Cause of death surprises researchers

A research team at the University of Michigan's College of Pharmacy set out looking for a way to kill a stubborn type of bacteria and they succeeded"”but not in the way they expected.

"We didn't get there the way we thought we'd get there, but in the end, we were right," said RonaldWoodard, chair of medicinal chemistry at the University of Michigan College of Pharmacy.

Some of the better-known Gram negatives are salmonella, gonorrhea, cholera and meningicoccal meningitis, along with the bacteria that caused the black plague.

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 said. 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 said.

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 colouring test, Gram-positive and Gram-negative bacteria look different. Gram-positive cells are smooth on the outside, while Gram-negative 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 with antibiotics, Woodard said.

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 said. 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-positive cells could not survive without this external structure. This shows that is not true," Woodard said. Though they didn't die, they were weakened, and that made the cells an easy target for antibiotics.

Because Woodard suspected he might be flying in the face of conventional wisdom on bacteria, he solicited second opinions from the Borstel Research Center in Germany, which does a good deal of work on Gram-negative bacteria. Scientists there were initially skeptical, he said, but eventually, Uwe Mamat and Buko Lindner from Borstel signed on to the project.

"Bugs are very smart," Woodard said. "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."