Microevolutionary Events Involving Narrow Host Plasmids Influences Local Fixation of Vancomycin-Resistance in Enterococcus Populations

Abstract

Vancomycin-resistance in enterococci (VRE) is associated with isolates within ST18, ST17, ST78 Enterococcus faecium (Efm) and ST6 Enterococcus faecalis (Efs) human adapted lineages. Despite of its global spread, vancomycin resistance rates in enterococcal populations greatly vary temporally and geographically. Portugal is one of the European countries where Tn1546 (vanA) is consistently found in a variety of environments. A comprehensive multi-hierarchical analysis of VRE isolates (75 Efm and 29 Efs) from Portuguese hospitals and aquatic surroundings (1996–2008) was performed to clarify the local dynamics of VRE. Clonal relatedness was established by PFGE and MLST while plasmid characterization comprised the analysis of known relaxases, rep initiator proteins and toxin-antitoxin systems (TA) by PCR-based typing schemes, RFLP comparison, hybridization and sequencing. Tn1546 variants were characterized by PCR overlapping/sequencing. Intra- and inter-hospital dissemination of Efm ST18, ST132 and ST280 and Efs ST6 clones, carrying rolling-circle (pEFNP1/pRI1) and theta-replicating (pCIZ2-like, Inc18, pHTβ-like, two pRUM-variants, pLG1-like, and pheromone-responsive) plasmids was documented. Tn1546 variants, mostly containing ISEf1 or IS1216, were located on plasmids (30–150 kb) with a high degree of mosaicism and heterogeneous RFLP patterns that seem to have resulted from the interplay between broad host Inc18 plasmids (pIP501, pRE25, pEF1), and narrow host RepA_N plasmids (pRUM, pAD1-like). TAs of Inc18 (ω-ε-ζ) and pRUM (Axe-Txe) plasmids were infrequently detected. Some plasmid chimeras were persistently recovered over years from different clonal lineages. This work represents the first multi-hierarchical analysis of VRE, revealing a frequent recombinatorial diversification of a limited number of interacting clonal backgrounds, plasmids and transposons at local scale. These interactions provide a continuous process of parapatric clonalization driving a full exploration of the local adaptive landscape, which might assure long-term maintenance of resistant clones and eventually fixation of Tn1546 in particular geographic areas.

The Plasmidome of Firmicutes: Impact on the Emergence and the Spread of Resistance to Antimicrobials

“…According to this typing system, RIP variants are designated as "rep" followed by a subindex number and are arbitrarily called Rep families. Although this system is very useful to enlarge the knowledge of scarcely explored plasmid diversity in contemporary isolates of enterococci and staphylococci, its application is limited to known plasmids, mainly AbR plasmids of these genera, as illustrated in various surveys and this study (59,66,68,71).…”

“…Such plasmids have been reported in S. aureus since their first detection in the early 1950s (84) and were eventually described in contemporary E. faecalis isolates (68,85). A second branch contains pRI1-like plasmids (rep 14 ), which correspond to plasmids of different enterococcal species (E. faecium, Enterococcus hirae, Enterococcus mundtii) isolated from foodborne animals and hospital patients (7,59,71,86). These plasmids can be mobilized by other AbR conjugative theta replicating plasmids present in the same cell (71,87), and it seems they are widely spread among enterococcal populations.…”

“…Because many open reading frames of pHT and pMG1 plasmids do not show significant homology with any reported proteins, they used to be categorized as a new type of highly efficient conjugative plasmids with a MOB P family REL. This plasmid group is represented by relatives of pHT (pHTα, pHTβ, and pHTγ) and pMG1, which have greatly facilitated the dissemination of resistance to glycopeptides (Tn1546_vanA) and high-level resistance to aminoglycosides (Tn4001-like elements) among human E. faecium and E. avium isolates from the United States and Japan (123,124) and, to a lesser extent, European countries (7,70,71,122).…”

“…Toxin-antitoxin systems associated with other plasmid families such as ες and relBE have been detected on members of this plasmid group, reflecting rearrangements with representatives of other plasmid families (7). Even though to date, only a few members of pheromone-responsive plasmids have been fully sequenced, typing surveys reveal a wide diversity of plasmids among populations, often containing RIPs, RELs, or regions from plasmids of different origins (68,71).…”

“…5 and 7), some plasmid chimeras being fixed and persistently recovered for years. For example, emblematic mosaics theta/RCR plasmids of staphylococci (e.g., cointegrates of RepA_N/pSK41 and Rep_1/ pUB110, which encode resistance to gentamicin) and E. faecalis (e.g., pAMα1) have both been selected in those lineages since the early 1970s (7,71,84).…”