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New Approach to Defeating Gram-Negative Bugs

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Ronald Woodard’s team set out looking for a way to kill a stubborn type of bacteria and they succeeded — but not in the way he expected. “We didn’t get there the way we thought we’d get there, but in the end, we were right,” says Woodard, chair of medicinal chemistry at the University of Michigan College of Pharmacy.

Woodard is senior author of an article describing the way he and his research team genetically modified Escherichia coli bacteria, to weaken its defenses. That article appeared in the recently released inaugural issue of the American Chemical Society’s journal, ACS Chemical Biology.

Some of the better-known gram-negatives are salmonella, gonorrhea, cholera and meningicoccal meningitis, along with the bacteria that caused the black plague. Woodard and his collaborators worked on E. coli in part because it is one of the more common gram-negative bacteria, and it is considered by researchers the gold standard of gram-negative bacteria.

After their genetic modifications, E. coli was killed with just a fraction of the antibiotic dose typically needed. It was 512 times more susceptible to Rifampin, 256 times more vulnerable to Novobiocin, and eight times more susceptible to Bacitracin, suggesting doses could be dramatically cut and still be effective, Woodard says. Antibiotics typically only effective against gram-positive bacteria could work against gram-negative bacteria if a compound can be designed to mimic this genetic modification, Woodard says.

Also, E. coli can typically withstand the bile salts found in the human digestive tract, but by weakening it, Woodard’s team found E. coli would die in the presence of normal levels of bile salts to which the bacteria would be exposed in the human gut.

Besides differing in how they respond to Gram’s coloring test, gram-positive and gramnegative bacteria look different. Gram-positive cells are smooth on the outside, while gramnegative cells have sugars and carbohydrates on the outside in structures that look like hairs. That exterior protection is part of what makes Gram-negative bacteria harder to kill antibiotics, Woodard explains.

Woodard’s team set out to genetically modify the cells to eliminate the key sugar to which the hair anchors on the outside of the cell.  “Unfortunately, the bug didn’t die,” Woodard reports. The researchers found that a “backup” gene from a different pathway also could form the anchor, so they knocked out that gene, as well. Initially the cell with both genomic knockouts did not survive without special nutritional supplements. Later, they were surprised to see that with different growth conditions, the cell began to grow again but without the hair-like structure. The cells survived — but they looked a lot like gram-positive cells, without all the sugars on the outside.

“We, as well as the entire scientific community, always thought gram-negative cells could not survive without this external structure. This shows that is not true,” Woodard says. Though they didn’t die, they were weakened, and that made the cells an easy target for antibiotics.

“Bugs are very smart,” Woodard says. “It’s not a matter of if a bug will become antibiotic resistant, but when. We have to work hard to get ahead of them.”

Woodard’s research is funded in part by a $2 million, five-year grant from the National Institutes of Health.

Source: University of Michigan

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