Aimee E. Belanger

One of the oldest unresolved microbiological phenomena is why only a small fraction of the diverse microbiological population grows on artificial media. The "uncultivable" microbial majority arguably represents our planet's largest unexplored pool of biological and chemical novelty. Previously we showed that species from this pool could be grown inside diffusion chambers incubated in situ, likely because diffusion provides microorganisms with their naturally occurring growth factors. Here we utilize this approach and develop a novel high-throughput platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. We have designed and tested an isolation chip (ichip) composed of several hundred miniature diffusion chambers, each inoculated with a single environmental cell. We show that microbial recovery in the ichip exceeds manyfold that afforded by standard cultivation, and the grown species are of significant phylogenetic novelty. The new method allows access to a large and diverse array of previously inaccessible microorganisms and is well suited for both fundamental and applied research.

The antimycobacterial compound ethambutol [Emb; dextro-2,2'-(ethylenediimino)-di-1-butanol] is used to treat tuberculosis as well as disseminated infections caused by Mycobacterium avium. The critical target for Emb lies in the pathway for the biosynthesis of cell wall arabinogalactan, but the molecular mechanisms for drug action and resistance are unknown. The cellular target for Emb was sought using drug resistance, via target overexpression by a plasmid vector, as a selection tool. This strategy led to the cloning of the M. avium emb region which rendered the otherwise susceptible Mycobacterium smegmatis host resistant to Emb. This region contains three complete open reading frames (ORFs), embR, embA, and embB. The translationally coupled embA and embB genes are necessary and sufficient for an Emb-resistant phenotype which depends on gene copy number, and their putative novel membrane proteins are homologous to each other. The predicted protein encoded by embR, which is related to known transcriptional activators from Streptomyces, is expendable for the phenotypic expression of Emb resistance, but an intact divergent promoter region between embR and embAB is required. An Emb-sensitive cell-free assay for arabinan biosynthesis shows that overexpression of embAB is associated with high-level Emb-resistant arabinosyl transferase activity, and that embR appears to modulate the in vitro level of this activity. These data suggest that embAB encode the drug target of Emb, the arabinosyl transferase responsible for the polymerization of arabinose into the arabinan of arabinogalactan, and that overproduction of this Emb-sensitive target leads to Emb resistance.

Bacterial glycogen is a polyglucose storage compound that is thought to prolong viability during stationary phase. However, a specific role for glycogen has not been determined. We have characterized SMEG53, a temperature-sensitive mutant of Mycobacterium smegmatis that contains a mutation in glgE, encoding a putative glucanase. This mutation causes exponentially growing SMEG53 cells to stop growing at 42 degrees C in response to high levels of glycogen accumulation. The mutation in glgE is also associated with an altered growth rate and colony morphology at permissive temperatures; the severity of these phenotypes correlates with the amount of glycogen accumulated by the mutant. Suppression of the temperature-sensitive phenotype, via a decrease in glycogen accumulation, is mediated by growth in certain media or multicopy expression of garA. The function of GarA is unknown, but the presence of a forkhead-associated domain suggests that this protein is a member of a serine-threonine kinase signal transduction pathway. Our results suggest that in M. smegmatis glycogen is continuously synthesized and then degraded by GlgE throughout exponential growth. In turn, this constant recycling of glycogen controls the downstream availability of carbon and energy. Thus, in addition to its conventional storage role, glycogen may also serve as a carbon capacitor for glycolysis during the exponential growth of M. smegmatis.

The use of macrolide antibiotics in food animals has the potential to select for macrolide-resistant strains of resident bacterial flora. This may include the animal pathogens that are the intended targets of macrolide antibiotic intervention and Campylobacter, common inhabitants of the intestinal tract of food animals that are zoonotic pathogens in man. Such Campylobacter strains are not only resistant to the macrolide antibiotics used in food animals, e.g. tylosin, tilmicosin and tulathromycin, but to the macrolide antibiotics used in human medicine, e.g. erythromycin, azithromycin and clarithromycin, as well. Retail meat is a possible source of Campylobacter and persons consuming the meat derived from macrolide-treated food animals could acquire infections due to macrolide-resistant strains of this organism. Erythromycin is sometimes used to treat human cases of campylobacteriosis and those infected with animal-derived macrolide-resistant Campylobacter may not respond to treatment. The actual risk to human health from the use of macrolide antibiotics in food animals has been difficult to determine because of a lack of information about the macrolide-resistant Campylobacter found on the farm and in the clinic. Recently, however, a plethora of new information has become available on this topic. This review discusses what is currently known about the selection of macrolide-resistant Campylobacter in food animals, the prevalence of macrolide-resistant Campylobacter on retail meat, the prevalence of animal-derived macrolide-resistant Campylobacter in the clinic and the human health consequences associated with macrolide-resistant Campylobacter infection. This work will emphasize the comprehensive body of data generated in Denmark and the US as part of government-sponsored research studies over the last 10 years. These scientific findings may allow informed decisions to be made in the future about how macrolide antibiotics should be used in food animals while still safeguarding human health.