But what might be the function of suicide in a unicellular organism? It is becoming increasingly apparent that bacteria live and die in complex communities that in many ways resemble a multicellular organism. Release of pheromones induces bacteria in a population to respond in concert by changing patterns of gene expression, "quorum sensing". Most bacterial species actually do not live as planktonic suspensions in vivo, but form complex biofilms, tightly knit communities of cells. From this perspective, programmed death of damaged cells may be beneficial to a multicellular community of clonal organisms. For example, suicide could limit the spread of a viral infection. In the case of serious damage by toxic factors, cells will autolyse and donate their nutrients to their neighbors, instead of draining resources from their kin in a futile attempt to repair.

While studying biofilm resistance of P. aeruginosa, we found that a small fraction of persistor cells is actually responsible for the survival of the population (Brooun et al., 2000). Our working hypothesis is that persistors are cells with disabled programmed death (Lewis, 2000, 2001). Finding genes responsible for persistence (tolerance to antibiotics) is the aim of our collaborative genomics project with Affymetrix and TIGR. We are using DNA arrays to detect changes in gene expression in the entire genome. These changes pinpoint genes likely involved in PCD. A complementary approach is to search for mutants in transposon insertion library that are affected in cell death. Finding of PCD genes will lead to a new approach to antibiotic development. A drug that activates PCD will be formulated with a conventional cidal antibiotic, resulting in eradication of recalcitrant infections.

2. Natural inhibitors of microbial resistance from medicinal plants.
Bacteria have an unusual ability to resist chemically unrelated antimicrobials, including ones they never encountered in nature. We found that this ability is largely due to the presence of Multidrug Resistance Pumps, membrane translocases that pump out antibiotics from the cell (Lomovskaya and Lewis, 1992). MDRs are found in all microorganisms, and we wondered whether organisms that produce antibiotics learned how to combat this resistance.

Medicinal plants make many different types of antimicrobials, but these are rather weak when tested in vitro. We found that this is due to their efflux by MDRs.
But why should plants keep on making antibiotics if microorganisms have MDR pumps that make these substances essentially ineffective? We hypothesized that plants are “smart” and have developed MDR inhibitors that act synergistically with antibiotics. We indeed discovered a compound that specifically disrupts this bacterial resistance mechanism. Barberry (Berberis fremontii) plants make an ineffective antibiotic berberine. We found that barberry also produces a substance, 5'-methoxyhydnocarpin (5'-MHC), that is a potent inhibitor of MDRs.

When combined with berberine, 5'-MHC has effective antimicrobial action against the human pathogen S. aureus (Stermitz et al., 2000). Importantly, 5'-MHC has no antimicrobial activity on its own. This is the first case of synergy among plant compounds documented at the molecular level. This finding provides an important precedent for the idea that synergistic interaction among different compounds (antimicrobial or not) explains the frequent failures to isolate single active substances from medicinal plants.
We are currently pursuing discovery of additional MDR inhibitors from medicinal plants that act against Gram negative bacteria and yeast. Plants have faced the problem of microbial multidrug resistance for far longer than we have, and their solution is apparently to use a combination of an antibiotic with an MDR inhibitor. Emulating Nature's strategy and potentiating antibiotics with MDR inhibitors can be an effective strategy against drug-resistant microorganisms.

3. Development of sterile surface materials.
We have recently developed a “sterile” surface material in collaboration with Professor Alexander Klibanov of MIT (Tiller et al., 2001). Antimicrobial action requires the active molecule to penetrate into the cell in order to reach its target. This would seemingly preclude creation of a surface to which antimicrobials are bound covalently and are therefore immobilized. Conventional designs include incorporation of free molecules into the material that will then release the antiseptic. We solved the problem of an active surface by reasoning that an antimicrobial molecule tethered covalently to the surface by a long polymer “thread” will retain its mobile properties and will be able to penetrate into cells and reach its targets. N-hexylated poly(4-vinylpyridine) coupled to the surface of amino-glass produced a polymer material that kills bacteria on contact. This approach promises to provide a new class of antiseptic materials for hospitals and home use to stem the spread of drug-resistant pathogens such as S. aureus and E. coli. The obvious advantages of this new type of polymer are stability – activity is not lost over time due to the release of the active component; and environmental friendliness – no toxic materials are leached out. Current work focuses on development this material into a product, and on finding additional effective agents for sterile surfaces.

4. Discovery of “uncultivable” microorganisms (in collaboration with Professor Slava Epstein).
It has been known for a good half a century that 99% of all microbial species from most environments are uncultivable and as such largely unavailable to scientists. Attempts to culture more species in the lab by manipulating growth media were unsuccessful. The riddle of uncultivable microorganisms has been recognized as the main challenge for basic and applied research in microbiology by the American Society for Microbiology (Young, 1997).
We have recently designed a chamber for growing uncultivable organisms. The first representatives of “uncultivables” are currently growing in our labs. This method opens the prospect of resolving the nature of uncultivability, and will allow us to build a collection of novel organisms to serve as a source for antibiotic discovery. Of especial interest is the prospect of discovering the missing microbial taxons. Bacteria come in several very large groups (like cyanobacteria) that diverged billions of years ago. About a third of these large groups do not have a single cultivable representative, and we know of their existence only from small pieces of DNA extracted from the environment.

Selected Publications

Lomovskaya, O. and Lewis, K. (1992) EMR, an Escherichia coli locus for multidrug resistance. Proc. Natl. Acad. Sci. USA 89: 8938-8942.

Lewis, K. (1994) Multidrug resistance pumps in bacteria: variations on a theme. Trends Biochem. Sci. 19: 119-123.

Lomovskaya, O., Lewis, K., and Matin, A. (1995) EmrR is a negative upstream regulator of the E. coli multidrug resistance pump EmrAB. J. Bacteriol. 177:2328-2334.

Ferrante, A., Augliera, J., Lewis, K., and Klibanov, M.A. (1995) Cloning of an organic solvent resistance gene in E. coli: the unexpected role of alkylhydroperoxide reductase. Proc. Natl. Acad. Sci. USA 92:7617-7621.

Hsieh, P.-C., Siegel, S.A., Rogers, B., Davis, D., and Lewis, K. (1998) Bacteria lacking a multidrug pump: a sensitive tool for drug discovery. Proc. Natl. Acad. Sci. USA. 95:6602-6606.

Lewis, K. (1998) Pathogen resistance as the origin of kin altruism. J. Theor. Biol. 43:359-363.

Lewis, K. (1999). Multidrug resistance: versatile drug sensors of bacterial cells. Current Biol. 9:R403-R407.

Brooun, A., Tomashek, J.J., and Lewis, K. (1999). Purification and ligand binding of EmrR, a regulator of a multidrug transporter. J. Bacteriol. 181:5131-5133.

Brooun, A., Liu, S., and Lewis, K. (2000). A Dose-Response Study of Antibiotic Resistance in Pseudomonas aeruginosa Biofilms. Antimicrob. Agents Chemother. 44:640-646.

Stermitz, F.R., P. Lorenz, J.N. Tawara, Zenewicz, L., and Lewis, K. (2000). Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin, a multidrug pump inhibitor. Proc. Natl. Acad. Sci. USA 97:1433-1437.

Featured in:
Chem. Engineer. News (2000). 78 (8):6-7. Plant may hold key to ultimate antibiotic.
Chem. Engineer. News (2000). 78 (51):24-31. Chemistry highlights 2000.

Stermitz, F. R., J. Tawara-Matsuda, P. Lorenz, P. Mueller, L. Zenewicz, and K. Lewis. (2000). 5'-Methoxyhydnocarpin and pheophorbide a: Berberis species components which potentiate berberine growth inhibition of resistant Staphylococcus aureus. J. Nat. Prod. 63:1146-1149.

Lewis, K. (2000). Programmed death in bacteria. Microbiol. Mol. Biol. Rev. 64:503-514.

Guz, N.R., Stermitz, F.R., Johnson, J.B., Beeson, T.D., Wilen, S., Hsiang, J-F., and Lewis, K. 2001. Flavonolignan and flavone inhibitors of a Staphylococcus aureus multidrug resistance (MDR) pump. Structure-activity relationships. J. Med. Chem. 44:261-268.

Lewis, K. (2001). The riddle of biofilm resistance. Antimicrob. Agents Chemother. 45:999-1007.

Lewis, K. (2001). In search of natural substrates and inhibitors of MDR pumps. J. Mol. Microbiol. Biotechnol. 3:247-254.

Tiller, J.C., Liao, C.J., Lewis, K., and Klibanov, A.M. (2001). Designing surfaces that kill bacteria on contact. Proc. Natl. Acad. Sci. U S A. 22:5981-5985.

Featured in:
Chem. Engineer. News (2001) 79:13. Designed Surface Kills Bacteria.

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