Insertion Sequence–Driven Diversification Creates a Globally Dispersed Emerging Multiresistant Subspecies of E. faecium

Abstract

Enterococcus faecium, an ubiquous colonizer of humans and animals, has evolved in the last 15 years from an avirulent commensal to the third most frequently isolated nosocomial pathogen among intensive care unit patients in the United States. E. faecium combines multidrug resistance with the potential of horizontal resistance gene transfer to even more pathogenic bacteria. Little is known about the evolution and virulence of E. faecium, and genomic studies are hampered by the absence of a completely annotated genome sequence. To further unravel its evolution, we used a mixed whole-genome microarray and hybridized 97 E. faecium isolates from different backgrounds (hospital outbreaks (n = 18), documented infections (n = 34) and asymptomatic carriage of hospitalized patients (n = 15), and healthy persons (n = 15) and animals (n = 21)). Supported by Bayesian posterior probabilities (PP = 1.0), a specific clade containing all outbreak-associated strains and 63% of clinical isolates was identified. Sequencing of 146 of 437 clade-specific inserts revealed mobile elements (n = 74), including insertion sequence (IS) elements (n = 42), phage genes (n = 6) and plasmid sequences (n = 26), hypothetical (n = 58) and membrane proteins (n = 10), and antibiotic resistance (n = 9) and regulatory genes (n = 11), mainly located on two contigs of the unfinished E. faecium DO genome. Split decomposition analysis, varying guanine cytosine content, and aberrant codon adaptation indices all supported acquisition of these genes through horizontal gene transfer with IS16 as the predicted most prominent insert (98% sensitive, 100% specific). These findings suggest that acquisition of IS elements has facilitated niche adaptation of a distinct E. faecium subpopulation by increasing its genome plasticity. Increased genome plasticity was supported by higher diversity indices (ratio of average genetic similarities of pulsed-field gel electrophoresis and multi locus sequence typing) for clade-specific isolates. Interestingly, the previously described multi locus sequence typing–based clonal complex 17 largely overlapped with this clade. The present data imply that the global emergence of E. faecium, as observed since 1990, represents the evolution of a subspecies with a presumably better adaptation than other E. faecium isolates to the constraints of a hospital environment.

Identification of resistance and virulence factors in an epidemic Enterobacter hormaechei outbreak strain

“…Labelling, hybridization and data acquisition were performed as previously described (Leavis et al, 2007;Paauw et al, 2008). A Bioprime system (Invitrogen) was used to label 0.5 mg DNA.…”

“…Inferior spots (empty or less than twice the Cy3 background) were excluded from normalization and data analysis (Leavis et al, 2007;Paauw et al, 2008). Each slide was independently normalized to correct for individual differences.…”

“…In our case, the graded output generated a range of values from 20.5 to 0.5 in increments of 0.05, with 20.5 corresponding to 5 % or less EPP and 0.5 corresponding to 95 % or more EPP. The E. cloacae complex core genome was defined as the set of genomic DNA fragments present in all isolates with a likelihood of .95 % (GACK data .20.5) (Leavis et al, 2007;Paauw et al, 2008).…”

“…In our case, the graded output generated a range of values from 20.5 to 0.5 in increments of 0.05, with 20.5 corresponding to 5 % or less EPP and 0.5 corresponding to 95 % or more EPP. The E. cloacae complex core genome was defined as the set of genomic DNA fragments present in all isolates with a likelihood of .95 % (GACK data .20.5) (Leavis et al, 2007;Paauw et al, 2008).For statistical analysis, the data were transformed to binary output, where 0 corresponded to 5 % or less EPP, 1 to 95 % or more EPP, and the values between 5 % and 95 % were discarded.Determination of R-plasmid pQC-related DNA fragments.Because the approximately 300 kb plasmid pQC appears to play an important role in the outbreaks, it was determined which DNA fragments were derived from pQC as previously described (Paauw et al, 2008). Subsequently, the pQC-derived DNA fragments were sequenced from both sides.…”

“…1) (Paauw et al, 2008). Associated DNA fragments were identified using paired Student's t-test (P,0.001) followed by false discovery rate (FDR) correction (Benjamini et al, 2001;Leavis et al, 2007). The sequences of the top 100 most strongly associated DNA fragments were sequenced from both sides.…”