Targeted plasmids, a biotechnological tool that opens up new paths against antimicrobial resistance

Targeted antibacterial plasmids (TAP) are emerging as a promising tool to decolonise the gut of multidrug-resistant bacteria without disturbing beneficial bacteria. This is the main conclusion of a study published in Nucleic Acid Research, conducted by the Barcelona Institute for Global Health (ISGlobal), a centre supported by the ”la Caixa” Foundation, in collaboration with the French INSERM institute of the Centre National de la Recherche Scientifique (CNRS) in France.

Antimicrobial resistance (AMR) is one of the major public health threats: in 2019, 3.57 million deaths were associated with resistant infections, nearly one million directly attributable to them. The lack of new effective antibiotics exacerbates this problem and necessitates the search for innovative alternatives.

The intestinal tract hosts a complex community of microorganisms known as the microbiota. It comprises commensal bacteria, which aid nutrient absorption and act as a barrier against pathogens, and potentially pathogenic bacteria, such as certain strains of Escherichia coli (E. coli). When multidrug-resistant bacteria to antibiotics colonise this environment, the risk of subsequent infections increases, particularly in immunocompromised individuals. The main challenge is to develop an antibacterial strategy capable of selectively eliminating antimicrobial-resistant strains without affecting other beneficial commensal species.

Targeted antibacterial plasmids and the CRISPR-Cas system

A plasmid is a small circular DNA molecule independent of the bacterial chromosome. Targeted antibacterial plasmids (TAP) are biotechnological tools designed to selectively combat antibiotic resistance. Unlike conventional antibiotics, which act on a broad spectrum of bacteria, TAPs recognise and attack only bacteria carrying a specific resistance gene.

To achieve this, they use bacterial conjugation, a natural DNA transfer mechanism, to enter the resistant bacterium. Once inside, TAPs express a CRISPR-Cas system programmed to identify a specific bacterial DNA sequence and induce the death of the resistant bacterium or cause it to lose antibiotic resistance.

Using TAPs to eliminate or resensitise strains of E. coli

“In this study, we assessed the potential of TAPs against certain multidrug-resistant bacterial strains: the E. coli producing ESBLs (Extended-Spectrum Beta-Lactamases), enzymes that render them resistant to most beta-lactam antibiotics, including penicillins and cephalosporins. These bacteria carry the blaCTX-M-15 gene, one of the most common resistance determinants,” explains Natalia Roson-Calero, ISGlobal researcher and co-author of the study.

The elimination or resensitisation of these strains is particularly relevant, as third-generation cephalosporin-resistant enterobacteria are classified as critical pathogens by the WHO due to their high disease burden and limited therapeutic options. The study focused particularly on the high-risk ST131 clone, one of the most prevalent and virulent lineages.

“When the TAP incorporated Cas9 – an enzyme acting as a molecular scissors – it induced double-strand breaks in the blaCTX-M-15 gene. If the gene was on the chromosome, the damage was lethal and the bacterium died rapidly. If it was on a plasmid, the cut caused the loss of that plasmid and, with it, the resistance,” explains Roson-Calero. “In some cases, this loss triggered toxin–antitoxin (TA) systems associated with the plasmid itself, which could also lead to bacterial death.”

However, the research team observed that TAPs based on another version of the enzyme, dCas9, did not cut the DNA. Instead, they blocked transcription of the blaCTX-M-15 gene, preventing production of the resistance-conferring protein. As a result, the bacterium regained antibiotic sensitivity without directly compromising its viability.

The goal: to eliminate only resistant strains

In mixed cultures, TAPs specifically eliminated E. coli carrying blaCTX-M-15 without affecting other bacterial species present. Moreover, in conjugation assays performed in human faeces (a complex microbial environment closer to the clinical situation), both TAP-Cas9 and TAP-dCas9 significantly suppressed cefotaxime-resistant E. coli.

“Our results reinforce the potential of TAPs as a selective gut decolonisation tool. Furthermore, they open the door to future microbiome-targeted editing strategies to prevent dissemination or infection by multidrug-resistant bacteria,” concludes Jordi Vila, Research Professor and investigator in ISGlobal’s Antimicrobial Resistance group.