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

Characterization of the conjugative modules of the pRAS4 plasmid of Aeromonas salmonicida reveals an environmental lineage

Conjugative plasmids of Aeromonas salmonicida, the causative agent of furunculosis in salmonids, have long been implicated in the dissemination of antimicrobial resistance in aquaculture. Among these, pRAS4 has remained the least characterized, with no complete published annotation and no molecular description of its conjugation machinery. In the present study, we report the complete sequence of pRAS4 from two A. salmonicida subsp. salmonicida strains, F878/91 and F830/91, isolated from Atlantic salmon (Salmo salar L.) showing characteristic signs of furunculosis on the Western coast of Norway in 1991. Mating experiments produced antibiotic-resistant transconjugants in Escherichia coli DH5α, and antimicrobial susceptibility testing of single-colony-purified transconjugants confirmed selective transfer of the tetracycline-resistance phenotype encoded by pRAS4. Domain based re-annotation using InterProScan identified two previously unassigned coding sequences as canonical components of the conjugation machinery: an 823-amino-acid coding sequence originally annotated as traN was reassigned as a TraG/VirD4-family type IV coupling protein (Pfam PF02534, E = 5.1 × 10−99), and a 366-amino-acid coding sequence originally labelled “hypothetical protein” was identified as a MobA/VirD2 family relaxase (Pfam PF03432, E = 4.7 × 10−14). Together with the canonical virB1–virB11 mating pair formation cluster and the mobC accessory relaxosome gene, these findings establish that pRAS4 encodes a complete and functional MOB_P/MPF_T conjugation system. PlasmidFinder analysis returned no replicon match for pRAS4, and BLASTp of the RepA protein returned no close match for the canonical IncU plasmid RepAs (pRAS1, pAr-32, pRA3). The closest sequence relatives of the pRAS4 RepA, type IV coupling protein and relaxase are environmental plasmids of Acidithiobacillus, Acidovorax, Microvirgula and related β- and γ-proteobacteria, and the pRAS4 backbone shares an extensive synteny with the plasmids pYKCT010 and pYK0414 from uncultured bacteria of Japanese river sediments. These findings indicate that pRAS4 is a self-transmissible R-plasmid carrying a previously uncharacterized environmental plasmid backbone, and the tetracycline Tet A efflux determinant flanked by Tn3 transposases represents a subsequent adaption under aquaculture associated antibiotic selection.

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

Genomic insights into the emergence, adaptation, and environmental dissemination of Enterococcus faecium as a multidrug-resistant pathogen: A One Health perspective.

Enterococcus faecium has become a prominent nosocomial pathogen, demonstrating significant multidrug resistance (MDR) and persistence in both hospital and environmental contexts. Genomic analyses indicate a highly adaptable genome characterized by a substantial accessory component enriched in antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs), including plasmids, transposons, integrative conjugative elements, and prophages. The acquisition and dissemination of resistance determinants, such as vancomycin (vanA, vanB), aminoglycoside, macrolide, tetracycline, and novel oxazolidinone resistance genes (optrA, poxtA), are predominantly facilitated by horizontal gene transfer mediated by these MGEs. Virulence factors, stress response regulators, and biofilm-associated genes augment E. faecium's survival in hospital settings and its colonization potential. Environmental reservoirs, such as wastewater, animal farms, and food products, facilitate interspecies gene exchange, underscoring the pathogen's significance within the broader One Health AMR network. This review brings together what we know about E. faecium's genome, focusing on its evolutionary adaptation, mobilome architecture, clinical and epidemiological importance, and spread in the environment. It gives us a complete picture of what we need to do to keep an eye on and control the disease in the future.

Widespread dissemination of the RepA_N-type megaplasmid conferring a carbohydrate utilization advantage in clonal complex 17 Enterococcus faecium in Taiwan

ABSTRACT Enterococcus faecium has become a major nosocomial pathogen, primarily driven by its ability to acquire mobile genetic elements carrying antimicrobial resistance or metabolic traits. Our previous study identified a megaplasmid conferring a high-level gentamicin resistance (HLGR), which can undergo recombination between intact and IS1216V-disrupted aac(6′)-Ie-aph(2″)-Ia genes, resulting in a non-HLGR phenotype. In this study, we further characterized the RepA_N-type megaplasmid in sequence type (ST)17 and ST78 E. faecium and examined its distribution among 465 isolates collected between 2011 and 2020 in Taiwan. Both sublineages carried gene clusters associated with carbohydrate utilization, albeit with differences in repertoires. Loss of the RepA_N megaplasmid in ST17 isolate E37 impaired growth on sucrose, confirming its contribution to carbohydrate metabolism. Screening of clinical isolates showed that repA_N and scrB (sucrose utilization) genes were highly prevalent (90.8% and 85.8%, respectively) in the 10 years, whereas IS1216V-inserted aac(6′)-Ie-aph(2″)-Ia decreased over time (84.6% in 2011 and 55.7% in 2020), paralleling a decline in HLGR rates (57.7% in 2011 and 45.9% in 2020). Among the 465 E. faecium isolates, ST17 and ST78, both belonging to clonal complex 17, were the most dominant sublineages. The expansion of ST78 vancomycin-resistant E. faecium since 2015 corresponded with the emergence of type III IS1216V-inserted aac(6′)-Ie-aph(2″)-Ia, while ST17, mainly carrying type II IS1216V insertion, remained stable throughout the decade. Collectively, these results indicate that the RepA_N-type megaplasmid was widely disseminated and may enhance metabolic adaptability in E. faecium, supporting its persistence in clinical environments. IMPORTANCE Enterococcus faecium is a major hospital-associated pathogen with high genomic plasticity. This study characterized a widespread RepA_N-type megaplasmid, which conferred both high-level gentamicin resistance and enhanced carbohydrate utilization. The co-selection of antimicrobial resistance and metabolic adaptability on a single mobile genetic element may underlie the evolutionary success of dominant clinical lineages such as sequence type (ST)17 and ST78 in Taiwan. These findings provide a framework for understanding how plasmids contribute to the persistence and spread of E. faecium in healthcare settings. Enterococcus faecium is a major hospital-associated pathogen with high genomic plasticity. This study characterized a widespread RepA_N-type megaplasmid, which conferred both high-level gentamicin resistance and enhanced carbohydrate utilization. The co-selection of antimicrobial resistance and metabolic adaptability on a single mobile genetic element may underlie the evolutionary success of dominant clinical lineages such as sequence type (ST)17 and ST78 in Taiwan. These findings provide a framework for understanding how plasmids contribute to the persistence and spread of E. faecium in healthcare settings.

Widespread immune systems protect bacteria against conjugative plasmids

Conjugative plasmids are a class of mobile genetic elements capable of efficient transfer between bacterial cells. Although they can introduce beneficial traits such as antibiotic resistance to recipients, they may also behave as genetic parasites. Bacteria would thus be expected to have evolved barriers to plasmid conjugation. However, the distribution of these barriers and their underlying mechanisms remain poorly understood. Here, we performed a large-scale analysis of 364 diverse strains of the opportunistic pathogen Acinetobacter baumannii as recipients of the broad-host-range conjugative plasmids R388 and RP4. Major variations in host susceptibilities to conjugation, with limited phylogenetic association, suggested multiple and fast-evolving plasmid-specific barriers. Functional genetic analyses revealed a role for core genes, pointing to epistasis or genetic background effects. This is illustrated by the previously unrecognized role of H-NS expression in alleviating conjugation barriers in a strain-dependent manner. Most importantly, we identified three novel immune systems protecting bacteria against conjugation by R388 and RP4. Their patchy distribution within the species, and that of their homologs across bacteria, indicate that they are part of a dynamic repertoire of immune systems against conjugation. While the Ishtar system promotes plasmid loss through putative HEPN nuclease domains, Namtar and Attar sense distinct components of the R388 type IV secretion system (T4SS) to trigger a non-proliferative, energetically depleted state, analogously to the abortive infection response of anti-phage defenses. Live imaging of conjugation showed Namtar halting cell division in Escherichia coli recipients, conferring population-level immunity against plasmid spread via horizontal and vertical transmission. The existence of immune systems specifically targeting T4SS components suggests that conjugative plasmids impose a selective disadvantage greater than previously thought. This work reveals an additional layer of bacterial immunity directed at a class of genetic elements driving dissemination of antibiotic resistance.

Specific exclusion of conjugative plasmids from the gut microflora