin, Dr. Handelsman and her colleagues investigated antibiotic-resistance genes among uncultured bacteria in an undisturbed soil environment. Their functional metagenomic analysis of a remote Alaskan soil uncovered a reservoir for beta-lactamases that function in E. coli, including divergent beta-lactamases and the first bifunctional beta-lactamase. These enzymes confer resistance to most beta-lactam antibiotics including penicillins, cephalosporins, and the monobactam aztreonam.
The authors concluded that their findings suggest, even without the selective pressure imposed by anthropogenic activity, that the soil microbial community in an unpolluted site harbors unique and ancient beta-lactam resistance determinants. Moreover, despite their evolutionary distance from previously known genes, the Alaskan beta-lactamases confer resistance on E. coli without manipulating its gene expression machinery.
Ed DeLong, Ph.D., professor in the biological engineering division and the department of civil and environmental engineering at MIT, notes in a 2007 commentary on metabolomics in MIT’s Technology Review, “Like the human genome sequence, the results of metagenomic analysis represent a type of ‘parts list’ that does not fully capture the functional properties, interrelationships, and dynamics of living microbial communities. They do, however,” he says, “begin to extend our analytical reach beyond the single organism. Population genomics, ‘community metabolism,’ and genomic comparisons of different microbial communities are all now possible.” Dr. DeLong’s laboratory focuses on investigating the structure, function, and ecological significance of natural microbial communities in natural settings.
And apart from their potential to discover new antibiotics to treat life-threatening microbial diseases, metagenomic approaches, Dr. DeLong says, enable direct assessment of community diversity and provide data sets relevant to both measuring and modeling biological process.
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