Genetic diversity among Enterococcus faecalis

Abstract

Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes.

Figure 1. Dendrogram created from serological typing strains using the E. faecalis MLST database efaecalis.mlst.net.

Multi-locus sequence typing (MLST) of E. faecalis isolates was based on sequences of internal gene fragments for 7 housekeeping genes. Each gene variation (for each of the seven genes) is assigned a unique allele number. The combination of the 7 allele numbers (allelic profile) for each strain defines the multi-locus sequence type, or ST. The relatedness of isolates based on sequence type is shown as an unrooted cladogram, determined by (UPGMA) analysis of the allelic profiles. Boxed isolates represent the most common serotypes found in human populations in previous studies , .

Figure 2. Dendogram of isolates aligned with capsule type, pathogenicity island segments, and antibiotic resistance traits.

MLST-based dendrogram showing genetic relationship of all E. faecalis isolates in this study. Small yellow highlights indicate a serotyping type strain, while black boxes designate the five most common serotypes . Arrows designate isolates used for comparative genomic microarray analysis. Abbreviations are defined as follows: ST = sequence type; CPS = capsule type; PAI = pathogenicity island fragment outlined as letter designations in Fig. S1 (A = nuc1; B = cylB; C = esp; D = hydrolase homolog similar to xylS; E = psaA homolog; F = gls-24 like); A red letter B indicates strains that readily transfer cytolysin via mating; Ab^R = antibiotic resistance; T = tetracycline resistance; TM = tetM+; TL = tetL+; E = ermB+; VA = vanA+; VB = vanB+; G = gentamicin resistant; C = cat+; A = blaZ+; CBH = bile salt hydrolase; GEL = gelatinase; CYL = cytolysin. More detailed strain information is listed in Table 1.

Figure 3. Dendrogram and composite virulence determinants among sequence types.

MLST-based dendrogram compiling data from isolates of the 51 identified sequence type lineages. PAI = number pathogenicity island genes present per lineage; Ab^R = number of antibiotic resistance determinants per lineage. Brackets encompass abundant clonal isolates and their single and double locus variants where applicable.

Figure 4. Virulence-associated phenotypes and corresponding genotype for all isolates.

Phenotypes were determined by microdilution assay for antibiotic resistance or enzyme-specific tests for auxiliary enzymatic traits. Genotypes were determined by PCR amplification and/or hybridization for genes known to encode each phenotype. A positive genotype is indicated by the presence of one or more genes known to produce a given phenotype (e.g. genotypically positive vancomycin strains may contain the vanA or vanB genes—a strain containing both would only be counted once).

Figure 5. Visualization of final absent and present calls for all probe sets across 8 distinct E. faecalis isolates.

Comparative genomic hybridization was performed on DNA from isolates V583, MMH594, JH1, HIP11704, D6, ARO1/DG, Com6, and Fly1 as described in materials and methods. Probe sets are ordered according to the E. faecalis V583 gene sequence and the Affymetrix library file. Absent probe sets are in white; present probe sets are in black. Clustering of the strains was based on complete linkage using the Pearson correlation coefficient for the Absent/Present calls (A = 0, P = 1). The same clustering pattern is generated when average signal intensity values are used instead of Absent/Present calls (not shown). The numbered regions on the left correspond to the following previously identified and unidentified mobile genetic regions from strain V583: 1) EF_0125-EF_0166, 2) EF_0302-EF_0355 (PHAGE01), 3) EF_0479-EF_0628 (V583 PAI), 4) EF_1275-EF_1293 (PHAGE02), 5) EF_1416-EF_1489 (PHAGE03), 6) EF_1847-EF_1897, 7) EF_1987-EF_2043 (PHAGE04), 8) EF_2084-EF_2145 (PHAGE05), 9) EF_2240-2282/EF_2335-2351, 10) EF_2284-EF_2334 (Tn/vanB), 11) EF_2512-EF_2545, 12) EF_2798-EF_2856 (PHAGE06), and 13) EF_2936-EF_2955 (PHAGE07) (see text).

Figure 6. Classification of core and dispensable sequences in 8 strains of Enterococcus faecalis.

Data from comparative genomic hybridization analysis of isolates V583, MMH594, JH1, HIP11704, D6, ARO1/DG, Com6, and Fly1 are organized by the number of strains for which each probe set is detected (top to bottom). Absent probe sets are in white; present probe sets are in black. Clustering of the strains was based on complete linkage using the Pearson correlation coefficient for the Absent/Present calls (A = 0, P = 1). Genetic elements were classified as part of the core genome if present in all strains tested. COG designations were obtained from the National Center for Biotechnology Information (NCBI). Some genes/probe sets represent more than one COG category.

Figure 7. Antibiotic determinants and pathogenicity island composition per sequence type lineage, aligned by capsule locus (cps) variation (CPS type).

The percentage of sequence type lineages positive for each antibiotic resistance and pathogenicity island listed in Fig 3 were alligned by CPS capsule type and arranged in descending order. Capsule types are invariant for strains within individual sequence type lineages. Details of capsule operon (cps) arrangements are outlined in Materials and Methods.