Antibiotic Resistant Genetic Elements Inactivated by CRISPR-Based Tool

Antibiotic resistance has steadily accelerated in recent years to become a global health crisis with estimates of more than 10 million worldwide deaths per year by 2050. Antibiotic-resistant bacteria are known to flourish in hospital settings, sewage treatment areas, animal husbandry locations and fish farms.  

In a new study published in npj Antimicrobials and Resistance titled, “A conjugal gene drive-like system efficiently suppresses antibiotic resistance in a bacterial population,” researchers from University of California, San Diego (UCSD) have developed a novel CRISPR-based technology for removing antibiotic resistant elements from populations of bacteria. The Pro-Active Genetics (Pro-AG) tool, named pPro-MobV, is a second-generation technology similar to gene drives, which are currently applied in insect populations to disrupt the spread of harmful properties, such as parasites that cause malaria. 

“With pPro-MobV we have brought gene-drive thinking from insects to bacteria as a population engineering tool,” said Ethan Bier, PhD, distinguished professor in the department of cell and developmental biology at UCSD and corresponding author of the study. “With this new CRISPR-based technology we can take a few cells and let them go to neutralize antibiotic resistance in a large target population.” 

In 2019, Bier’s lab collaborated with Victor Nizet, MD, distinguished professor at UCSD School of Medicine, to develop the initial Pro-AG concept, in which a genetic cassette is introduced and copied between the genomes of bacteria to inactivate their antibiotic-resistant components. The cassette launches into an antibiotic resistant gene to restore sensitivity of the bacteria to antibiotic treatments. 

In the new study, Bier and his colleagues developed a system that spreads the antibiotic CRISPR cassette components via conjugal transfer. Results showed that this next-generation pPro-MobV system can exploit a naturally created bacterial mating tunnel between cells to spread the key disabling elements.  

The process was demonstrated in bacterial biofilms, communities of microorganisms that can contaminate various surfaces and are difficult to remove under conventional cleaning methods. Biofilms are created in the majority of infections that lead to serious disease, as they create a protective layer of cells that is difficult for antibiotics to penetrate. The new technology carries potential in health care settings, environmental remediation, and microbiome engineering. 

Bier says the biofilm context for combatting antibiotic resistance is particularly important since this is one of the most challenging forms of bacterial growth to overcome in the clinic or in enclosed environments such as aquafarm ponds and sewage treatment plants.  

The researchers also found that components of the active genetic system could be carried and delivered by bacteriophages, or viruses that infect bacteria. The researchers envision pPro-MobV elements to be deployed in conjunction with engineered phage viruses. Additionally, this genetic platform can incorporate homology-based deletion as a safety measure to remove the gene cassette if desired. 

“This technology is one of the few ways that I’m aware of that can actively reverse the spread of antibiotic-resistant genes, rather than just slowing or coping with their spread,” said Justin Meyer, PhD, a professor in the department of ecology, behavior, and evolution at UCSD and co-author on the study.