By Sean Henahan, Access Excellence

Caption: Streptococcus pyogenes (electron micrograph)

Researchers at Yale University biologists report they have succeeded in preventing the expression of genes that make bacteria resistant to two widely used antibiotics, chloramphenicol and ampicillin. This manipulation appears to restore the bacteria's sensitivity to the antibiotics in laboratory cultures.

"Although the path from our experiments to a practical therapeutic tool may be a very long and costly one, this method could restore the full usefulness of today's front-line antibiotics, thus bypassing the tremendous expense of developing new antibiotics," said Nobel laureate Sidney Altman.
 
Professor Altman received the Nobel Prize in 1989 in recognition of his discovery that RNA is not just a passive carrier of genetic code, but also can be an enzyme that actively engages in chemical reactions. He and colleagues at Yale used laboratory techniques derived from this discovery to explore the genetic mechanisms of drug resistance.

The Yale biologists restored the sensitivity of E. coli bacteria to either chloramphenicol or ampicillin by creating   synthetic genes coding for strings of RNA and introducing them into the bacteria via small circular pieces of DNA called plasmids. Plasmids are known to carry genes that cause bacteria to become drug resistant in the first place.

Once inside the bacteria, the synthetic genes produced small strings of RNA nucleotides called External Guide Sequences (EGS). These EGSs are engineered to bind to targeted messenger RNA. Once the EGS molecules attach to their target in a specific virus or bacteria, they cause an RNA enzyme called RNase P to destroy the mRNA to which they are bound. The EGS molecules are then freed to repeat the process.

The EGS technology can be used to seek out and destroy the mRNAs associated with particular diseases, or in this case,  the mRNAs associated with resistance to specific drugs. In animal research, EGS have shown promise against hepatitis. EGS molecules that target viruses that cause hepatitis B, hepatitis C, psoriasis and other inflammatory diseases are also in development.

In this current study, drug sensitivity was restored in virtually all bacteria in laboratory test cultures. The research also showed that both boosting the ratio of EGSs to target mRNAs and increasing the number of different target sites on the mRNA enhanced the method's efficiency in restoring drug sensitivity, and also prevented a return to drug resistance.

"We've been working on enzymes at Yale for 25 years or more, and it was only recently that we found some potential practical value from the research," noted Professor Altman, who has been working on drug-resistant bacteria for the past six years. "You can never predict when basic investigations will yield important practical discoveries, which underscores the importance of continued support for non-applied research."

THE COMING PLAGUE?

Health authorities have watched with growing alarm over the past 20 years as the number and variety of drug-resistant bacteria has continued to increase. Typically, physicians must turn to stronger and more expensive antibiotics to treat resistant organisms. Of particular concern has been the increase in hospital cases that appear resistant to all known treatments.

For example, haemophilus influenzae, which causes meningitis, used to be treated routinely with ampicillin, but about 20 percent of cases today are resistant to the antibiotic. Infections caused by surgery and other hospital procedures also are showing greater drug resistance. The problem has been exacerbated by the availability of antibiotics without prescriptions in many parts of the world, says Robert S. Baltimore, professor of pediatrics (infectious diseases) at the Yale School of Medicine.

The next step for researchers will be to find a practical way to restore drug sensitivity, regardless of the specific drug or infection involved. To do this, they will have to determine the best method of getting EGSs inside the bacteria, Professor Altman said. Instead of using plasmids as he did, which would  require exposing patients to a second type of bacteria, researchers most likely will find a chemical package that can readily enter the target bacteria. Then the method must be tested in animals and humans.

It is a relatively simple matter to design the EGS sequence itself, noted Altman, because the methods are "all pretty well worked out." In fact, the specific EGS that will  restore sensitivity to a specific drug can be designed in a matter of hours or days,  and then produced with a machine called an RNA synthesizer. The entire process of finding and testing the effectiveness of a specific EGS takes only a few weeks or  months compared to years required for the development of most antibiotics.

The research appears in the Aug. 5 issue of the Proceedings of the National Academy of Sciences.