Yale Breakthrough in Destroying Drug-Resistant Bacteria Focuses on Thwarting Genes that Cause Resistance

Yale University biologists for the first time have succeeded in preventing the expression of genes that make bacteria resistant to two widely used antibiotics – chloramphenicol and ampicillin – thus restoring the bacteria’s sensitivity to the antibiotics in laboratory cultures. If successful in animal and human studies, the general method could help avert a worldwide health crisis in treating widespread diseases such as tuberculosis, meningitis and pneumonia, which are becoming increasingly drug resistant.

“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, who announced the finding in the Aug. 5 issue of the Proceedings of the National Academy of Sciences.

Professor Altman, along with senior research scientist Cecilia Guerrier-Takada and postdoctoral fellow Reza Salavati, used laboratory techniques based on research leading up to his 1989 Nobel prize-winning discovery that RNA is not just a passive carrier of genetic code, but also can be an enzyme that actively engages in chemical reactions. The discovery triggered a new branch of genetic engineering aimed at treating lethal viruses and drug-resistant bacteria, as well as repairing genetic defects.

In the past 15 years, physicians have noted a significant increase in drug-resistant bacteria, often requiring the use of more expensive antibiotics accompanied by more negative side-effects, according to Robert S. Baltimore, professor of pediatrics infectious diseases at the Yale School of Medicine.

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, he said. Infections caused by surgery and other hospital procedures also are showing greater drug resistance. Dr. Baltimore said the problem has been exacerbated by the availability of antibiotics without prescriptions in many parts of the world.

Restoring Drug Sensitivity

To restore the sensitivity of E. coli bacteria to either chloramphenicol or ampicillin, the Yale biologists crafted synthetic genes coding for strings of RNA and introduced them into the bacteria via small circular pieces of DNA called plasmids. Plasmids sometimes 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 . Nucleotides are the basic chemical building blocks of genetic material. EGSs are engineered to bind to targeted “messenger RNA” mRNA , a family of compounds that play a key role in controlling body chemistry. 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’s therapeutic value lies in the fact that it can be used to seek out and destroy the mRNAs associated with particular diseases – or the mRNAs associated with resistance to specific drugs. In fact, an EGS already has proved effective against hepatitis in animal experiments at Innovir Laboratories Inc., which has an exclusive license from Yale to develop Professor Altman’s patented discoveries. Innovir is developing EGS molecules that target viruses that cause hepatitis B, hepatitis C, psoriasis and other inflammatory diseases, as well as drug-resistant infections.

Researchers also can use EGSs to inactivate mRNA molecules in a highly selective way to gain a better understanding of how cellular chemistry functions.

In this 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. Research was funded by the National Institute of General Medical Sciences.

“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.”

Next Step for Researchers

The next step in finding a practical way to restore drug sensitivity, regardless of the specific drug or infection involved, is to find the best method of getting EGSs inside 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, he added, 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.