Widespread immune systems protect bacteria against conjugative plasmids

Stage-Specific Strategies to Limit the Spread of Antibiotic Resistance in Bacteria with a Focus on Conjugation

Horizontal gene transfer (HGT) is the main reason for antibiotic resistance evolution, enabling bacteria to obtain resistance determinants far more rapidly than through point mutations alone. Among HGT mechanisms, conjugation plays a particularly critical role for its efficiency, broad host range, and capacity in disseminating complex resistance plasmids across microbial communities. Despite its clinical significance, conjugation has remained an under-explored target for antimicrobial intervention. This research aims to provide an integrated analysis of bacterial conjugation within the broader context of HGT, emphasizing its evolutionary persistence and mechanistic vulnerabilities. Evidence is synthesized from molecular biology, experimental evolution, and population genetics to uphold the argument for the maintenance of plasmid-mediated resistance through a balance of transfer efficiency, compensatory adaptations, and post-transfer stabilization mechanisms. Building on this foundation, a stage-specific framework is proposed for disrupting conjugation by targeting pre-transfer cell-cell contact, peri-transfer DNA processing, and post-transfer plasmid maintenance. Multiple intervention strategies are reviewed and evaluated in this study, including conjugation inhibitors, relaxase-targeting compounds, CRISPR-based barriers, plasmid curing compounds, restriction-modification systems, as well as the plasmid addiction modules. Collectively, these approaches demonstrate that plasmid persistence is an evolvable trait that can be subjected to targeted interference. Overall, this study highlights conjugation-specific interfering strategies as a potential opportunity in slowing down resistance dissemination and preserving the efficacy of existing antibiotics.

Specific exclusion of conjugative plasmids from the gut microflora

The rise of dangerous antimicrobial resistance (AMR), especially to the newer carbapenem antibiotics, is largely driven by the spread of conjugative plasmids between bacteria. These plasmids rapidly disseminate among common gut organisms like Escherichia coli and Klebsiella pneumoniae, which together account for around half of lethal sepsis and septic shock. Current AMR control measures, such as surveillance and isolation, are often ineffective, and AMR is often detected for the first time when infection is well established. Natural plasmid entry-exclusion systems (EES) protect bacterial populations from repeated entry by plasmids that are already established, or indeed by any plasmid with a related cognate EES, and we demonstrate here the exploitation of this mechanism for therapeutic purposes. We combined exclusion genes from three major AMR plasmid types (IncM, IncL, and IncC) into an efficient conjugative plasmid backbone and showed that this engineered ‘probiotic plasmid’ prevents invasive AMR plasmids from entering bacterial populations in vitro and in vivo, in the mouse gut. This offers a promising new strategy to control invasive AMR plasmids and prevent AMR acquisition in high-risk settings, such as in hospitals or AMR-endemic regions. Graphical abstract

Identification of essential genes for conjugative transfer in antimicrobial resistance-associated pELF-type linear plasmids of opportunistic pathogen Enterococcus faecium

The study findings revealed that pELF-type plasmids utilize highly minimized conjugation machinery, which is similar to unusual systems previously identified in other gram-positive bacteria, such as Streptomyces.

Distinct plasmid- and host-encoded mechanisms drive small plasmid copy number-mediated heteroresistance in Escherichia coli

Advances in understanding of plasmid biology through the application of transposon insertion sequencing

Plasmids are extra-chromosomal DNA molecules capable of autonomous replication, stable inheritance in a bacterial population, and horizontal transfer to other bacteria. Plasmids can harbour auxiliary genetic material that contributes to host bacterial fitness, the most prominent example being antimicrobial resistance (AMR) determinants, which remain the greatest threat to modern medicine. Since their discovery in the early 1950s, plasmids have been extensively studied due to their diversity, their capacity to spread between bacterial hosts, and their ability to carry and disseminate multiple AMR genes simultaneously. Recent advances in sequencing technology have transformed plasmid research, with transposon–insertion sequencing (TIS) enabling simultaneous analysis of millions of mutants and providing unprecedented scale, speed and resolution for studying plasmid biology. Here, we briefly outline a recommended methodology for generating plasmid transposon mutant libraries, which can be combined with TIS to investigate plasmid replication, maintenance and conjugation. We further summarise data from nine comprehensive plasmid TIS studies to date on five distinct plasmids, discuss alternative uses for plasmid libraries, challenges and future perspectives.

Intergeneric conjugative transfer of plasmid-associated antibiotic resistance genes from environmental Aeromonas spp. to gram-negative recipient strains