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Background <p>The epidemiology of vancomycin-resistant <i>Enterococcus faecium</i> (VREfm) varies across different countries, with a steady global increase. In Portugal, however, epidemiological data on clinical VREfm have been scarce since the early 2000s. This long-term study investigates VREfm isolates from human infections collected at a Porto hospital between 2010 and 2021.</p> Methods <p>Two hundred VREfm isolates, mostly urinary (39%) were characterized by antimicrobial susceptibility testing to 8 antibiotics, chlorhexidine susceptibility, and PCR-based detection of vancomycin-resistance genes, virulence markers, plasmid replicases, and the <i>bac43</i>/T8 gene. Whole-genome sequencing, by Illumina, was used to assess clonal diversity (MLST, cgMLST, SNP phylogeny) and genomic content of antimicrobial resistance (AMR) genes, bacteriocins (76 genes) and putative virulence markers (35 genes). The plasmidome size and replicase initiation proteins of selected isolates was further improved by incorporating nanopore sequencing which enabled hybrid assemblies.</p> Results <p>All isolates were multidrug resistant; 98% carried <i>vanA</i>, while two (1%) were resistant to linezolid (G2576T mutation). Chlorhexidine susceptibility remained stably low over time (MICs: 2–4&#xa0;mg/L). The population was polyclonal, with a shift from ST18-like lineages to ST80 and ST117 dominance. While ARGs and virulence markers showed no clear association with clonal waves, bacteriocin profiles did, with <i>bac</i>43 becoming increasingly prevalent. ST117-CT24 emerged as the most persistent clone. The plasmidome comprised stable Rep3-like mobilizable plasmids carrying bacteriocins (<i>bac</i>43, <i>bac</i>AS5), RepA_N mega-plasmids harboring virulence/AMR/bacteriocins, and highly plastic medium-to-large <i>vanA</i> plasmids with diverse replicase initiation proteins. Strikingly, linear <i>vanA</i> plasmids (repUS78_pZY2) appeared in the most recent isolates, paralleling findings in vancomycin-variable <i>E. faecium</i> from the same hospital and VREfm from other countries.</p> Conclusions <p>Our findings reveal dynamic clonal shifts, novel plasmid architectures, and the key role of bacteriocins in shaping clonal success in a WHO priority pathogen. Furthermore, we highlight the need for AMR surveillance frameworks to consider factors beyond conventional prevalence metrics and core-genome comparisons. Integrating intra-species genomic heterogeneity and non-traditional evolutionary indicators will be essential to more accurately predict, track, and ultimately mitigate the dissemination of multidrug-resistant human pathogens.</p>

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Twelve years of genomic surveillance of vancomycin-resistant Enterococcus faecium: emergence of linear vanA and bacteriocin-carrying plasmids challenging infection control

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月5日

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  • Ana C. Almeida-Santos,
  • Ana P. Tedim,
  • Bárbara Duarte,
  • Ana P. Pereira,
  • Luís M. Silva,
  • Júlio Teixeira,
  • Ana P. Castro,
  • Louise Roer,
  • Anette M. Hammerum,
  • Henrik Hasman,
  • Teresa M. Coque,
  • Carla Novais,
  • Ana R. Freitas,
  • Luísa Peixe

摘要

Background

The epidemiology of vancomycin-resistant Enterococcus faecium (VREfm) varies across different countries, with a steady global increase. In Portugal, however, epidemiological data on clinical VREfm have been scarce since the early 2000s. This long-term study investigates VREfm isolates from human infections collected at a Porto hospital between 2010 and 2021.

Methods

Two hundred VREfm isolates, mostly urinary (39%) were characterized by antimicrobial susceptibility testing to 8 antibiotics, chlorhexidine susceptibility, and PCR-based detection of vancomycin-resistance genes, virulence markers, plasmid replicases, and the bac43/T8 gene. Whole-genome sequencing, by Illumina, was used to assess clonal diversity (MLST, cgMLST, SNP phylogeny) and genomic content of antimicrobial resistance (AMR) genes, bacteriocins (76 genes) and putative virulence markers (35 genes). The plasmidome size and replicase initiation proteins of selected isolates was further improved by incorporating nanopore sequencing which enabled hybrid assemblies.

Results

All isolates were multidrug resistant; 98% carried vanA, while two (1%) were resistant to linezolid (G2576T mutation). Chlorhexidine susceptibility remained stably low over time (MICs: 2–4 mg/L). The population was polyclonal, with a shift from ST18-like lineages to ST80 and ST117 dominance. While ARGs and virulence markers showed no clear association with clonal waves, bacteriocin profiles did, with bac43 becoming increasingly prevalent. ST117-CT24 emerged as the most persistent clone. The plasmidome comprised stable Rep3-like mobilizable plasmids carrying bacteriocins (bac43, bacAS5), RepA_N mega-plasmids harboring virulence/AMR/bacteriocins, and highly plastic medium-to-large vanA plasmids with diverse replicase initiation proteins. Strikingly, linear vanA plasmids (repUS78_pZY2) appeared in the most recent isolates, paralleling findings in vancomycin-variable E. faecium from the same hospital and VREfm from other countries.

Conclusions

Our findings reveal dynamic clonal shifts, novel plasmid architectures, and the key role of bacteriocins in shaping clonal success in a WHO priority pathogen. Furthermore, we highlight the need for AMR surveillance frameworks to consider factors beyond conventional prevalence metrics and core-genome comparisons. Integrating intra-species genomic heterogeneity and non-traditional evolutionary indicators will be essential to more accurately predict, track, and ultimately mitigate the dissemination of multidrug-resistant human pathogens.