Researchers from the Antimicrobial Resistance (AMR) Interdisciplinary Research Group (IRG) at Singapore-MIT Alliance for Research and Technology (SMART), MIT’s research enterprise in Singapore, and Nanyang Technological University (NTU) have developed a tool using CRISPRi technology that can help understand and prevent biofilm development, drug resistance, and other physiological behaviours of bacteria such as Enterococcus faecalis.

E. faecalis, a bacteria found in the human gut, is one of the most prevalent causes of hospital-associated infections and can lead to a variety of multidrug-resistant, life-threatening infections including bacteraemia (bloodstream infection), endocarditis (infection of the heart), catheter-associated urinary tract infection and wound infections.

However, current methods for understanding and preventing E. faecalis biofilm formation and development are labour-intensive and time-consuming. The SMART AMR research team designed an easily modifiable genetic technique that allows rapid and efficient silencing of bacteria genes to prevent infections.

In a paper titled “Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in Enterococcus faecalis” published in the journal mBio, the researchers explain the scalable dual-vector nisin-inducible CRISPRi system which can identify genes that allow bacteria like E. faecalis to form biofilms, cause infections, acquire antibiotic resistance, and evade the host immune system. The team combined CRISPRi technology with rapid DNA assembly under controllable promoters, which enables rapid silencing of single or multiple genes, to investigate nearly any aspect of enterococcal biology.

“Infections caused by E. faecalis are usually antibiotic tolerant and more difficult to treat, rendering them a significant public health threat,” says Dr Irina Afonina, Postdoctoral Associate at SMART AMR and lead author of the paper. “Identifying the genes that are involved in these bacterial processes can help us discover new drug targets or propose antimicrobial strategies to effectively treat such infections and overcome antimicrobial resistance.”

The tool enables rapid and efficient investigation of a wide range of aspects of enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication. The method can be scaled up to simultaneously silence multiple bacterial genes or perform full-genome studies.

Image caption- SMART Postdoc Dr Irina Afonina and NTU PhD Student Jerome Chua use CRISPRi technology to understand biofilm formation in Enterococcus faecalis.