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Battling Bacterial Resistance—Shooting for the Genes
By Lone Frank

Featured Article.

As scientists all over the world worry about how to turn the rising tide of antibiotic resistance among bacteria, a Danish team is joining the battle with a new strategy. They are hitting the microbes in their genes and the weapon of choice is called PNA.

Structure of PNA-DNA duplex from NMR analysis.

Researchers at the Danish biotech start-up Pantheco and their collaborators at the University of Copenhagen may be first to use so-called antisense technology against bacteria. The antisense principle is to interrupt a disease process by preventing cells from synthesizing harmful proteins produced by our own body or an outside attacker like a virus or microbe. Antisense works at the genetic level as modified RNA or DNA molecules are specifically designed to bind and inactivate mRNA in the cell.

The Danish team is working with a peculiar compound PNA—peptide nucleic acid—which was developed in Peter Nielsen's laboratory at the University of Copenhagen. "The molecule is best described as a cross between protein and DNA," explains Nielsen. PNA presents the double helical structure and the characteristic base pairing found in nucleic acids, but instead of the naturally-occurring sugar phosphate the PNA backbone is protein-like.

Experts agree that the spreading of microbial antibiotic resistance is an alarming and global problem

Nielsen and his Pantheco colleagues have come a long way with a new PNA oligomer—a short PNA sequence—that is targeted to bind and inactivate a bacterial gene essential for proliferation. "The PNA is chemically modified to penetrate into cells and, when mice are injected, it effectively cures an E. coli infection," says biochemist Jeppe Christensen of Pantheco. Researchers evaluate the antibacterial effect using a traditional animal model of peritoneal infection; the new PNA oligo measures up to the very strong antibiotics Gentamycin and Ampicillin. PNA has potential against more problematic organisms than E. coli. In test tube experiments with bacterial cultures different PNAs finish off 90 clinical bacterial isolates with varying resistance patterns including several multiresistant strains.

The combined results evoked enthusiasm among antibacterial researchers when recently presented at the Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) in Toronto, Canada. "This work is very promising," says microbiologist Niels Frimodt Møller of Statens Serum Institute in Copenhagen. "I'm particularly impressed to see one dose of a compound completely eradicate an otherwise lethal infection in mice without any apparent side effects," he adds, but underlines that effects of long-term use must be studied.

Experts agree that the spreading of microbial antibiotic resistance is an alarming and global problem. "We see strong geographical variation but the bottom line is that all types of bacteria are developing resistance to the available drugs," says chief clinical microbiologist Michael Tvede of Copenhagen University Hospital. He cites the heavy use of broad-spectrum drugs, particularly in the United States, Southern and Eastern Europe, as the biggest current worry. In the US, almost one third of pneumonia-causing pneumococcus isolates do not respond to penicillin. And multiresistant Staphylococcus aureus, which typically infects wounds, is reported in many hospitals.

"There is an urgent need to develop completely new classes of antibacterial drugs," says Møller. Today's antibiotics are mostly naturally-occurring compounds originally produced by fungi as a defense against microbes and bacteria very efficiently evolve enzymes that inactivate these compounds. "When new chemical variants of known antibiotics are used in the clinic resistance develops quickly", explains Møller. Resistance is spread when the responsible genes are shuttled between bacterial cells on small circular plasmid DNA molecules.

PNA seems to offer a significant advantage over traditional drugs. "Because we are using a radically new strategy targeting the genetic level, it is highly unlikely that the already established resistance mechanisms will play a role," explains Nielsen. He adds that there are no known enzymes that can break down PNA. Tvede is encouraged by the results so far, but he cautions that historically, bacteria have developed resistance to everything we have fought them with. "All new drugs need to be monitored carefully," he says.

The next step for the PNA team is to develop oligos that can be clinically tested in humans against infections with Gram Negative bacteria including E. coli and others, which cause most urinary tract and blood-borne infections. "The common Gram Negative infections show increasing resistance to traditional drugs and a new supplement would be welcomed by clinicians," says Tvede. Further ahead lies the challenge of less common but potentially very serious infections, says Christensen. "In parallel we are working on PNA against the multiresistant Staphylococcus bacteria that present a serious problem in relation to wound infections."

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Schou, C., Hansen, H.F., Nielsen, PE, Kristiansen, E. & Giwercman, B. Antibiotic effects of PNA (peptide nucleic acid) antisense compounds against multiresistant Echerichia coli. Abstract presented at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Toronto, Ontario, Canada, September 17-20, 2000.
Nielsen, PE. Antisense peptide nucleic acids. Curr Opin Mol Ther 2, 282-287 (June 2000).

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