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Gaining Ground In The Race Against Antibiotic Resistance

September 22, 2005
Howard Hughes Medical Institute
Antibiotic resistance puts humans in an escalating "arms race" with bacteria, as scientists try to develop new antibiotics faster than the bacteria can evolve new resistance strategies. Now researchers are reproducing in the lab the natural evolution of the bacterial enzymes that confer resistance, which could give drug designers a sneak peek at the next generation of resistance.

In this close-up of the metallo-beta-lactamase enzyme's active site (in red), the two essential metal ions (blue spheres) and a beta-lactam antibiotic (colored sticks) are highlighted. The activity of this enzyme has been optimized by in vitro evolution resulting from exposure to large amounts of antibiotics. Over the black background (on top), a large number of antibiotic molecules are depicted.
Credit: Image : Pablo E. Tomatis

Antibiotic resistance has put humans in an escalating 'armsrace' with infectious bacteria, as scientists try to develop newantibiotics faster than the bacteria can evolve new resistancestrategies. But now, researchers have a new strategy that may give thema leg up in the race—reproducing in the lab the natural evolution ofthe bacterial enzymes that confer resistance.

A team ofscientists in Argentina and Mexico identified mutations that increasedthe efficiency of a bacterial enzyme that renders penicillin andcephalosporin antibiotics useless. The results could lead to moreeffective enzyme inhibitors by giving drug designers a sneak peek atthe next generation of resistance.

Alejandro Vila, a HowardHughes Medical Institute international research scholar, and colleaguesat the University of Rosario's Institute of Molecular and CellularBiology in Argentina and at the Biotechnology Institute of the NationalAutonomous University of Mexico report their findings in the earlyonline edition of the Proceedings of the National Academy of Sciencesthe week of September 19, 2005.

Staying one step ahead ofresistance with new antibiotics and treatments for infections is a hugechallenge because bacteria evolve quickly to evade them. When thescientists introduced random mutations into the gene for a bacterialresistance enzyme and grew the bacteria on increasing concentrations ofantibiotics, it took only a few days of test tube evolution to increasedrug resistance. Eventually, they found four mutations in the evolvedenzyme that allowed the bacteria to survive on a drug dose 64 timeshigher than the dose that kills bacteria hosting the un-evolved enzyme.

“Wewere mimicking what is going on in the doctor's clinic—puttingselection pressure on the enzyme by giving higher doses of antibiotic,”said Vila. “The only ones to survive will be those that have an enzymethat can work more efficiently.”

The researchers conducted theirexperiments using a drug called cephalexin, a member of the widelyprescribed cephalosporin class of antibiotics. These drugs and thepenicillins, which share a common chemical backbone called the β-lactamring, work by disrupting the bacterial cell wall. Bacteria have evolvedenzymes called β-lactamases, which chop the β-lactam ring in half,inactivating the drugs. An inhibitor for one type of lactamase hasalready been marketed as part of a 'package drug' with amoxicillin tofight resistance.

But the lactamase enzyme that Vila's groupstudied is in a different class that is causing an emerging problemaround the world. This class, the metallo-β-lactamases, is morethreatening, said Vila, because it is effective against a broaderspectrum of antibiotics, such as carbapenems. However, it alsorepresents a younger set of enzymes that are still evolving, and thatenabled the scientists to observe that evolution in fast-forward.

Thegroup used a lactamase gene from the Bacillus cereus soil bacteria andtested it in the laboratory strain E. coli. The gene is very similar tolactamase genes found in disease-causing bacteria such as Pseudomonasand Acinetobacter—common culprits in resistant, hard-to-treat hospitalinfections. And it is almost identical to a lactamase gene found inBacillus anthracis, which causes anthrax.

Together, the fourmutations identified by the group increased the enzyme's efficiency atinactivating cephalexin seven-fold. The mutations influenced theenzyme's active site, where the chopping of antibiotic molecules takesplace. One of the mutations has already been found in nature, in alactamase from Pseudomonas.

In some cases, there is a tradeoffassociated with antibiotic resistance: the bacteria's success infighting a particular antibiotic can cause it to lose efficiency ininactivating other antibiotics. But that was not the case here.

“Thisevolved enzyme works better against cephalexin and with the sameefficiency on other antibiotics, as well,” said Vila. In fact, themutant enzyme inactivated seven other cephalosporins as efficiently asor better than the original enzyme. “So it hasn't lost anything, andthe outcome is that the bacteria has increased its range of resistance.This is a huge concern in the clinic.”

To date, there are noknown inhibitors of metallo-β-lactamases, but directed evolution couldhelp in their design, Vila said, by giving drug makers a reliableprediction of what the next generation of enzymes will look like.“Since we were able to reproduce the natural evolution in the testtube, you could generate a more efficient lactamase to use as a target,so that your inhibitor would be one step ahead.”

This would givescience an edge in the resistance race, and it might help slow thevicious cycle enough to develop antibiotics impervious to lactamases.

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