Novel Approaches for Detecting Gene Amplification Events
Abstract
Antibiotic resistant bacteria have become an important health concern for members of the U.S. military, as well as civilian support personnel, deployed throughout the world. It is well documented that bacteria can become resistant to antibiotics by acquiring new genes that impart drug resistance directly from other bacteria sharing the same environment. Specifically, bacteria can make direct contact with one another to spread drug resistance genes via horizontal gene transfer. As a consequence, drug resistance can spread throughout the bacterial populations that inhabit our gut, hence rendering antibiotic therapy ineffective. In addition, increased resistance to specific antibiotics can result from processes that amplify the number of copies of drug resistance genes. While both of these processes have been known for decades, improved mechanistic details of how they occur in nature are still needed based on the premise that understanding how these processes work can lead to new strategies to prevent the acquisition and spread of antibiotic resistance. To achieve this goal, we have modified a genetic system that is now commonly used to make specific changes in gene sequence to develop a highly sensitive system to detect the transfer of antibiotic resistance genes from one microorganism to another, as well as gene amplification events, in living cells. The system takes advantage of a specific protein (Cas9) that can be directed to any specific DNA sequence within a bacterial cells. We have modified the gene that encodes the Cas9 protein to encode a protein that is fused to green fluorescent protein (GFP). Cells expressing Cas9-GFP are then imaged by fluorescence microscopy to detect the presence of specific DNA sequences that indicate horizontal gene transfer or gene amplification events have occurred. The detection of these events on a microscopic level is aided by the use of microfluidic “chips” that allow bacteria to be individually imaged at high resolution so that changes in genetic composition can be detected in real time. We are furtherer modifying the Cas9-GFP detection system for use in vitro to detect antibiotic resistance genes directly from a variety of sources, such as the human gut, soil or water. More rapid detection of antibiotic resistance genes will be useful for health care workers to identify the most effective strategies to treat bacterial infections, as well as to prevent the spread of drug resistance through vulnerable populations.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- May 26, 2016
- Source ID
- HDTRA11510053
Entities
People
- Gregory J. Phillips
Organizations
- Defense Threat Reduction Agency
- Iowa State University