Molecular characterization of a widespread, pathogenic, and antibiotic resistance-receptive Enterococcus faecalis lineage and dissemination of its putative pathogenicity island - PubMed (original) (raw)

Molecular characterization of a widespread, pathogenic, and antibiotic resistance-receptive Enterococcus faecalis lineage and dissemination of its putative pathogenicity island

Sreedhar R Nallapareddy et al. J Bacteriol. 2005 Aug.

Abstract

Enterococcus faecalis, a common cause of endocarditis and known for its capacity to transfer antibiotic resistance to other pathogens, has recently emerged as an important, multidrug-resistant nosocomial pathogen. However, knowledge of its lineages and the potential of particular clones of this species to disseminate and cause disease is limited. Using a nine-gene multilocus sequence typing (MLST) scheme, we identified an evolving and widespread clonal complex of E. faecalis that has caused outbreaks and life-threatening infections. Moreover, this unusual clonal complex was found to contain isolates of unexpected relatedness, including the first known U.S. vancomycin-resistant enterococcus (E. faecalis strain V583), the first known penicillinase-producing (Bla(+)) E. faecalis isolate, and the previously described widespread clone of penicillinase producers, a trait found in <0.1% of E. faecalis isolates. All members of this clonal cluster (designated as BVE for Bla(+) Van(r) endocarditis) were found to contain a previously described putative pathogenicity island (PAI). Further analysis of this PAI demonstrated its dissemination worldwide, albeit with considerable variability, confirmed its association with clinical isolates, and found a common insertion site in different clonal lineages. PAI deletions, MLST, and the uncommon resistances were used to predict the evolution of the BVE clonal cluster. The finding of a virulent and highly successful clonal complex of E. faecalis with different members resistant to the primary therapies of choice, ampicillin and vancomycin, has important implications for the evolution of virulence and successful lineages and for public health monitoring and control.

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Figures

FIG. 1.

Chromosomal locations of the nine MLST loci in the E. faecalis V583 genome (28). The arrowheads inside the circle represent the open reading frame orientations of each locus (not drawn to scale). Antigenic gene arrowheads are shaded in black, and housekeeping gene arrowheads are shaded in gray. The arrowhead of dnaA (coding for chromosomal replication factor) positioned at nucleotide 1 is not shaded. The distance between any two loci ranged from 103 kb to 1,247 kb. The putative PAI region is boxed.

FIG. 2.

Allelic variation of the MLST loci sequenced for analysis. Allelic variations of ace, efaA, and salA were as previously published (23) and hence are not shown here. For these three genes, alleles A to I of the previous study (23) were redefined as alleles 1 to 9 for this study. The nucleotides present in each of the variable sites of allele 1 (E. faecalis OG1RF) are shown. Only those sites that differ are shown for the other alleles. The position of each variable site within the sequenced fragment is shown by the number above the nucleotide, read vertically.

FIG. 3.

Analysis of an unusual E. faecalis clonal cluster by MLST and PFGE. (A) Clustering of four clonally related STs using BURST analysis. The central circle denotes the predominant type among isolates tested using a nine-gene MLST scheme, and each surrounding circle indicates one allele difference. A dashed straight line denotes a double locus difference. (B) PFGE fingerprints. The PFGE phylogenetic tree was based on the unweighted pair group method. Tolerance of up to 5% shift in the band position was used. Isolates are generally referred to by their previously published designations. Year and place of isolation and ST type are shown. Isolates from outbreaks are marked with an asterisk. V583 is the first vancomycin-resistant enterococcus isolated in the United States, and HH-22 is the first-known Bla+ isolate of E. faecalis. E228 and E366, isolated a year apart from the same hospital and differing by three bands, are represented by E228.

FIG. 4.

Schematic presentation of hybridization profiles for potential virulence-related genes and PAI genes. (A) Virulence-related gene (non-PAI) profile and ace B repeat number profile of 21 MLST-analyzed isolates. The 10 genes (of ef0080, ef0089, ef0786, ef1091, ef1092, ef1093, ef1269, ef1818, ef2224, and ef3191) present in all 21 isolates are not shown. The STs with the 23.9-kb deletion involving fsrB are marked with a superscript “a” in the fsrB data. (B) Determination of the PAI insertion site by PCR using primers within and outside the PAI of V583. The double arrow denotes an expected-size PCR fragment with primers PAIout plus ef0481forward or ef0482forward. STs yielding an ∼1.7-kb-larger PCR product or an ∼1-kb-larger PCR product are marked with superscript “b” and “c,” respectively. (C) Hybridization results with PAI-associated intragenic probes, representing 18 genes dispersed over the entire PAI region. The superscript numbers on ST and the +/− symbols denote the number of isolates of that type. The integrated plasmid region (ef0506 to ef0485) (28) is boxed. (D) Distribution of three PAI-associated genes among 341 clinical isolates, 58 nosocomial stool isolates, 33 community-derived stool isolates, and 22 animal isolates.

FIG. 5.

Predicted path of evolution of the BVE clonal complex based on MLST, deletions in the PAI region, year of isolation, and the presence of uncommon resistance genes, blaZ and vanB. Isolates of the BVE clonal complex contained all 14 tested potential virulence genes (Fig. 4). Isolates of each ST within the clonal complex are grouped by oval shading, and SLVs are denoted by overlapping shaded ovals. Arrows with continuous and dashed lines represent the predicted and alternative evolutionary paths. Putative ancestral isolates are boxed with a dashed line. Five partial deletions (ΔΑ to ΔΕ) of the PAI of E. faecalis strain MMH594, including that previously described for V583 (34), are shown. VanB, vancomycin resistance encoded by the vanB gene.

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Molecular characterization of a widespread, pathogenic, and antibiotic resistance-receptive Enterococcus faecalis lineage and dissemination of its putative pathogenicity island - PubMed (original) (raw)