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Emergence of Epidemic Multidrug-Resistant Enterococcus faecium from Animal and Commensal Strains

Emergence of Epidemic Multidrug-Resistant Enterococcus faecium from Animal and Commensal Strains RESEARCH ARTICLE Emergence of Epidemic Multidrug-Resistant Enterococcus faecium from Animal and Commensal Strains a,b a,b,c b b b b François Lebreton, Willem van Schaik, Abigail Manson McGuire, Paul Godfrey, Allison Griggs, Varun Mazumdar, d d b b b b b c Jukka Corander, Lu Cheng, Sakina Saif, Sarah Young, Qiandong Zeng, Jennifer Wortman, Bruce Birren, Rob J. L. Willems, b a,b Ashlee M. Earl, Michael S. Gilmore Departments of Ophthalmology, Microbiology and Immunobiology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA ; The b c Broad Institute, Cambridge, Massachusetts, USA ; Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands ; Department of Mathematics and Statistics, University of Helsinki, Helsinki, Finland F.L., W.V.S., and A.M.M. contributed equally to this article. ABSTRACT Enterococcus faecium, natively a gut commensal organism, emerged as a leading cause of multidrug-resistant hospital-acquired infection in the 1980s. As the living record of its adaptation to changes in habitat, we sequenced the genomes of 51 strains, isolated from various ecological environments, to understand how E. faecium emerged as a leading hospital patho- gen. Because of the scale and diversity of the sampled strains, we were able to resolve the lineage responsible for epidemic, multidrug-resistant human infection from other strains and to measure the evolutionary distances between groups. We found that the epidemic hospital-adapted lineage is rapidly evolving and emerged approximately 75 years ago, concomitant with the introduction of antibiotics, from a population that included the majority of animal strains, and not from human commensal lines. We further found that the lineage that included most strains of animal origin diverged from the main human commensal line approximately 3,000 years ago, a time that corresponds to increasing urbanization of humans, development of hygienic practices, and domestication of animals, which we speculate contributed to their ecological separation. Each bifurcation was accompanied by the acquisition of new metabolic capabilities and colonization traits on mobile elements and the loss of function and genome remodeling associated with mobile element insertion and movement. As a result, diversity within the species, in terms of sequence divergence as well as gene content, spans a range usually associated with speciation. IMPORTANCE Enterococci, in particular vancomycin-resistant Enterococcus faecium, recently emerged as a leading cause of hospital-acquired infection worldwide. In this study, we examined genome sequence data to understand the bacterial adapta- tions that accompanied this transformation from microbes that existed for eons as members of host microbiota. We observed changes in the genomes that paralleled changes in human behavior. An initial bifurcation within the species appears to have oc- curred at a time that corresponds to the urbanization of humans and domestication of animals, and a more recent bifurcation parallels the introduction of antibiotics in medicine and agriculture. In response to the opportunity to fill niches associated with changes in human activity, a rapidly evolving lineage emerged, a lineage responsible for the vast majority of multidrug-resistant E. faecium infections. Received 17 July 2013 Accepted 23 July 2013 Published 20 August 2013 Citation Lebreton F, van Schaik W, Manson McGuire A, Godfrey P, Griggs A, Mazumdar V, Corander J, Cheng L, Saif S, Young S, Zeng Q, Wortman J, Birren B, Willems RJL, Earl AM, Gilmore MS. 2013. Emergence of epidemic multidrug-resistant Enterococcus faecium from animal and commensal strains. mBio 4(4):e00534-13. doi:10.1128/mBio.00534-13. Editor Larry McDaniel, University of Mississippi Medical Center Copyright © 2013 Lebreton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Michael S. Gilmore, michael_gilmore@meei.harvard.edu. ntibiotic resistance is a leading threat to human health world- cocci have begun to transmit vancomycin resistance to Awide that substantially increases the cost of health care (1). methicillin-resistant Staphylococcus aureus (12). Enterococci emerged in the 1970s and 1980s as leading causes of Previously, we examined a limited sampling of human com- antibiotic-resistant infection of the bloodstream, urinary tract, mensal and hospital isolates of E. faecium and found that by aver- and surgical wounds (1), contributing to 10,000 to 25,000 deaths age nucleotide identity analysis (ANI), some differed by more per year in the USA (2). Resistance to antibiotics is common than 5%, crossing the threshold used for species identity (13). among enterococci (1), and vancomycin-resistant Enterococ- Since variation was noted among hospital strains (13–16) and cus faecium now represents up to 80% of E. faecium isolates in since little was known about strains from the gastrointestinal (GI) some hospitals (3). Agricultural practices have promoted the tracts of domestic and other animals, it was of interest to deter- emergence of antibiotic resistance (4–6). The use of avoparcin in mine the scope of diversity within the species and to precisely animal feed in Europe and elsewhere appears to have contributed define these populations and their origins. We therefore charac- to the proliferation of vancomycin resistance (7–11), and entero- terized the breadth of the species by sequencing and comparing July/August 2013 Volume 4 Issue 4 e00534-13 mbio.asm.org 1 Lebreton et al. FIG 1 goeBURST analysis of 2,273 E. faecium entries in the E. faecium MLST database (http://efaecium.mlst.net), which can be grouped into 773 sequence types (STs) (brown circles), based upon MLST relatedness. STs included in this study are highlighted in purple. the genomes of 51 strains, sampling all areas of the existing mul- household pets (19). Two strains (EnGen0002 and 1_231_408) tilocus sequence type (MLST) phylogeny (Fig. 1). possess hybrid genomes, consisting of a background genome of clade A1, into which 195 kb to 740 kb DNA from a clade B donor RESULTS have recombined (Fig. S4). To understand the forces that gave rise to the observed clade Phylogenomic reconstruction of E. faecium divergence. We de- structure in the context of human activity, we estimated the time termined the nucleotide sequences of the genomes of 51 E. faecium at which these bifurcations occurred, using Bayesian evolutionary strains of different MLST types (see Table S1 in the supplemental analysis on sampled phylogenetic trees (BEAST) (20). To limit the material), which were obtained from diverse ecological environ- confounding effect of recombination, detectable signatures of re- ments (see Fig. S1 in the supplemental material) on five conti- combination were removed from the analysis using BRATNext- nents, and isolated over the last 60 years (Fig. S1). A single nucle- Gen (21). Concerned that differing stresses in different habitats otide polymorphism (SNP)-based phylogenetic tree, which could affect mutation rate, we calculated inferred rates of muta- compared these strains to each other and to an additional 22 tion for each clade separately. A significantly higher mutation rate strains from GenBank (Table S1), was generated based on varia- was found for strains in the hospital-adapted clade A1 (4.9 10 tion in 1,344 shared single-copy orthologous groups (ortho- groups) (Fig. 2). This tree confirmed the deep divide between  0.3 10 substitutions per nucleotide per year) than for sister 6 6 clades (clades A and B) (13, 16). Most (5/7) strains isolated from clade A2 (3.6  10  0.6  10 substitutions per nucleotide the feces of nonhospitalized humans cluster in clade B. We were per year). The mutation rate for clade B was intermediate at 1.3 5 5 able to resolve the epidemic hospital strains (clade A1) from a 10  0.2  10 substitutions per nucleotide per year, a rate mixed group of animal strains and sporadic human infection iso- that is similar to those recently reported for Staphylococcus aureus lates (clade A2). This clade structure was independently recapitu- (22, 23). lated based on cluster analysis of (i) shared gene content (Fig. S2) To determine whether the calculated mutation rate differences and (ii) gene synteny (Fig. S3). reflected historic events or whether they are still experimentally Clade A1 strains account for the vast majority of human infec- detectable, the rate of mutation to fosfomycin resistance was mea- tion (Fig. 2) and include sequence types (STs) from the clonal sured for 10 randomly selected strains from each clade. Resistance complex 17 (CC17) genogroup (e.g., sequence type 17 [ST17], was verified for stability by passage in the absence of selection, ST117, and ST78 [18]) associated with hospital ward outbreaks followed by retesting. Clade A1 strains yielded spontaneous around the globe (see Table S1 in the supplemental material). fosfomycin-resistant variants at a rate about an order of magni- Interestingly, the three clade A1 strains of animal origin are from tude higher than strains of either clade A2 or clade B (Fig. 3), pet dogs, consistent with known links between hospital strains and paralleling the results of BEAST analysis. Therefore, mutation 2 mbio.asm.org July/August 2013 Volume 4 Issue 4 e00534-13 Emergence of Multidrug-Resistant Enterococcus faecium FIG 2 RAxML SNP-based tree based on the concatenated alignments of DNA sequences of 1,344 single-copy core genes in 73 E. faecium genomes. Bootstrap- ping was performed with 1,000 replicates. The origins of the strains are indicated. The dates for the split between the clades, estimated by a BEAST analysis, are indicated (ya, years ago). The infectivity score reflects the number of strains of a particular ST, in the MLST database, isolated from infection. The clades are color coded as follows: clade B in dark blue, clade A1 in red, and clade A2 in gray. rates for each clade inferred by BEAST were used to estimate the have significantly larger overall average genome size (2,843 159 time of divergence between clades A1, A2, and B. This placed the genes; 2.98  0.15 Mb) than strains of either clade A2 (2,597 time of the initial split between clade A and clade B at 2,776  153 genes; 2.75 0.14 Mb) or clade B (2,718 120 genes; 2.84 818 years ago and that between clade A1 and clade A2 at 74  0.1 Mb) (Fig. 4A), indicating that perpetuating cycles of infection 30 years ago (Fig. 2). and survival in the hospital are associated with acquisition of new Gene content differences. Gene gain and loss make funda- functions. Clade A1 strains also have larger core genomes (1,945 mental contributions to new habitat adaptation and the emer- genes) than strains of clade A2 (1,724 genes) or clade B (1,805 gence of new lineages (24). Strains from clade A1 were found to genes), which is consistent with a very recent emergence of this July/August 2013 Volume 4 Issue 4 e00534-13 mbio.asm.org 3 Lebreton et al. lineage (i.e., little time for divergence between strains to occur) (Fig. 4C). In contrast, the pan-genome of clade A2 is larger (6,343 genes) than those of clade A1 and B (5,663 and 5,551 genes, re- spectively) (Fig. 4B), which is consistent with the diverse origins of strains from this clade. In comparison to other opportunists, the E. faecium genome is relatively open (see Fig. S5 in the supplemen- tal material). Previously, the genomes of hospital strains of the sister species, Enterococcus faecalis, were found to differ from commensal organ- isms largely as the result of mobile element acquisition (13), which was associated with the absence of CRISPR (clustered regularly interspaced short palindromic repeat) protection (25). It was, therefore, of interest to determine the extent to which mobile elements drove the divergence of E. faecium clades. Mobile ele- ments were identified using PHAST (26) for phages, SIGI-HMM (27) for genomic islands, and BLAST for repA orthologs in plasmid-related contigs (28). Clade A1 was found to be enriched in mobile elements, including plasmids (5.4  1.9 plasmids/ge- nome in clade A1, compared to 2.7 2.2 and 1.5 1.1 plasmids/ genome in clade A2 and B strains, respectively), integrated phages (1.6  0.9 phages/genome, compared to 0.7  0.7 and 0.9  0.8 phages/genome in clade A2 and B strains, respectively) and other FIG 3 Frequency of fosfomycin resistance was determined in triplicate for 10 randomly selected strains from each E. faecium clade (clade A1 [red], A2 genomic islands (36  26 kb of island-associated sequence/ge- [gray], and B [dark blue]). Each symbol represents the average value for one nome, compared to 14  10 and 17  11 kb of island-associated strain, and the clade average  standard deviation (error bars) for the 10 sequence/genome in clade A2 and B strains, respectively) strains per clade are indicated. (Fig. 4D). Because the genome sequences generated in the present study were of high quality, yielding a small number of scaffolds FIG 4 (A) Genome size comparison for E. faecium clade A1 (red), A2 (gray), and B (dark blue). (B and C) Pan-genome (B) and core genome (C) are shown for increasing values of the number of sequenced E. faecium genomes within each clade. Circles represent the number of new or core genes present when a particular genome is added to each subset. Black bars represent median values. The curve for the estimation of the size of the E. faecium pan-genome for each clade is a least-squares power law fit through medians. The size of the core genome within each clade was estimated by fitting an exponential curve through medians. (D) Heat map showing the enrichment in genetic mobile elements in E. faecium genomes within each clade (clade A1 [red], A2, [gray], and B [light blue]). Horizontal boxes represent strains, which are ordered within clades as in Fig. 2 (rotated 90°). The aggregate length (kb) of islands was used to compare content in each clade (ranging from 4 kb to 99 kb; median, 17 kb), whereas the numbers of putative plasmids (ranging from 0 to 9; median, 3) or phage elements (ranging from 0 to 4; median, 1) are represented. The heat map reflects the 10th percentile (light gray), 50th percentile (medium gray), and 90th percentile (black). The “” symbol in a box indicates genome sequence for which the length of genomic islands could not be determined using the SIGI-HMM algorithm (27). 4 mbio.asm.org July/August 2013 Volume 4 Issue 4 e00534-13 Emergence of Multidrug-Resistant Enterococcus faecium FIG 5 Summary of clade-specific antibiotic resistance genes, insertion sequences (IS), and select defenses against horizontal gene transfer. Each box represents a strain, arranged by clade as shown in Fig. 2. The “” symbol in a box indicates genome sequence with an assembly quality that precluded identification of the indicated feature. An asterisk in a box indicates hybrid genomes that contain CRISPR-cas on recombined fragments. CRISPR and type IV restriction- modification (RM) systems are included in the miscellaneous (Misc.) category. (see https://olive.broadinstitute.org/projects/work_package_1 strains include a putative choloylglycine bile hydrolase related to /downloads), we were able to quantify and determine the rate of that known to be important in the pathogenesis of Listeria infec- occurrence and location of IS elements. IS element occurrence tion (31), which may enable E. faecium to colonize regions of the ranges from a low of 2.6 per Mbp (clade B strain EnGen0047) to a intestine more proximal to the bile duct. high of 50.7 IS elements per Mbp (clade A2 strain EnGen0024). Genes representing 138 orthogroups were found to be en- Three IS elements (ISEnfa3,ISSpn10, and IS16) are highly en- riched in clade B strains compared to clade A strains. These largely riched in clade A1 and are found outside this clade only in a single occur in 24 clusters of contiguous genes but this time with few clade A2 strain (EnGen0024) and the clade A1/B hybrid strain, signatures of mobile elements. Gene groups C33, C35, C37, C43, EnGen0002 (Fig. 5). On average, strains of clade A1 harbored a C44, C45, C51, and C54 and a single gene (EfmE980_2866) have total of 391 kb of mobile element DNA, and clade A harbored an predicted roles in carbon metabolism, highlighting the differential average of 332 kb. Clade B strains contained an average of 340 kb use of carbohydrates by strains of each clade (Table 1; see Table S2 of mobile element DNA. in the supplemental material). Cluster 50 encodes a cysteine- To identify functional differences and remaining differences in containing DnaJ-like chaperone, adjacent to a putative metallo- gene content not restricted to mobile elements, we next identified -lactamase class protein that is likely to be involved in the ho- orthogroups present in 80% of genomes of one clade but in meostasis of glutathione pools (since these commensal strains of 20% of strains from a comparator (see Table S2 in the supple- E. faecium do not inactivate -lactams), involved in maintenance mental material). Contiguous groups of genes were identified and of protein structure. A main driver of clade divergence, therefore, associated with the mobile elements identified above where pos- appears to stem from residence in different ecological environ- sible. To begin to understand the ecological forces that led to the ments that have selected for the systematic exchange of phospho- initial bifurcation between clades A and B, we identified genes transferase system (PTS) systems, with strains of clade A acquiring occurring in most clade A (A1 plus A2) strains but that were rare new PTS systems on mobile elements and deleting obsolete PTS in clade B and vice versa. We found 66 orthogroups enriched at the systems from the clade B chromosome. level of 80% in clade A and 20% in clade B and 138 ortho- Interestingly, cluster 39, which is enriched in clade B, contains groups enriched in clade B versus clade A (Table S2). Genes en- four genes that are predicted to form an agr-like quorum-sensing riched in clade A strains largely occurred in 12 clusters of contig- system (32), along with another Mga-type regulator that may uous genes (cluster 2 [C2], C8, C10, C11, C12, C17, C19, C20, connect quorum sensing to carbohydrate utilization (Table 1; C21, C22, C23, and C24), with 8 clusters occurring in identifiable see Table S2 in the supplemental material) (33). Unexpectedly, mobile elements. Cluster 10, 11, 12, and 24 genes encode func- cluster 53, with an apparent 98-amino-acid secretion target tions related to altered carbohydrate utilization (Table 1 and Ta- (EfmE980_2510), which also is enriched in clade B, appears to ble S2). Cluster 19 genes include ABC transporters putatively re- encode a type VII secretion system. Both agr (32) and type VII lated to antibiotic transport. Other genes enriched in clade A secretion systems (34, 35) have been studied for their contribution strains, with predicted roles in adapting to different habitats, in- to infection pathogenesis, but the pattern of differential presence clude genes encoding a putative membrane-bound metallopro- observed here highlights potentially important roles in commen- tease in cluster 17 that likely confers resistance to a cognate bac- salism as well. teriocin (29), and an LPXTG-anchored collagen adhesin in cluster It was also of interest to examine differential gene presence in 21 that may relate to colonization and niche selection (30). Indi- clades A1 and A2. In hospital epidemic clade A1, 48 genes were vidual genes showing an enrichment in clade A versus clade B identified as differentially present, with 37 genes occurring in 6 July/August 2013 Volume 4 Issue 4 e00534-13 mbio.asm.org 5 Lebreton et al. TABLE 1 Enrichment of functional gene clusters in E. faecium clades A A1 A2 B A1 Cluster vs vs vs vs vs Putative function of the cluster or gene of interest B B B A A2 10 PTS system, N-acetylglucosamine-specific 12 PTS system, glucitol/sorbitol-specific 11 Alternate pathways for glycolysis and gluconeogenesis 24 Starch, xylose and sucrose utilization 19 ABC transporter and regulatory proteins 20 ABC transporter of unknown function 23 ABC transporter 17 Bacteriocin self-immunity protease 21 Surface proteins 22 Hexapaptide transferase, LysR substrate binding domain Putative toxin-antitoxin system Unknown 27 PTS system, Glucose/mannose and GlcNAc, ManNAc and Neu5Ac 1 PTS system, Lactose/Cellobiose specific 3 PTS system, glucose specific 18 Enterocin A immunity, Class II bacteriocin 15 ABC transporter of unknown function 5 Regulatory genes, HTH DNA binding domain 14 IS66 family transposase 25 Putative toxin-antitoxin system IS605- and IS200-like 26 Unknown 4 Unknown 9 Unknown 6 PTS system, mannose/fructose/sorbose specific 16 Glycosyl hydrolase, Sugar uptake systems 7 Phage integrase and excisionase 13 Unknown 28 Transcriptionnal regulator, LPXTG cell wall anchor protein DNA binding regulators 33 PTS system, sorbose specific 35 PTS system, maltose specific 36 PTS system, fructose/sorbose specific 43 PTS system sucrose/amylose specific 44 PTS system Lactose/Cellobiose specific 45 PTS system associated Lactose/Cellobiose/maltose 46 Exopolysaccharide biosynhtesis, glycosyltransferase 51 Mga regulators 54 Mga regulator 42 LacG, ABC transporter 30 DNA binding regulator, phospholipase, ABC transporter 31 Putative peptidase, DNA binding regulator 39 AgrABC o peron 37 Chitinase C1, Chitin binding protein, DNA binding regulator 41 GadR/MutR family transcriptionnal regulator, 50 DnaJ chaperone, Metallo-beta-lactamase class Efflux pump MtrF, beta-Ala-Xaa dipeptidase 53 Putative type VII secretion system 48 Oligopeptide transport system and permease 47 Unknown 49 Unknown 38 Unknown 40 Unknown 32 Unknown 52 Unknown Differentially occurring clusters of genes associated with chromosomal DNA (black), putative ICE elements (integrative and conjugative elements) (dark gray), plasmids (medium gray), or phages (light gray). Clusters functionally associated with carbohydrate uptake and utilization are indicated in blue type. No genes are differentially enriched in the genomes of strains in clade A2 compared to clade A1. HTH, helix-turn-helix. distinct clusters associated with mobile elements (Table 1; see Ta- was lost from clade B by strains of clade A. C6 is known to play an ble S2 in the supplemental material). Interestingly, the split be- important role in GI tract colonization following antibiotic treat- tween clades A1 and A2 is also associated with the gain of pathways ment (36). It is interesting that clade A1 recovered this ability, and for carbohydrate utilization. Clade A1 strains acquired an appar- this observation suggests that it may relate to human colonization. ent mobile element of 13 genes (C6 [Table 1]) encoding enzymes Cluster C16 is also differentially enriched in clade A1 and contrib- for uptake and utilization of fructose, sorbose, and mannose. This utes to carbohydrate utilization. No orthogroups were enriched in appears to be functionally related to a cluster (C36) that earlier clade A2 versus clade A1. 6 mbio.asm.org July/August 2013 Volume 4 Issue 4 e00534-13 Emergence of Multidrug-Resistant Enterococcus faecium We identified additional genes that show enrichment in clade rangement occurred within the phage sequence. Larger inversions A1 compared to clade B. Gene clusters 1, 3, and 27 putatively in other areas of clade A1 and A2 genomes were also observed, encode proteins for PTS systems and enzymes for the interconver- including a 1.2-Mbp inversion in both EnGen0046 and En- sion and metabolism of lactose/cellobiose, glucose, mannose, Gen0049, and again appear to be driven by recombination within N-acetylneuraminate, N-acetylmannosamine, and other sialic ac- phages present at the boundaries. Most genome rearrangements ids. Clusters 1 and 27 are associated with mobile elements. Cluster observed in E. faecium can be linked to the occurrence of mobile 18 (C18), which is also enriched in clade A1 compared to clade B, genetic elements at the boundaries. Select novel rearrangements encodes a three-gene operon for a class II bacteriocin that may be were arbitrarily verified by PCR, and the accuracy of assembly was a colonization factor (Table 1; see Table S2 in the supplemental verified in each case. material). In addition to mediating inversions and recombinations, in- Bifurcation of clade A parallels the proliferation of resis- troduction and proliferation of IS elements in a bacterial popula- tance. To understand the role that antibiotics played as a driver of tion can facilitate adaptation to new niches as the result of obsolete clade formation, we examined the differential presence of resis- gene inactivation (1). We identified 133 instances of IS element- tance genes (see Table S3 in the supplemental material). Two re- mediated gene inactivation in E. faecium (see Table S4 in the sup- sistance genes [aac(6’)-li conferring resistance to kanamycin and plemental material). The number of IS-mediated gene inactiva- bacA conferring bacitracin resistance] are part of the core E. fae- tion events was highest in clade A1 genomes and lowest in clade B cium genome. The ubiquitous presence of aac(6’)-li has been ob- strains. In clade A1 strains, we found a strong enrichment for served before and contributes to the intrinsic resistance of E. fae- disruption of a core gene encoding a putative major facilitator cium to several aminoglycosides (37). The bacA gene may be superfamily (MFS) transporter (EFAU004_02447 in strain responsible for intrinsic resistance to bacitracin observed among AUS0004) (Table S4). E. faecium (38). Seven strains analyzed were isolated in the 1950s Since compromised defense was associated with the evolution and 1960s, allowing for the identification of genes associated with of hospital epidemic strains of E. faecalis (25), it was of interest to some of the earliest known acquired resistances to occur in E. fae- examine more closely the relationship between the presence of a cium. Strains EnGen0025, EnGen0027, EnGen0031, EnGen0032, CRISPR-Cas system and mobile element content. We therefore and E1636 were isolated between 1957 and 1965; these strains fall examined the 73 E. faecium genome sequences studied for the into clade A2. Each of these strains also possesses the fusA fusidic presence of CRISPR-cas using CRISPRfinder (39). Only 7 E. fae- acid resistance gene. Additionally, strains EnGen0025, En- cium genomes carried cas genes (Fig. 5), and in 5 of these (strains Gen0027, EnGen0031, and E1636 possess the msrC gene, which Com12, EnGen0002, EnGen0056, 1_141_733, and 1_231_408), a confers erythromycin resistance. Strain EnGen0025 additionally CRISPR array could be readily identified immediately down- acquired the aminoglycoside resistance genes ant(6’)-la (confer- stream. In strains 1_231_408 and EnGen0056, where spacers ring resistance to streptomycin) and aph(3=)-III (conferring resis- could be matched to known genes, one was derived from a phage tance to several aminoglycosides, including neomycin and genta- that is a common lysogen in E. faecium genomes (present in 39 out micin B), ermB, and tetM. As shown in Fig. 2, this strain (the fifth of 73 genomes). Interestingly, this phage is absent from these 2 strain from the top of clade A2) is closely related to the clade A1 genomes, suggesting CRISPR-Cas functionality. Notably, all branch point and presumably the clade A1 founder. strains that carry cas genes are either found in a distinct subgroup Other resistances exhibit clear clade specificity (Fig. 5; see Ta- within clade B or are hybrid strains 1_231_408 and EnGen0002 ble S3 in the supplemental material). Vancomycin resistance is that acquired the cas genes and its associated CRISPR-locus from completely absent from clade B. Vancomycin resistance occurs the clade B parent (see Fig. S4 in the supplemental material). Apart mainly in clade A1 but also occurs in clade A2. Aminoglycoside from the CRISPR defense, we observed a gene encoding a putative resistance genes ant(6’)-la and aph(3=)-III are completely absent type IV methyl-directed restriction enzyme in strains of both clade from clade B strains, but they occur in most clade A1 isolates. B and A2, but not in clade A1 genomes (Fig. 5). Interestingly, in clade B, the msrC resistance gene correlates per- Evidence of varying selection in genomes from each clade. fectly with the presence of a CRISPR element. We have not found We examined polymorphisms in shared genes to detect genes un- prior mention of the occurrence of several resistance genes in der particularly strong selection in the different habitats occupied E. faecium, including the aadD cassette, which confers resistance by strains of each clade. Because of the clade structure, we used a to tobramycin and kanamycin, in a single genome (strain En- tree-based approach (40) to compare the ratios of nonsynony- Gen0035). We also observed genes lnuB, ermG, and ermT (that mous to synonymous base changes (dN/dS ratio). We removed likely confer various degrees of resistance to the macrolides- potentially confounding (41) recombined fragments using BRAT- lincosamides-streptogramin B [MLS] class of antibiotics), tetC NextGen (21). Genes under positive selection were identified (conferring resistance to tetracycline), and fosB (conferring resis- when the dN/dS ratio in the clade of interest (foreground) was tance to fosfomycin) in E. faecium. observed to be significantly higher than the dN/dS ratio in the Clade structure is reflected in E. faecium genome organiza- comparator genomes (background) (see Table S4B in the supple- tion. The Aus0004 genome possesses a previously identified mental material). No genes were found to be under positive selec- 683-kb inversion around the replication termination site (17). tion in clade B compared to clades A, A1, and A2, likely reflecting Similar inversions appear to have occurred several times indepen- the fact that clade B strains had long-fixed beneficial mutations in dently (since boundaries were not strictly identical) in strains of this particular niche before the emergence of the A clade. Only clades A1 and A2 (i.e., in strains EnGen0007 and EnGen0025), but four genes were found to be under differential positive selection not in strains of clade B (see Fig. S6 in the supplemental material). pressure in clade A compared to clade B, two of which were anno- This inversion is bounded by different phages in different strains, tated as having roles in amino acid transport and metabolism and it appears that the recombination responsible for this rear- (Table S4B). Interestingly, in strains of the hospital-adapted clade July/August 2013 Volume 4 Issue 4 e00534-13 mbio.asm.org 7 Lebreton et al. A1, a penicillin binding protein transpeptidase and the D-alanyl- in Gram-negative bacteria has been linked to the emergence of D-alanine ligase were under differential positive selection com- antibiotic-resistant lineages that are pathogenic to humans (50– pared to strains of both clades A2 and clade B (Table S4B). Finally, 52). In Gram-positive bacteria, hypermutating populations of an MFS transporter involved in carbohydrate transport and me- pathogenic Streptococcus pneumoniae and Staphylococcus aureus tabolism in clade A1 and an N-acetylglucosamine transferase in have been observed (53, 54). In E. faecium, polymorphisms in clade A2 were found to be under positive selection pressure, pro- mutS and mutL (which encode DNA mismatch repair proteins) viding independent support for the importance of differential car- have been noted (55), but the polymorphisms are not associated bohydrate utilization as a determinant of clade structure, as in- with differential mutation rates in different clades. Higher muta- ferred from gene gain/loss patterns described above. tion rates have been associated with microbes recently experienc- ing a host switch (e.g., Mycoplasma gallisepticum, 0.8 10 to 1.2 DISCUSSION 10 substitutions per site per year [61]) and with the emer- Speciation results from expansion into new ecological niches and gence of pathogenic lineages (52), possibly including E. faecium subsequent isolation from the founder population (42) and is ac- strains of the CC17 genogroup (56). It appears that the epidemic companied by changes in the genome stemming from mutation, hospital clade A1 emerged because of its ability to acquire mobile recombination (43), and horizontal gene transfer (44). All of these elements, its ability to utilize carbohydrates of nondietary origin, processes have contributed to the current population structure of and its hypermutability. E. faecium and its emergence as a leading multidrug-resistant hos- Previously, the average nucleotide identity of eight E. faecium pital pathogen. strains was determined to range between 93.5 and 95.6% when Quantification of mutation rates for strains in each E. faecium comparing strains from clades A and B (13), and clade A and B clade allowed us to estimate that the first bifurcation in the E. fae- strains would be considered to be distinct species by existing cri- cium population took place approximately 3,000 years ago, sub- teria (57, 58). The identification of hybrid clade A1/B strains stantially sooner than previously suggested (16). Although it is (strains EnGen0002 and 1_231_408) show that the ecological difficult to know the ecological drivers of this split with precision, niches of human-infecting hospital strains and human commen- the timing suggests that it relates to increasing insulation between sal strains do occasionally overlap. The emergence of the distinct the flora of humans and animals, which likely stemmed from in- clade structure in E. faecium parallels anthropogenic changes in creased urbanization, increased domestication of animals provid- urbanization and animal domestication and, more recently, the ing restricted and specialized diets (45, 46), and increasing use of introduction of antibiotics into agriculture and medicine. The net hygienic measures (47, 48). This bifurcation was associated with a effect of these forces is the emergence of a rapidly evolving lineage, wholesale loss and replacement of carbohydrate utilization path- which has crossed a degree of divergence usually associated with ways, mediated largely by acquisition on mobile elements by speciation. strains of clade A. Many of the clade B pathways lost by clade A MATERIALS AND METHODS strains relate to the utilization of complex carbohydrates from Bacterial strains. Strains selected for genome analysis were drawn from dietary sources, and the pathways lost were replaced by pathways those representing diverse points within the known phylogenic structure, on mobile elements associated with the utilization of amino sug- as determined by MLST (Fig. 1), and are listed in Table S1 in the supple- ars, such as those occurring on epithelial cell surfaces and in mu- mental material. DNA was purified from each E. faecium strain as de- cin, suggesting a possible shift from a lifestyle dependent mainly scribed before (13) for DNA sequence analysis. Methods for DNA se- on host diet (clade B) to one increasingly dependent on host se- quencing, genome assembly, and bioinformatic analysis are provided in cretions (clade A). In addition to carbohydrate utilization path- Supplemental Methods at https://olive.broadinstitute.org/projects/work ways, there was a substantial shift in genes encoding Mga-type _package_1/downloads, along with details of the genome sequences. helix-turn-helix regulators, which in Streptococcus pyogenes con- nect expression of niche-specific genes with carbohydrate metab- SUPPLEMENTAL MATERIAL olism (33). Supplemental material for this article may be found at http://mbio.asm.org The second split in the E. faecium population, the split between /lookup/suppl/doi:10.1128/mBio.00534-13/-/DCSupplemental. Figure S1, JPG file, 0.5 MB. clade A1 and clade A2, appears to have occurred approximately Figure S2, JPG file, 1.4 MB. 75 years ago, coinciding precisely with the introduction of antibi- Figure S3, JPG file, 1.5 MB. otics in both clinical medicine and agriculture. However, this split Figure S4, JPG file, 2.6 MB. may not have been directly driven by the usage of antibiotics, as Figure S5, JPG file, 0.7 MB. antibiotics are used both in farming and in human medicine. The Figure S6, JPG file, 6.2 MB. ability to rapidly acquire new traits on mobile elements, including Table S1, DOCX file, 0.1 MB. Table S2, PDF file, 0.6 MB. carbohydrate utilization pathways as well as resistance to antibi- Table S3, PDF file, 0.1 MB. otics, appears to be an intrinsic trait of clade A1 and clade A2. Table S4, DOCX file, 0.1 MB. Although clade A1 strains now cause the vast majority of infec- tions (Fig. 2), early clinical isolates from the 1950s and 1960s do ACKNOWLEDGMENTS not cluster in clade A1. The earliest isolation of a strain associated This project was funded in part by the National Institute of Allergy and with an MLST type occurring in clade A1, occurred in 1982 (49). Infectious Diseases, National Institutes of Health, Department of Health That isolate already possessed high-level resistance to gentamicin and Human Services, under contract HHSN272200900018C. Portions of and carried the esp gene. this work were also supported by NIH/NIAID grants AI083214 (Harvard- Interestingly, we found that the recently emergent hospital- wide Program on Antibiotic Resistance), and AI072360. W.V.S. and adapted clade A1 is hypermutable, as reflected in the inferred rate R.J.L.W. were supported by the European Union Seventh Framework of mutation in the genomes, and experimentally. Hypermutation Programme (FP7-HEALTH-2011-single-stage) “Evolution and Transfer 8 mbio.asm.org July/August 2013 Volume 4 Issue 4 e00534-13 Emergence of Multidrug-Resistant Enterococcus faecium of Antibiotic Resistance” (EvoTAR) under grant agreement number WP, Corander J. 2012. Restricted gene flow among hospital subpopula- tions of Enterococcus faecium. mBio 3(4):e00151-12. 15. van Schaik W, Top J, Riley DR, Boekhorst J, Vrijenhoek JEP, Schap- We acknowledge Lucia Alvarado and Clint Howarth for data submis- endonk CME, Hendrickx APA, Nijman IJ, Bonten MJM, Tettelin H, sions, Susanna Hamilton and Sinead Chapman for project management, Willems RJL. 2010. Pyrosequencing-based comparative genome analysis Chris Desjardins for helpful discussions, and Matthew Laird for help with of the nosocomial pathogen Enterococcus faecium and identification of a IslandViewer. large transferable pathogenicity island. BMC Genomics 11:239. 16. Galloway-Peña J, Roh JH, Latorre M, Qin X, Murray BE. 2012. ADDENDUM IN PROOF Genomic and SNP analyses demonstrate a distant separation of the hos- pital and community-associated clades of Enterococcus faecium. PLoS One Following submission we were made aware that others recently described a 7:e30187. doi: 10.1371/journal.pone.0030187. split, between human and bovine populations of S. aureus, datable by BEAST 17. Lam MMC, Seemann T, Bulach DM, Gladman SL, Chen H, Haring V, analysis, to approximately 5,000 years ago (L. A. Weinert, J. J. Welch, M. A. Moore RJ, Ballard S, Grayson ML, Johnson PDR, Howden BP, Stinear Suchard, P. Lemey, A. Rambaut, and J. R. Fitzgerald, Biol Lett. 8:829-832, TP. 2012. Comparative analysis of the first complete Enterococcus faecium 2012). genome. J. Bacteriol. 194:2334 –2341. 18. Willems RJ, van Schaik W. 2009. Transition of Enterococcus faecium from REFERENCES commensal organism to nosocomial pathogen. Future Microbiol. 1. Gilmore MS, Lebreton F, van Schaik W. 2013. Genomic transition of 4:1125–1135. enterococci from gut commensals to leading causes of multidrug-resistant 19. De Regt MJA, van Schaik W, van Luit-Asbroek M, Dekker HAT, van hospital infection in the antibiotic era. Curr. Opin. Microbiol. 16:10 –16. Duijkeren E, Koning CJM, Bonten MJM, Willems RJL. 2012. Hospital 2. McKinnell JA, Kunz DF, Chamot E, Patel M, Shirley RM, Moser SA, and community ampicillin-resistant Enterococcus faecium are evolution- Baddley JW, Pappas PG, Miller LG. 2012. Association between arily closely linked but have diversified through niche adaptation. PLoS vancomycin-resistant enterococci bacteremia and ceftriaxone usage. In- One 7:e30319. doi: 10.1371/journal.pone.0030319. fect. Control Hosp. Epidemiol. 33:718 –724. 20. Drummond AJ, Suchard MA, Xie D, Rambaut A. 2012. Bayesian phy- 3. Arias CA, Murray BE. 2008. Emergence and management of drug- logenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29: resistant enterococcal infections. Expert Rev. Anti Infect. Ther. 1969 –1973. 6:637– 655. 21. Marttinen P, Hanage WP, Croucher NJ, Connor TR, Harris SR, Bentley 4. Smillie CS, Smith MB, Friedman J, Cordero OX, David LA, Alm EJ. SD, Corander J. 2012. Detection of recombination events in bacterial 2011. Ecology drives a global network of gene exchange connecting the genomes from large population samples. Nucleic Acids Res. 40:e6. doi: human microbiome. Nature 480:241–244. 10.1093/nar/gkr928. 5. Harrison EM, Paterson GK, Holden MTG, Larsen J, Stegger M, Larsen 22. Holden MTG, Hsu LY, Kurt K, Weinert LA, Mather AE, Harris SR, AR, Petersen A, Skov RL, Christensen JM, Bak Zeuthen A, Heltberg O, Strommenger B, Layer F, Witte W, de Lencastre H, Skov R, Westh H, Harris SR, Zadoks RN, Parkhill J, Peacock SJ, Holmes MA. 2013. Whole Zemlicková H, Coombs G, Kearns AM, Hill RLR, Edgeworth J, Gould genome sequencing identifies zoonotic transmission of MRSA isolates I, Gant V, Cooke J, Edwards GF, McAdam PR, Templeton KE, McCann with the novel mecA homologue mecC. EMBO Mol. Med. 5:509 –515. A, Zhou Z, Castillo-Ramírez S, Feil EJ, Hudson LO, Enright MC, 6. Price LB, Stegger M, Hasman H, Aziz M, Larsen J, Andersen PS, Balloux F, Aanensen DM, Spratt BG, Fitzgerald JR, Parkhill J, Achtman Pearson T, Waters AE, Foster JT, Schupp J, Gillece J, Driebe E, Liu CM, M, Bentley SD, Nübel U. 2013. A genomic portrait of the emergence, Springer B, Zdovc I, Battisti A, Franco A, Zmudzki J, Schwarz S, Butaye evolution, and global spread of a methicillin-resistant Staphylococcus au- P, Jouy E, Pomba C, Porrero MC, Ruimy R, Smith TC, Robinson DA, reus pandemic. Genome Res. 23:653– 664. Weese JS, Arriola CS, Yu F, Laurent F, Keim P, Skov R, Aarestrup FM. 23. Nübel U, Dordel J, Kurt K, Strommenger B, Westh H, Shukla SK, 2012. Staphylococcus aureus CC398: host adaptation and emergence of Žemliková H, Leblois R, Wirth T, Jombart T, Balloux F, Witte W. methicillin resistance in livestock. mBio 3(1):e00305-11. 2010. A timescale for evolution, population expansion, and spatial spread 7. Acar J, Casewell M, Freeman J, Friis C, Goossens H. 2000. Avoparcin of an emerging clone of methicillin-resistant Staphylococcus aureus. PLoS and virginiamycin as animal growth promoters: a plea for science in Pathog. 6:e1000855. doi:10.1371/journal.ppat.1000855. decision-making. Clin. Microbiol. Infect. 6:477– 482. 24. Dagan T, Martin W. 2007. Ancestral genome sizes specify the minimum 8. Collignon PJ. 1999. Vancomycin-resistant enterococci and use of avopar- rate of lateral gene transfer during prokaryote evolution. Proc. Natl. Acad. cin in animal feed: is there a link? Med. J. Aust. 171:144 –146. Sci. USA 104:870 – 875. 9. Bager F, Madsen M, Christensen J, Aarestrup FM. 1997. Avoparcin used 25. Palmer KL, Gilmore MS. 2010. Multidrug-resistant enterococci lack as a growth promoter is associated with the occurrence of vancomycin- CRISPR-cas. mBio 1(4):e00227-10. resistant Enterococcus faecium on Danish poultry and pig farms. Prev. Vet. 26. Zhou Y, Liang Y, Lynch KH, Dennis JJ, Wishart DS. 2011. PHAST: a fast Med. 31:95–112. phage search tool. Nucleic Acids Res. 39:W347–W352. 10. Lauderdale TL, Shiau YR, Wang HY, Lai JF, Huang IW, Chen PC, Chen 27. Waack S, Keller O, Asper R, Brodag T, Damm C, Fricke WF, Surovcik HY, Lai SS, Liu YF, Ho M. 2007. Effect of banning vancomycin analogue K, Meinicke P, Merkl R. 2006. Score-based prediction of genomic islands avoparcin on vancomycin-resistant enterococci in chicken farms in Tai- in prokaryotic genomes using hidden Markov models. BMC Bioinformat- wan. Environ. Microbiol. 9:819 – 823. ics 7:142. 11. Willems RJL, Top J, van Santen M, Robinson DA, Coque TM, Baquero 28. Jensen LB, Garcia-Migura L, Valenzuela AJS, Løhr M, Hasman H, F, Grundmann H, Bonten MJM. 2005. Global spread of vancomycin- Aarestrup FM. 2010. A classification system for plasmids from entero- resistant Enterococcus faecium from distinct nosocomial genetic complex. cocci and other Gram-positive bacteria. J. Microbiol. Methods 80:25– 43. Emerg. Infect. Dis. 11:821– 828. 29. Kjos M, Borrero J, Opsata M, Birri DJ, Holo H, Cintas LM, Snipen L, 12. Kos VN, Desjardins CA, Griggs A, Cerqueira G, Van Tonder A, Holden Hernández PE, Nes IF, Diep DB. 2011. Target recognition, resistance, MTG, Godfrey P, Palmer KL, Bodi K, Mongodin EF, Wortman J, immunity and genome mining of class II bacteriocins from Gram-positive Feldgarden M, Lawley T, Gill SR, Haas BJ, Birren B, Gilmore MS. 2012. bacteria. Microbiology 157:3256 –3267. Comparative genomics of vancomycin-resistant Staphylococcus aureus 30. Hendrickx APA, van Luit-Asbroek M, Schapendonk CME, van Wamel strains and their positions within the clade most commonly associated WJB, Braat JC, Wijnands LM, Bonten MJM, Willems RJL. 2009. SgrA, with methicillin-resistant S. aureus hospital-acquired infection in the a nidogen-binding LPXTG surface adhesin implicated in biofilm forma- United States. mBio 3(3):e00112-12. tion, and EcbA, a collagen binding MSCRAMM, are two novel adhesins of 13. Palmer KL, Godfrey P, Griggs A, Kos VN, Zucker J, Desjardins C, hospital-acquired Enterococcus faecium. Infect. Immun. 77:5097–5106. Cerqueira G, Gevers D, Walker S, Wortman J, Feldgarden M, Haas B, 31. Dussurget O, Cabanes D, Dehoux P, Lecuit M, Buchrieser C, Glaser P, Birren B, Gilmore MS. 2012. Comparative genomics of enterococci: variation in Enterococcus faecalis, clade structure in E. faecium, and defin- Cossart P, European Listeria Genome Consortium. 2002. Listeria mono- ing characteristics of E. gallinarum and E. casseliflavus. mBio 3(1):e00318- cytogenes bile salt hydrolase is a PrfA-regulated virulence factor involved in 11. the intestinal and hepatic phases of listeriosis. Mol. Microbiol. 45: 14. Willems RJL, Top J, van Schaik W, Leavis H, Bonten M, Sirén J, Hanage 1095–1106. July/August 2013 Volume 4 Issue 4 e00534-13 mbio.asm.org 9 Lebreton et al. 32. Novick RP, Geisinger E. 2008. Quorum sensing in staphylococci. Annu. BE. 2009. Analysis of clonality and antibiotic resistance among early clin- Rev. Genet. 42:541–564. ical isolates of Enterococcus faecium in the United States. J. Infect. Dis. 33. Hondorp ER, McIver KS. 2007. The Mga virulence regulon: infection 200:1566 –1573. where the grass is greener. Mol. Microbiol. 66:1056 –1065. 50. LeClerc JE, Li B, Payne WL, Cebula TA. 1996. High mutation frequen- 34. Simeone R, Bottai D, Brosch R. 2009. ESX/type VII secretion systems and cies among Escherichia coli and Salmonella pathogens. Science 274: their role in host-pathogen interaction. Curr. Opin. Microbiol. 12:4 –10. 1208 –1211. 35. Chen YH, Anderson M, Hendrickx APA, Missiakas D. 2012. Charac- 51. Jolivet-Gougeon A, Kovacs B, Le Gall-David S, Le Bars H, Bousarghin terization of EssB, a protein required for secretion of ESAT-6 like proteins L, Bonnaure-Mallet M, Lobel B, Guillé F, Soussy CJ, Tenke P. 2011. in Staphylococcus aureus. BMC Microbiol. 12:219. Bacterial hypermutation: clinical implications. J. Med. Microbiol. 60: 36. Zhang X, Top J, de Been M, Bierschenk D, Rogers M, Leendertse M, 563–573. Bonten MJ, van der Poll T, Willems RJ, van Schaik W. 2013. Identifi- 52. Maciá MD, Blanquer D, Togores B, Sauleda J, Pérez JL, Oliver A. 2005. cation of a genetic determinant in clinical Enterococcus faecium strains that Hypermutation is a key factor in development of multiple-antimicrobial contributes to intestinal colonization during antibiotic treatment. J. In- resistance in Pseudomonas aeruginosa strains causing chronic lung infec- fect. Dis. 207:1780 –1786. tions. Antimicrob. Agents Chemother. 49:3382–3386. 37. Costa Y, Galimand M, Leclercq R, Duval J, Courvalin P. 1993. Char- 53. del Campo R, Morosini MI, de la Pedrosa EG, Fenoll A, Muñoz- acterization of the chromosomal aac(6=)-Ii gene specific for Enterococcus Almagro C, Máiz L, Baquero F, Cantón R, Spanish Pneumococcal faecium. Antimicrob. Agents Chemother. 37:1896 –1903. Infection Study Network. 2005. Population structure, antimicrobial re- 38. Bywater R, McConville M, Phillips I, Shryock T. 2005. The susceptibility sistance, and mutation frequencies of Streptococcus pneumoniae isolates to growth-promoting antibiotics of Enterococcus faecium isolates from from cystic fibrosis patients. J. Clin. Microbiol. 43:2207–2214. pigs and chickens in Europe. J. Antimicrob. Chemother. 56:538 –543. 54. Prunier AL, Malbruny B, Laurans M, Brouard J, Duhamel JF, Leclercq 39. Grissa I, Vergnaud G, Pourcel C. 2007. CRISPRFinder: a web tool to R. 2003. High rate of macrolide resistance in Staphylococcus aureus strains identify clustered regularly interspaced short palindromic repeats. Nucleic from patients with cystic fibrosis reveals high proportions of hypermut- Acids Res. 35:W52–W57. able strains. J. Infect. Dis. 187:1709 –1716. 40. Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phy- 55. Willems RJ, Top J, Smith DJ, Roper DI, North SE, Woodford N. 2003. logenetic analyses with thousands of taxa and mixed models. Bioinformat- Mutations in the DNA mismatch repair proteins MutS and MutL of ics 22:2688 –2690. oxazolidinone-resistant or -susceptible Enterococcus faecium. Antimicrob. 41. Anisimova M, Nielsen R, Yang Z. 2003. Effect of recombination on the Agents Chemother. 47:3061–3066. accuracy of the likelihood method for detecting positive selection at amino 56. Ruiz-Garbajosa P, Top J, Coque TM, Cantón R, Bonten MJ, Baquero F, acid sites. Genetics 164:1229 –1236. Willems RJ. 2008. Abstr. 18th Eur. Cong. Clin Microbiol. Infect. Dis., 42. Cohan FM. 2001. Bacterial species and speciation. Syst. Biol. 50:513–524. abstr. P2043. 43. Fraser C, Hanage WP, Spratt BG. 2007. Recombination and the nature of 57. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, bacterial speciation. Science 315:476 – 480. Tiedje JM. 2007. DNA-DNA hybridization values and their relationship 44. Wiedenbeck J, Cohan FM. 2011. Origins of bacterial diversity through to whole-genome sequence similarities. Int. J. Syst. Evol. Microbiol. 57: horizontal genetic transfer and adaptation to new ecological niches. FEMS 81–91. Microbiol. Rev. 35:957–976. 58. Konstantinidis KT, Tiedje JM. 2005. Genomic insights that advance the 45. Petroutsa EI, Manolis SK. 2010. Reconstructing Late Bronze Age diet in species definition for prokaryotes. Proc. Natl. Acad. Sci. U. S. A. 102: mainland Greece using stable isotope analysis. J. Archaeol. Sci. 37: 2567–2572. 614 – 620. 59. Tettelin H, Riley D, Cattuto C, Medini D. 2008. Comparative genomics: 46. Jay M, Richards MP. 2006. Diet in the Iron Age cemetery population at the bacterial pan-genome. Curr. Opin. Microbiol. 11:472– 477. Wetwang Slack, East Yorkshire, UK: carbon and nitrogen stable isotope 60. Chang D, Zhu Y, Zou Y, Fang X, Li T, Wang J, Guo Y, Su L, Xia J, Yang evidence. J. Archaeol. Sci. 33:653– 662. R, Fang C, Liu C. 2012. Draft genome sequence of Enterococcus faecium 47. McEvedy C, Jones R. 1978. Atlas of world population history. Penguin strain LCT-EF90. J. Bacteriol. 194:3556 –3557. Books, Middlesex, United Kingdom. 61. Delaney NF, Balenger S, Bonneaud C, Marx CJ, Hill GE, Ferguson-Noel 48. Osborne R, Cunliffe B. 2005. Mediterranean urbanization 800 – 600 BC. N, Tsai P, Rodrigo A, Edwards SV. 2012. Ultrafast evolution and loss of Oxford University Press, Oxford, United Kingdom. CRISPRs following a host shift in a novel wildlife pathogen, Mycoplasma 49. Galloway-Peña JR, Nallapareddy SR, Arias CA, Eliopoulos GM, Murray gallisepticum. PLoS Genet. 8:e1002511. 10 mbio.asm.org July/August 2013 Volume 4 Issue 4 e00534-13 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png mBio Pubmed Central

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Abstract

RESEARCH ARTICLE Emergence of Epidemic Multidrug-Resistant Enterococcus faecium from Animal and Commensal Strains a,b a,b,c b b b b François Lebreton, Willem van Schaik, Abigail Manson McGuire, Paul Godfrey, Allison Griggs, Varun Mazumdar, d d b b b b b c Jukka Corander, Lu Cheng, Sakina Saif, Sarah Young, Qiandong Zeng, Jennifer Wortman, Bruce Birren, Rob J. L. Willems, b a,b Ashlee M. Earl, Michael S. Gilmore Departments of Ophthalmology, Microbiology and Immunobiology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA ; The b c Broad Institute, Cambridge, Massachusetts, USA ; Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands ; Department of Mathematics and Statistics, University of Helsinki, Helsinki, Finland F.L., W.V.S., and A.M.M. contributed equally to this article. ABSTRACT Enterococcus faecium, natively a gut commensal organism, emerged as a leading cause of multidrug-resistant hospital-acquired infection in the 1980s. As the living record of its adaptation to changes in habitat, we sequenced the genomes of 51 strains, isolated from various ecological environments, to understand how E. faecium emerged as a leading hospital patho- gen. Because of the scale and diversity of the sampled strains, we were able to resolve the lineage responsible for epidemic, multidrug-resistant human infection from other strains and to measure the evolutionary distances between groups. We found that the epidemic hospital-adapted lineage is rapidly evolving and emerged approximately 75 years ago, concomitant with the introduction of antibiotics, from a population that included the majority of animal strains, and not from human commensal lines. We further found that the lineage that included most strains of animal origin diverged from the main human commensal line approximately 3,000 years ago, a time that corresponds to increasing urbanization of humans, development of hygienic practices, and domestication of animals, which we speculate contributed to their ecological separation. Each bifurcation was accompanied by the acquisition of new metabolic capabilities and colonization traits on mobile elements and the loss of function and genome remodeling associated with mobile element insertion and movement. As a result, diversity within the species, in terms of sequence divergence as well as gene content, spans a range usually associated with speciation. IMPORTANCE Enterococci, in particular vancomycin-resistant Enterococcus faecium, recently emerged as a leading cause of hospital-acquired infection worldwide. In this study, we examined genome sequence data to understand the bacterial adapta- tions that accompanied this transformation from microbes that existed for eons as members of host microbiota. We observed changes in the genomes that paralleled changes in human behavior. An initial bifurcation within the species appears to have oc- curred at a time that corresponds to the urbanization of humans and domestication of animals, and a more recent bifurcation parallels the introduction of antibiotics in medicine and agriculture. In response to the opportunity to fill niches associated with changes in human activity, a rapidly evolving lineage emerged, a lineage responsible for the vast majority of multidrug-resistant E. faecium infections. Received 17 July 2013 Accepted 23 July 2013 Published 20 August 2013 Citation Lebreton F, van Schaik W, Manson McGuire A, Godfrey P, Griggs A, Mazumdar V, Corander J, Cheng L, Saif S, Young S, Zeng Q, Wortman J, Birren B, Willems RJL, Earl AM, Gilmore MS. 2013. Emergence of epidemic multidrug-resistant Enterococcus faecium from animal and commensal strains. mBio 4(4):e00534-13. doi:10.1128/mBio.00534-13. Editor Larry McDaniel, University of Mississippi Medical Center Copyright © 2013 Lebreton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Michael S. Gilmore, michael_gilmore@meei.harvard.edu. ntibiotic resistance is a leading threat to human health world- cocci have begun to transmit vancomycin resistance to Awide that substantially increases the cost of health care (1). methicillin-resistant Staphylococcus aureus (12). Enterococci emerged in the 1970s and 1980s as leading causes of Previously, we examined a limited sampling of human com- antibiotic-resistant infection of the bloodstream, urinary tract, mensal and hospital isolates of E. faecium and found that by aver- and surgical wounds (1), contributing to 10,000 to 25,000 deaths age nucleotide identity analysis (ANI), some differed by more per year in the USA (2). Resistance to antibiotics is common than 5%, crossing the threshold used for species identity (13). among enterococci (1), and vancomycin-resistant Enterococ- Since variation was noted among hospital strains (13–16) and cus faecium now represents up to 80% of E. faecium isolates in since little was known about strains from the gastrointestinal (GI) some hospitals (3). Agricultural practices have promoted the tracts of domestic and other animals, it was of interest to deter- emergence of antibiotic resistance (4–6). The use of avoparcin in mine the scope of diversity within the species and to precisely animal feed in Europe and elsewhere appears to have contributed define these populations and their origins. We therefore charac- to the proliferation of vancomycin resistance (7–11), and entero- terized the breadth of the species by sequencing and comparing July/August 2013 Volume 4 Issue 4 e00534-13 mbio.asm.org 1 Lebreton et al. FIG 1 goeBURST analysis of 2,273 E. faecium entries in the E. faecium MLST database (http://efaecium.mlst.net), which can be grouped into 773 sequence types (STs) (brown circles), based upon MLST relatedness. STs included in this study are highlighted in purple. the genomes of 51 strains, sampling all areas of the existing mul- household pets (19). Two strains (EnGen0002 and 1_231_408) tilocus sequence type (MLST) phylogeny (Fig. 1). possess hybrid genomes, consisting of a background genome of clade A1, into which 195 kb to 740 kb DNA from a clade B donor RESULTS have recombined (Fig. S4). To understand the forces that gave rise to the observed clade Phylogenomic reconstruction of E. faecium divergence. We de- structure in the context of human activity, we estimated the time termined the nucleotide sequences of the genomes of 51 E. faecium at which these bifurcations occurred, using Bayesian evolutionary strains of different MLST types (see Table S1 in the supplemental analysis on sampled phylogenetic trees (BEAST) (20). To limit the material), which were obtained from diverse ecological environ- confounding effect of recombination, detectable signatures of re- ments (see Fig. S1 in the supplemental material) on five conti- combination were removed from the analysis using BRATNext- nents, and isolated over the last 60 years (Fig. S1). A single nucle- Gen (21). Concerned that differing stresses in different habitats otide polymorphism (SNP)-based phylogenetic tree, which could affect mutation rate, we calculated inferred rates of muta- compared these strains to each other and to an additional 22 tion for each clade separately. A significantly higher mutation rate strains from GenBank (Table S1), was generated based on varia- was found for strains in the hospital-adapted clade A1 (4.9 10 tion in 1,344 shared single-copy orthologous groups (ortho- groups) (Fig. 2). This tree confirmed the deep divide between  0.3 10 substitutions per nucleotide per year) than for sister 6 6 clades (clades A and B) (13, 16). Most (5/7) strains isolated from clade A2 (3.6  10  0.6  10 substitutions per nucleotide the feces of nonhospitalized humans cluster in clade B. We were per year). The mutation rate for clade B was intermediate at 1.3 5 5 able to resolve the epidemic hospital strains (clade A1) from a 10  0.2  10 substitutions per nucleotide per year, a rate mixed group of animal strains and sporadic human infection iso- that is similar to those recently reported for Staphylococcus aureus lates (clade A2). This clade structure was independently recapitu- (22, 23). lated based on cluster analysis of (i) shared gene content (Fig. S2) To determine whether the calculated mutation rate differences and (ii) gene synteny (Fig. S3). reflected historic events or whether they are still experimentally Clade A1 strains account for the vast majority of human infec- detectable, the rate of mutation to fosfomycin resistance was mea- tion (Fig. 2) and include sequence types (STs) from the clonal sured for 10 randomly selected strains from each clade. Resistance complex 17 (CC17) genogroup (e.g., sequence type 17 [ST17], was verified for stability by passage in the absence of selection, ST117, and ST78 [18]) associated with hospital ward outbreaks followed by retesting. Clade A1 strains yielded spontaneous around the globe (see Table S1 in the supplemental material). fosfomycin-resistant variants at a rate about an order of magni- Interestingly, the three clade A1 strains of animal origin are from tude higher than strains of either clade A2 or clade B (Fig. 3), pet dogs, consistent with known links between hospital strains and paralleling the results of BEAST analysis. Therefore, mutation 2 mbio.asm.org July/August 2013 Volume 4 Issue 4 e00534-13 Emergence of Multidrug-Resistant Enterococcus faecium FIG 2 RAxML SNP-based tree based on the concatenated alignments of DNA sequences of 1,344 single-copy core genes in 73 E. faecium genomes. Bootstrap- ping was performed with 1,000 replicates. The origins of the strains are indicated. The dates for the split between the clades, estimated by a BEAST analysis, are indicated (ya, years ago). The infectivity score reflects the number of strains of a particular ST, in the MLST database, isolated from infection. The clades are color coded as follows: clade B in dark blue, clade A1 in red, and clade A2 in gray. rates for each clade inferred by BEAST were used to estimate the have significantly larger overall average genome size (2,843 159 time of divergence between clades A1, A2, and B. This placed the genes; 2.98  0.15 Mb) than strains of either clade A2 (2,597 time of the initial split between clade A and clade B at 2,776  153 genes; 2.75 0.14 Mb) or clade B (2,718 120 genes; 2.84 818 years ago and that between clade A1 and clade A2 at 74  0.1 Mb) (Fig. 4A), indicating that perpetuating cycles of infection 30 years ago (Fig. 2). and survival in the hospital are associated with acquisition of new Gene content differences. Gene gain and loss make funda- functions. Clade A1 strains also have larger core genomes (1,945 mental contributions to new habitat adaptation and the emer- genes) than strains of clade A2 (1,724 genes) or clade B (1,805 gence of new lineages (24). Strains from clade A1 were found to genes), which is consistent with a very recent emergence of this July/August 2013 Volume 4 Issue 4 e00534-13 mbio.asm.org 3 Lebreton et al. lineage (i.e., little time for divergence between strains to occur) (Fig. 4C). In contrast, the pan-genome of clade A2 is larger (6,343 genes) than those of clade A1 and B (5,663 and 5,551 genes, re- spectively) (Fig. 4B), which is consistent with the diverse origins of strains from this clade. In comparison to other opportunists, the E. faecium genome is relatively open (see Fig. S5 in the supplemen- tal material). Previously, the genomes of hospital strains of the sister species, Enterococcus faecalis, were found to differ from commensal organ- isms largely as the result of mobile element acquisition (13), which was associated with the absence of CRISPR (clustered regularly interspaced short palindromic repeat) protection (25). It was, therefore, of interest to determine the extent to which mobile elements drove the divergence of E. faecium clades. Mobile ele- ments were identified using PHAST (26) for phages, SIGI-HMM (27) for genomic islands, and BLAST for repA orthologs in plasmid-related contigs (28). Clade A1 was found to be enriched in mobile elements, including plasmids (5.4  1.9 plasmids/ge- nome in clade A1, compared to 2.7 2.2 and 1.5 1.1 plasmids/ genome in clade A2 and B strains, respectively), integrated phages (1.6  0.9 phages/genome, compared to 0.7  0.7 and 0.9  0.8 phages/genome in clade A2 and B strains, respectively) and other FIG 3 Frequency of fosfomycin resistance was determined in triplicate for 10 randomly selected strains from each E. faecium clade (clade A1 [red], A2 genomic islands (36  26 kb of island-associated sequence/ge- [gray], and B [dark blue]). Each symbol represents the average value for one nome, compared to 14  10 and 17  11 kb of island-associated strain, and the clade average  standard deviation (error bars) for the 10 sequence/genome in clade A2 and B strains, respectively) strains per clade are indicated. (Fig. 4D). Because the genome sequences generated in the present study were of high quality, yielding a small number of scaffolds FIG 4 (A) Genome size comparison for E. faecium clade A1 (red), A2 (gray), and B (dark blue). (B and C) Pan-genome (B) and core genome (C) are shown for increasing values of the number of sequenced E. faecium genomes within each clade. Circles represent the number of new or core genes present when a particular genome is added to each subset. Black bars represent median values. The curve for the estimation of the size of the E. faecium pan-genome for each clade is a least-squares power law fit through medians. The size of the core genome within each clade was estimated by fitting an exponential curve through medians. (D) Heat map showing the enrichment in genetic mobile elements in E. faecium genomes within each clade (clade A1 [red], A2, [gray], and B [light blue]). Horizontal boxes represent strains, which are ordered within clades as in Fig. 2 (rotated 90°). The aggregate length (kb) of islands was used to compare content in each clade (ranging from 4 kb to 99 kb; median, 17 kb), whereas the numbers of putative plasmids (ranging from 0 to 9; median, 3) or phage elements (ranging from 0 to 4; median, 1) are represented. The heat map reflects the 10th percentile (light gray), 50th percentile (medium gray), and 90th percentile (black). The “” symbol in a box indicates genome sequence for which the length of genomic islands could not be determined using the SIGI-HMM algorithm (27). 4 mbio.asm.org July/August 2013 Volume 4 Issue 4 e00534-13 Emergence of Multidrug-Resistant Enterococcus faecium FIG 5 Summary of clade-specific antibiotic resistance genes, insertion sequences (IS), and select defenses against horizontal gene transfer. Each box represents a strain, arranged by clade as shown in Fig. 2. The “” symbol in a box indicates genome sequence with an assembly quality that precluded identification of the indicated feature. An asterisk in a box indicates hybrid genomes that contain CRISPR-cas on recombined fragments. CRISPR and type IV restriction- modification (RM) systems are included in the miscellaneous (Misc.) category. (see https://olive.broadinstitute.org/projects/work_package_1 strains include a putative choloylglycine bile hydrolase related to /downloads), we were able to quantify and determine the rate of that known to be important in the pathogenesis of Listeria infec- occurrence and location of IS elements. IS element occurrence tion (31), which may enable E. faecium to colonize regions of the ranges from a low of 2.6 per Mbp (clade B strain EnGen0047) to a intestine more proximal to the bile duct. high of 50.7 IS elements per Mbp (clade A2 strain EnGen0024). Genes representing 138 orthogroups were found to be en- Three IS elements (ISEnfa3,ISSpn10, and IS16) are highly en- riched in clade B strains compared to clade A strains. These largely riched in clade A1 and are found outside this clade only in a single occur in 24 clusters of contiguous genes but this time with few clade A2 strain (EnGen0024) and the clade A1/B hybrid strain, signatures of mobile elements. Gene groups C33, C35, C37, C43, EnGen0002 (Fig. 5). On average, strains of clade A1 harbored a C44, C45, C51, and C54 and a single gene (EfmE980_2866) have total of 391 kb of mobile element DNA, and clade A harbored an predicted roles in carbon metabolism, highlighting the differential average of 332 kb. Clade B strains contained an average of 340 kb use of carbohydrates by strains of each clade (Table 1; see Table S2 of mobile element DNA. in the supplemental material). Cluster 50 encodes a cysteine- To identify functional differences and remaining differences in containing DnaJ-like chaperone, adjacent to a putative metallo- gene content not restricted to mobile elements, we next identified -lactamase class protein that is likely to be involved in the ho- orthogroups present in 80% of genomes of one clade but in meostasis of glutathione pools (since these commensal strains of 20% of strains from a comparator (see Table S2 in the supple- E. faecium do not inactivate -lactams), involved in maintenance mental material). Contiguous groups of genes were identified and of protein structure. A main driver of clade divergence, therefore, associated with the mobile elements identified above where pos- appears to stem from residence in different ecological environ- sible. To begin to understand the ecological forces that led to the ments that have selected for the systematic exchange of phospho- initial bifurcation between clades A and B, we identified genes transferase system (PTS) systems, with strains of clade A acquiring occurring in most clade A (A1 plus A2) strains but that were rare new PTS systems on mobile elements and deleting obsolete PTS in clade B and vice versa. We found 66 orthogroups enriched at the systems from the clade B chromosome. level of 80% in clade A and 20% in clade B and 138 ortho- Interestingly, cluster 39, which is enriched in clade B, contains groups enriched in clade B versus clade A (Table S2). Genes en- four genes that are predicted to form an agr-like quorum-sensing riched in clade A strains largely occurred in 12 clusters of contig- system (32), along with another Mga-type regulator that may uous genes (cluster 2 [C2], C8, C10, C11, C12, C17, C19, C20, connect quorum sensing to carbohydrate utilization (Table 1; C21, C22, C23, and C24), with 8 clusters occurring in identifiable see Table S2 in the supplemental material) (33). Unexpectedly, mobile elements. Cluster 10, 11, 12, and 24 genes encode func- cluster 53, with an apparent 98-amino-acid secretion target tions related to altered carbohydrate utilization (Table 1 and Ta- (EfmE980_2510), which also is enriched in clade B, appears to ble S2). Cluster 19 genes include ABC transporters putatively re- encode a type VII secretion system. Both agr (32) and type VII lated to antibiotic transport. Other genes enriched in clade A secretion systems (34, 35) have been studied for their contribution strains, with predicted roles in adapting to different habitats, in- to infection pathogenesis, but the pattern of differential presence clude genes encoding a putative membrane-bound metallopro- observed here highlights potentially important roles in commen- tease in cluster 17 that likely confers resistance to a cognate bac- salism as well. teriocin (29), and an LPXTG-anchored collagen adhesin in cluster It was also of interest to examine differential gene presence in 21 that may relate to colonization and niche selection (30). Indi- clades A1 and A2. In hospital epidemic clade A1, 48 genes were vidual genes showing an enrichment in clade A versus clade B identified as differentially present, with 37 genes occurring in 6 July/August 2013 Volume 4 Issue 4 e00534-13 mbio.asm.org 5 Lebreton et al. TABLE 1 Enrichment of functional gene clusters in E. faecium clades A A1 A2 B A1 Cluster vs vs vs vs vs Putative function of the cluster or gene of interest B B B A A2 10 PTS system, N-acetylglucosamine-specific 12 PTS system, glucitol/sorbitol-specific 11 Alternate pathways for glycolysis and gluconeogenesis 24 Starch, xylose and sucrose utilization 19 ABC transporter and regulatory proteins 20 ABC transporter of unknown function 23 ABC transporter 17 Bacteriocin self-immunity protease 21 Surface proteins 22 Hexapaptide transferase, LysR substrate binding domain Putative toxin-antitoxin system Unknown 27 PTS system, Glucose/mannose and GlcNAc, ManNAc and Neu5Ac 1 PTS system, Lactose/Cellobiose specific 3 PTS system, glucose specific 18 Enterocin A immunity, Class II bacteriocin 15 ABC transporter of unknown function 5 Regulatory genes, HTH DNA binding domain 14 IS66 family transposase 25 Putative toxin-antitoxin system IS605- and IS200-like 26 Unknown 4 Unknown 9 Unknown 6 PTS system, mannose/fructose/sorbose specific 16 Glycosyl hydrolase, Sugar uptake systems 7 Phage integrase and excisionase 13 Unknown 28 Transcriptionnal regulator, LPXTG cell wall anchor protein DNA binding regulators 33 PTS system, sorbose specific 35 PTS system, maltose specific 36 PTS system, fructose/sorbose specific 43 PTS system sucrose/amylose specific 44 PTS system Lactose/Cellobiose specific 45 PTS system associated Lactose/Cellobiose/maltose 46 Exopolysaccharide biosynhtesis, glycosyltransferase 51 Mga regulators 54 Mga regulator 42 LacG, ABC transporter 30 DNA binding regulator, phospholipase, ABC transporter 31 Putative peptidase, DNA binding regulator 39 AgrABC o peron 37 Chitinase C1, Chitin binding protein, DNA binding regulator 41 GadR/MutR family transcriptionnal regulator, 50 DnaJ chaperone, Metallo-beta-lactamase class Efflux pump MtrF, beta-Ala-Xaa dipeptidase 53 Putative type VII secretion system 48 Oligopeptide transport system and permease 47 Unknown 49 Unknown 38 Unknown 40 Unknown 32 Unknown 52 Unknown Differentially occurring clusters of genes associated with chromosomal DNA (black), putative ICE elements (integrative and conjugative elements) (dark gray), plasmids (medium gray), or phages (light gray). Clusters functionally associated with carbohydrate uptake and utilization are indicated in blue type. No genes are differentially enriched in the genomes of strains in clade A2 compared to clade A1. HTH, helix-turn-helix. distinct clusters associated with mobile elements (Table 1; see Ta- was lost from clade B by strains of clade A. C6 is known to play an ble S2 in the supplemental material). Interestingly, the split be- important role in GI tract colonization following antibiotic treat- tween clades A1 and A2 is also associated with the gain of pathways ment (36). It is interesting that clade A1 recovered this ability, and for carbohydrate utilization. Clade A1 strains acquired an appar- this observation suggests that it may relate to human colonization. ent mobile element of 13 genes (C6 [Table 1]) encoding enzymes Cluster C16 is also differentially enriched in clade A1 and contrib- for uptake and utilization of fructose, sorbose, and mannose. This utes to carbohydrate utilization. No orthogroups were enriched in appears to be functionally related to a cluster (C36) that earlier clade A2 versus clade A1. 6 mbio.asm.org July/August 2013 Volume 4 Issue 4 e00534-13 Emergence of Multidrug-Resistant Enterococcus faecium We identified additional genes that show enrichment in clade rangement occurred within the phage sequence. Larger inversions A1 compared to clade B. Gene clusters 1, 3, and 27 putatively in other areas of clade A1 and A2 genomes were also observed, encode proteins for PTS systems and enzymes for the interconver- including a 1.2-Mbp inversion in both EnGen0046 and En- sion and metabolism of lactose/cellobiose, glucose, mannose, Gen0049, and again appear to be driven by recombination within N-acetylneuraminate, N-acetylmannosamine, and other sialic ac- phages present at the boundaries. Most genome rearrangements ids. Clusters 1 and 27 are associated with mobile elements. Cluster observed in E. faecium can be linked to the occurrence of mobile 18 (C18), which is also enriched in clade A1 compared to clade B, genetic elements at the boundaries. Select novel rearrangements encodes a three-gene operon for a class II bacteriocin that may be were arbitrarily verified by PCR, and the accuracy of assembly was a colonization factor (Table 1; see Table S2 in the supplemental verified in each case. material). In addition to mediating inversions and recombinations, in- Bifurcation of clade A parallels the proliferation of resis- troduction and proliferation of IS elements in a bacterial popula- tance. To understand the role that antibiotics played as a driver of tion can facilitate adaptation to new niches as the result of obsolete clade formation, we examined the differential presence of resis- gene inactivation (1). We identified 133 instances of IS element- tance genes (see Table S3 in the supplemental material). Two re- mediated gene inactivation in E. faecium (see Table S4 in the sup- sistance genes [aac(6’)-li conferring resistance to kanamycin and plemental material). The number of IS-mediated gene inactiva- bacA conferring bacitracin resistance] are part of the core E. fae- tion events was highest in clade A1 genomes and lowest in clade B cium genome. The ubiquitous presence of aac(6’)-li has been ob- strains. In clade A1 strains, we found a strong enrichment for served before and contributes to the intrinsic resistance of E. fae- disruption of a core gene encoding a putative major facilitator cium to several aminoglycosides (37). The bacA gene may be superfamily (MFS) transporter (EFAU004_02447 in strain responsible for intrinsic resistance to bacitracin observed among AUS0004) (Table S4). E. faecium (38). Seven strains analyzed were isolated in the 1950s Since compromised defense was associated with the evolution and 1960s, allowing for the identification of genes associated with of hospital epidemic strains of E. faecalis (25), it was of interest to some of the earliest known acquired resistances to occur in E. fae- examine more closely the relationship between the presence of a cium. Strains EnGen0025, EnGen0027, EnGen0031, EnGen0032, CRISPR-Cas system and mobile element content. We therefore and E1636 were isolated between 1957 and 1965; these strains fall examined the 73 E. faecium genome sequences studied for the into clade A2. Each of these strains also possesses the fusA fusidic presence of CRISPR-cas using CRISPRfinder (39). Only 7 E. fae- acid resistance gene. Additionally, strains EnGen0025, En- cium genomes carried cas genes (Fig. 5), and in 5 of these (strains Gen0027, EnGen0031, and E1636 possess the msrC gene, which Com12, EnGen0002, EnGen0056, 1_141_733, and 1_231_408), a confers erythromycin resistance. Strain EnGen0025 additionally CRISPR array could be readily identified immediately down- acquired the aminoglycoside resistance genes ant(6’)-la (confer- stream. In strains 1_231_408 and EnGen0056, where spacers ring resistance to streptomycin) and aph(3=)-III (conferring resis- could be matched to known genes, one was derived from a phage tance to several aminoglycosides, including neomycin and genta- that is a common lysogen in E. faecium genomes (present in 39 out micin B), ermB, and tetM. As shown in Fig. 2, this strain (the fifth of 73 genomes). Interestingly, this phage is absent from these 2 strain from the top of clade A2) is closely related to the clade A1 genomes, suggesting CRISPR-Cas functionality. Notably, all branch point and presumably the clade A1 founder. strains that carry cas genes are either found in a distinct subgroup Other resistances exhibit clear clade specificity (Fig. 5; see Ta- within clade B or are hybrid strains 1_231_408 and EnGen0002 ble S3 in the supplemental material). Vancomycin resistance is that acquired the cas genes and its associated CRISPR-locus from completely absent from clade B. Vancomycin resistance occurs the clade B parent (see Fig. S4 in the supplemental material). Apart mainly in clade A1 but also occurs in clade A2. Aminoglycoside from the CRISPR defense, we observed a gene encoding a putative resistance genes ant(6’)-la and aph(3=)-III are completely absent type IV methyl-directed restriction enzyme in strains of both clade from clade B strains, but they occur in most clade A1 isolates. B and A2, but not in clade A1 genomes (Fig. 5). Interestingly, in clade B, the msrC resistance gene correlates per- Evidence of varying selection in genomes from each clade. fectly with the presence of a CRISPR element. We have not found We examined polymorphisms in shared genes to detect genes un- prior mention of the occurrence of several resistance genes in der particularly strong selection in the different habitats occupied E. faecium, including the aadD cassette, which confers resistance by strains of each clade. Because of the clade structure, we used a to tobramycin and kanamycin, in a single genome (strain En- tree-based approach (40) to compare the ratios of nonsynony- Gen0035). We also observed genes lnuB, ermG, and ermT (that mous to synonymous base changes (dN/dS ratio). We removed likely confer various degrees of resistance to the macrolides- potentially confounding (41) recombined fragments using BRAT- lincosamides-streptogramin B [MLS] class of antibiotics), tetC NextGen (21). Genes under positive selection were identified (conferring resistance to tetracycline), and fosB (conferring resis- when the dN/dS ratio in the clade of interest (foreground) was tance to fosfomycin) in E. faecium. observed to be significantly higher than the dN/dS ratio in the Clade structure is reflected in E. faecium genome organiza- comparator genomes (background) (see Table S4B in the supple- tion. The Aus0004 genome possesses a previously identified mental material). No genes were found to be under positive selec- 683-kb inversion around the replication termination site (17). tion in clade B compared to clades A, A1, and A2, likely reflecting Similar inversions appear to have occurred several times indepen- the fact that clade B strains had long-fixed beneficial mutations in dently (since boundaries were not strictly identical) in strains of this particular niche before the emergence of the A clade. Only clades A1 and A2 (i.e., in strains EnGen0007 and EnGen0025), but four genes were found to be under differential positive selection not in strains of clade B (see Fig. S6 in the supplemental material). pressure in clade A compared to clade B, two of which were anno- This inversion is bounded by different phages in different strains, tated as having roles in amino acid transport and metabolism and it appears that the recombination responsible for this rear- (Table S4B). Interestingly, in strains of the hospital-adapted clade July/August 2013 Volume 4 Issue 4 e00534-13 mbio.asm.org 7 Lebreton et al. A1, a penicillin binding protein transpeptidase and the D-alanyl- in Gram-negative bacteria has been linked to the emergence of D-alanine ligase were under differential positive selection com- antibiotic-resistant lineages that are pathogenic to humans (50– pared to strains of both clades A2 and clade B (Table S4B). Finally, 52). In Gram-positive bacteria, hypermutating populations of an MFS transporter involved in carbohydrate transport and me- pathogenic Streptococcus pneumoniae and Staphylococcus aureus tabolism in clade A1 and an N-acetylglucosamine transferase in have been observed (53, 54). In E. faecium, polymorphisms in clade A2 were found to be under positive selection pressure, pro- mutS and mutL (which encode DNA mismatch repair proteins) viding independent support for the importance of differential car- have been noted (55), but the polymorphisms are not associated bohydrate utilization as a determinant of clade structure, as in- with differential mutation rates in different clades. Higher muta- ferred from gene gain/loss patterns described above. tion rates have been associated with microbes recently experienc- ing a host switch (e.g., Mycoplasma gallisepticum, 0.8 10 to 1.2 DISCUSSION 10 substitutions per site per year [61]) and with the emer- Speciation results from expansion into new ecological niches and gence of pathogenic lineages (52), possibly including E. faecium subsequent isolation from the founder population (42) and is ac- strains of the CC17 genogroup (56). It appears that the epidemic companied by changes in the genome stemming from mutation, hospital clade A1 emerged because of its ability to acquire mobile recombination (43), and horizontal gene transfer (44). All of these elements, its ability to utilize carbohydrates of nondietary origin, processes have contributed to the current population structure of and its hypermutability. E. faecium and its emergence as a leading multidrug-resistant hos- Previously, the average nucleotide identity of eight E. faecium pital pathogen. strains was determined to range between 93.5 and 95.6% when Quantification of mutation rates for strains in each E. faecium comparing strains from clades A and B (13), and clade A and B clade allowed us to estimate that the first bifurcation in the E. fae- strains would be considered to be distinct species by existing cri- cium population took place approximately 3,000 years ago, sub- teria (57, 58). The identification of hybrid clade A1/B strains stantially sooner than previously suggested (16). Although it is (strains EnGen0002 and 1_231_408) show that the ecological difficult to know the ecological drivers of this split with precision, niches of human-infecting hospital strains and human commen- the timing suggests that it relates to increasing insulation between sal strains do occasionally overlap. The emergence of the distinct the flora of humans and animals, which likely stemmed from in- clade structure in E. faecium parallels anthropogenic changes in creased urbanization, increased domestication of animals provid- urbanization and animal domestication and, more recently, the ing restricted and specialized diets (45, 46), and increasing use of introduction of antibiotics into agriculture and medicine. The net hygienic measures (47, 48). This bifurcation was associated with a effect of these forces is the emergence of a rapidly evolving lineage, wholesale loss and replacement of carbohydrate utilization path- which has crossed a degree of divergence usually associated with ways, mediated largely by acquisition on mobile elements by speciation. strains of clade A. Many of the clade B pathways lost by clade A MATERIALS AND METHODS strains relate to the utilization of complex carbohydrates from Bacterial strains. Strains selected for genome analysis were drawn from dietary sources, and the pathways lost were replaced by pathways those representing diverse points within the known phylogenic structure, on mobile elements associated with the utilization of amino sug- as determined by MLST (Fig. 1), and are listed in Table S1 in the supple- ars, such as those occurring on epithelial cell surfaces and in mu- mental material. DNA was purified from each E. faecium strain as de- cin, suggesting a possible shift from a lifestyle dependent mainly scribed before (13) for DNA sequence analysis. Methods for DNA se- on host diet (clade B) to one increasingly dependent on host se- quencing, genome assembly, and bioinformatic analysis are provided in cretions (clade A). In addition to carbohydrate utilization path- Supplemental Methods at https://olive.broadinstitute.org/projects/work ways, there was a substantial shift in genes encoding Mga-type _package_1/downloads, along with details of the genome sequences. helix-turn-helix regulators, which in Streptococcus pyogenes con- nect expression of niche-specific genes with carbohydrate metab- SUPPLEMENTAL MATERIAL olism (33). Supplemental material for this article may be found at http://mbio.asm.org The second split in the E. faecium population, the split between /lookup/suppl/doi:10.1128/mBio.00534-13/-/DCSupplemental. Figure S1, JPG file, 0.5 MB. clade A1 and clade A2, appears to have occurred approximately Figure S2, JPG file, 1.4 MB. 75 years ago, coinciding precisely with the introduction of antibi- Figure S3, JPG file, 1.5 MB. otics in both clinical medicine and agriculture. However, this split Figure S4, JPG file, 2.6 MB. may not have been directly driven by the usage of antibiotics, as Figure S5, JPG file, 0.7 MB. antibiotics are used both in farming and in human medicine. The Figure S6, JPG file, 6.2 MB. ability to rapidly acquire new traits on mobile elements, including Table S1, DOCX file, 0.1 MB. Table S2, PDF file, 0.6 MB. carbohydrate utilization pathways as well as resistance to antibi- Table S3, PDF file, 0.1 MB. otics, appears to be an intrinsic trait of clade A1 and clade A2. Table S4, DOCX file, 0.1 MB. Although clade A1 strains now cause the vast majority of infec- tions (Fig. 2), early clinical isolates from the 1950s and 1960s do ACKNOWLEDGMENTS not cluster in clade A1. The earliest isolation of a strain associated This project was funded in part by the National Institute of Allergy and with an MLST type occurring in clade A1, occurred in 1982 (49). Infectious Diseases, National Institutes of Health, Department of Health That isolate already possessed high-level resistance to gentamicin and Human Services, under contract HHSN272200900018C. Portions of and carried the esp gene. this work were also supported by NIH/NIAID grants AI083214 (Harvard- Interestingly, we found that the recently emergent hospital- wide Program on Antibiotic Resistance), and AI072360. W.V.S. and adapted clade A1 is hypermutable, as reflected in the inferred rate R.J.L.W. were supported by the European Union Seventh Framework of mutation in the genomes, and experimentally. Hypermutation Programme (FP7-HEALTH-2011-single-stage) “Evolution and Transfer 8 mbio.asm.org July/August 2013 Volume 4 Issue 4 e00534-13 Emergence of Multidrug-Resistant Enterococcus faecium of Antibiotic Resistance” (EvoTAR) under grant agreement number WP, Corander J. 2012. Restricted gene flow among hospital subpopula- tions of Enterococcus faecium. mBio 3(4):e00151-12. 15. van Schaik W, Top J, Riley DR, Boekhorst J, Vrijenhoek JEP, Schap- We acknowledge Lucia Alvarado and Clint Howarth for data submis- endonk CME, Hendrickx APA, Nijman IJ, Bonten MJM, Tettelin H, sions, Susanna Hamilton and Sinead Chapman for project management, Willems RJL. 2010. Pyrosequencing-based comparative genome analysis Chris Desjardins for helpful discussions, and Matthew Laird for help with of the nosocomial pathogen Enterococcus faecium and identification of a IslandViewer. large transferable pathogenicity island. BMC Genomics 11:239. 16. Galloway-Peña J, Roh JH, Latorre M, Qin X, Murray BE. 2012. ADDENDUM IN PROOF Genomic and SNP analyses demonstrate a distant separation of the hos- pital and community-associated clades of Enterococcus faecium. PLoS One Following submission we were made aware that others recently described a 7:e30187. doi: 10.1371/journal.pone.0030187. split, between human and bovine populations of S. aureus, datable by BEAST 17. Lam MMC, Seemann T, Bulach DM, Gladman SL, Chen H, Haring V, analysis, to approximately 5,000 years ago (L. A. Weinert, J. J. Welch, M. A. Moore RJ, Ballard S, Grayson ML, Johnson PDR, Howden BP, Stinear Suchard, P. Lemey, A. Rambaut, and J. R. Fitzgerald, Biol Lett. 8:829-832, TP. 2012. Comparative analysis of the first complete Enterococcus faecium 2012). genome. J. Bacteriol. 194:2334 –2341. 18. Willems RJ, van Schaik W. 2009. Transition of Enterococcus faecium from REFERENCES commensal organism to nosocomial pathogen. Future Microbiol. 1. Gilmore MS, Lebreton F, van Schaik W. 2013. Genomic transition of 4:1125–1135. enterococci from gut commensals to leading causes of multidrug-resistant 19. De Regt MJA, van Schaik W, van Luit-Asbroek M, Dekker HAT, van hospital infection in the antibiotic era. Curr. Opin. Microbiol. 16:10 –16. Duijkeren E, Koning CJM, Bonten MJM, Willems RJL. 2012. Hospital 2. McKinnell JA, Kunz DF, Chamot E, Patel M, Shirley RM, Moser SA, and community ampicillin-resistant Enterococcus faecium are evolution- Baddley JW, Pappas PG, Miller LG. 2012. Association between arily closely linked but have diversified through niche adaptation. PLoS vancomycin-resistant enterococci bacteremia and ceftriaxone usage. In- One 7:e30319. doi: 10.1371/journal.pone.0030319. fect. Control Hosp. Epidemiol. 33:718 –724. 20. Drummond AJ, Suchard MA, Xie D, Rambaut A. 2012. Bayesian phy- 3. Arias CA, Murray BE. 2008. Emergence and management of drug- logenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29: resistant enterococcal infections. Expert Rev. Anti Infect. Ther. 1969 –1973. 6:637– 655. 21. Marttinen P, Hanage WP, Croucher NJ, Connor TR, Harris SR, Bentley 4. Smillie CS, Smith MB, Friedman J, Cordero OX, David LA, Alm EJ. SD, Corander J. 2012. Detection of recombination events in bacterial 2011. Ecology drives a global network of gene exchange connecting the genomes from large population samples. Nucleic Acids Res. 40:e6. doi: human microbiome. Nature 480:241–244. 10.1093/nar/gkr928. 5. Harrison EM, Paterson GK, Holden MTG, Larsen J, Stegger M, Larsen 22. Holden MTG, Hsu LY, Kurt K, Weinert LA, Mather AE, Harris SR, AR, Petersen A, Skov RL, Christensen JM, Bak Zeuthen A, Heltberg O, Strommenger B, Layer F, Witte W, de Lencastre H, Skov R, Westh H, Harris SR, Zadoks RN, Parkhill J, Peacock SJ, Holmes MA. 2013. Whole Zemlicková H, Coombs G, Kearns AM, Hill RLR, Edgeworth J, Gould genome sequencing identifies zoonotic transmission of MRSA isolates I, Gant V, Cooke J, Edwards GF, McAdam PR, Templeton KE, McCann with the novel mecA homologue mecC. EMBO Mol. Med. 5:509 –515. A, Zhou Z, Castillo-Ramírez S, Feil EJ, Hudson LO, Enright MC, 6. Price LB, Stegger M, Hasman H, Aziz M, Larsen J, Andersen PS, Balloux F, Aanensen DM, Spratt BG, Fitzgerald JR, Parkhill J, Achtman Pearson T, Waters AE, Foster JT, Schupp J, Gillece J, Driebe E, Liu CM, M, Bentley SD, Nübel U. 2013. A genomic portrait of the emergence, Springer B, Zdovc I, Battisti A, Franco A, Zmudzki J, Schwarz S, Butaye evolution, and global spread of a methicillin-resistant Staphylococcus au- P, Jouy E, Pomba C, Porrero MC, Ruimy R, Smith TC, Robinson DA, reus pandemic. Genome Res. 23:653– 664. Weese JS, Arriola CS, Yu F, Laurent F, Keim P, Skov R, Aarestrup FM. 23. Nübel U, Dordel J, Kurt K, Strommenger B, Westh H, Shukla SK, 2012. Staphylococcus aureus CC398: host adaptation and emergence of Žemliková H, Leblois R, Wirth T, Jombart T, Balloux F, Witte W. methicillin resistance in livestock. mBio 3(1):e00305-11. 2010. A timescale for evolution, population expansion, and spatial spread 7. Acar J, Casewell M, Freeman J, Friis C, Goossens H. 2000. Avoparcin of an emerging clone of methicillin-resistant Staphylococcus aureus. PLoS and virginiamycin as animal growth promoters: a plea for science in Pathog. 6:e1000855. doi:10.1371/journal.ppat.1000855. decision-making. Clin. Microbiol. Infect. 6:477– 482. 24. Dagan T, Martin W. 2007. Ancestral genome sizes specify the minimum 8. Collignon PJ. 1999. Vancomycin-resistant enterococci and use of avopar- rate of lateral gene transfer during prokaryote evolution. Proc. Natl. Acad. cin in animal feed: is there a link? Med. J. Aust. 171:144 –146. Sci. USA 104:870 – 875. 9. Bager F, Madsen M, Christensen J, Aarestrup FM. 1997. Avoparcin used 25. Palmer KL, Gilmore MS. 2010. Multidrug-resistant enterococci lack as a growth promoter is associated with the occurrence of vancomycin- CRISPR-cas. mBio 1(4):e00227-10. resistant Enterococcus faecium on Danish poultry and pig farms. Prev. Vet. 26. Zhou Y, Liang Y, Lynch KH, Dennis JJ, Wishart DS. 2011. PHAST: a fast Med. 31:95–112. phage search tool. Nucleic Acids Res. 39:W347–W352. 10. Lauderdale TL, Shiau YR, Wang HY, Lai JF, Huang IW, Chen PC, Chen 27. Waack S, Keller O, Asper R, Brodag T, Damm C, Fricke WF, Surovcik HY, Lai SS, Liu YF, Ho M. 2007. Effect of banning vancomycin analogue K, Meinicke P, Merkl R. 2006. Score-based prediction of genomic islands avoparcin on vancomycin-resistant enterococci in chicken farms in Tai- in prokaryotic genomes using hidden Markov models. BMC Bioinformat- wan. Environ. Microbiol. 9:819 – 823. ics 7:142. 11. Willems RJL, Top J, van Santen M, Robinson DA, Coque TM, Baquero 28. Jensen LB, Garcia-Migura L, Valenzuela AJS, Løhr M, Hasman H, F, Grundmann H, Bonten MJM. 2005. Global spread of vancomycin- Aarestrup FM. 2010. A classification system for plasmids from entero- resistant Enterococcus faecium from distinct nosocomial genetic complex. cocci and other Gram-positive bacteria. J. Microbiol. Methods 80:25– 43. Emerg. Infect. Dis. 11:821– 828. 29. Kjos M, Borrero J, Opsata M, Birri DJ, Holo H, Cintas LM, Snipen L, 12. Kos VN, Desjardins CA, Griggs A, Cerqueira G, Van Tonder A, Holden Hernández PE, Nes IF, Diep DB. 2011. Target recognition, resistance, MTG, Godfrey P, Palmer KL, Bodi K, Mongodin EF, Wortman J, immunity and genome mining of class II bacteriocins from Gram-positive Feldgarden M, Lawley T, Gill SR, Haas BJ, Birren B, Gilmore MS. 2012. bacteria. Microbiology 157:3256 –3267. Comparative genomics of vancomycin-resistant Staphylococcus aureus 30. Hendrickx APA, van Luit-Asbroek M, Schapendonk CME, van Wamel strains and their positions within the clade most commonly associated WJB, Braat JC, Wijnands LM, Bonten MJM, Willems RJL. 2009. SgrA, with methicillin-resistant S. aureus hospital-acquired infection in the a nidogen-binding LPXTG surface adhesin implicated in biofilm forma- United States. mBio 3(3):e00112-12. tion, and EcbA, a collagen binding MSCRAMM, are two novel adhesins of 13. Palmer KL, Godfrey P, Griggs A, Kos VN, Zucker J, Desjardins C, hospital-acquired Enterococcus faecium. Infect. Immun. 77:5097–5106. Cerqueira G, Gevers D, Walker S, Wortman J, Feldgarden M, Haas B, 31. Dussurget O, Cabanes D, Dehoux P, Lecuit M, Buchrieser C, Glaser P, Birren B, Gilmore MS. 2012. Comparative genomics of enterococci: variation in Enterococcus faecalis, clade structure in E. faecium, and defin- Cossart P, European Listeria Genome Consortium. 2002. Listeria mono- ing characteristics of E. gallinarum and E. casseliflavus. mBio 3(1):e00318- cytogenes bile salt hydrolase is a PrfA-regulated virulence factor involved in 11. the intestinal and hepatic phases of listeriosis. Mol. Microbiol. 45: 14. Willems RJL, Top J, van Schaik W, Leavis H, Bonten M, Sirén J, Hanage 1095–1106. July/August 2013 Volume 4 Issue 4 e00534-13 mbio.asm.org 9 Lebreton et al. 32. Novick RP, Geisinger E. 2008. Quorum sensing in staphylococci. Annu. BE. 2009. Analysis of clonality and antibiotic resistance among early clin- Rev. Genet. 42:541–564. ical isolates of Enterococcus faecium in the United States. J. Infect. Dis. 33. Hondorp ER, McIver KS. 2007. The Mga virulence regulon: infection 200:1566 –1573. where the grass is greener. Mol. Microbiol. 66:1056 –1065. 50. LeClerc JE, Li B, Payne WL, Cebula TA. 1996. High mutation frequen- 34. Simeone R, Bottai D, Brosch R. 2009. ESX/type VII secretion systems and cies among Escherichia coli and Salmonella pathogens. Science 274: their role in host-pathogen interaction. Curr. Opin. Microbiol. 12:4 –10. 1208 –1211. 35. Chen YH, Anderson M, Hendrickx APA, Missiakas D. 2012. Charac- 51. Jolivet-Gougeon A, Kovacs B, Le Gall-David S, Le Bars H, Bousarghin terization of EssB, a protein required for secretion of ESAT-6 like proteins L, Bonnaure-Mallet M, Lobel B, Guillé F, Soussy CJ, Tenke P. 2011. in Staphylococcus aureus. BMC Microbiol. 12:219. Bacterial hypermutation: clinical implications. J. Med. Microbiol. 60: 36. Zhang X, Top J, de Been M, Bierschenk D, Rogers M, Leendertse M, 563–573. Bonten MJ, van der Poll T, Willems RJ, van Schaik W. 2013. Identifi- 52. Maciá MD, Blanquer D, Togores B, Sauleda J, Pérez JL, Oliver A. 2005. cation of a genetic determinant in clinical Enterococcus faecium strains that Hypermutation is a key factor in development of multiple-antimicrobial contributes to intestinal colonization during antibiotic treatment. J. In- resistance in Pseudomonas aeruginosa strains causing chronic lung infec- fect. Dis. 207:1780 –1786. tions. Antimicrob. Agents Chemother. 49:3382–3386. 37. Costa Y, Galimand M, Leclercq R, Duval J, Courvalin P. 1993. Char- 53. del Campo R, Morosini MI, de la Pedrosa EG, Fenoll A, Muñoz- acterization of the chromosomal aac(6=)-Ii gene specific for Enterococcus Almagro C, Máiz L, Baquero F, Cantón R, Spanish Pneumococcal faecium. Antimicrob. Agents Chemother. 37:1896 –1903. Infection Study Network. 2005. Population structure, antimicrobial re- 38. Bywater R, McConville M, Phillips I, Shryock T. 2005. The susceptibility sistance, and mutation frequencies of Streptococcus pneumoniae isolates to growth-promoting antibiotics of Enterococcus faecium isolates from from cystic fibrosis patients. J. Clin. Microbiol. 43:2207–2214. pigs and chickens in Europe. J. Antimicrob. Chemother. 56:538 –543. 54. Prunier AL, Malbruny B, Laurans M, Brouard J, Duhamel JF, Leclercq 39. Grissa I, Vergnaud G, Pourcel C. 2007. CRISPRFinder: a web tool to R. 2003. High rate of macrolide resistance in Staphylococcus aureus strains identify clustered regularly interspaced short palindromic repeats. Nucleic from patients with cystic fibrosis reveals high proportions of hypermut- Acids Res. 35:W52–W57. able strains. J. Infect. Dis. 187:1709 –1716. 40. Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phy- 55. Willems RJ, Top J, Smith DJ, Roper DI, North SE, Woodford N. 2003. logenetic analyses with thousands of taxa and mixed models. Bioinformat- Mutations in the DNA mismatch repair proteins MutS and MutL of ics 22:2688 –2690. oxazolidinone-resistant or -susceptible Enterococcus faecium. Antimicrob. 41. Anisimova M, Nielsen R, Yang Z. 2003. Effect of recombination on the Agents Chemother. 47:3061–3066. accuracy of the likelihood method for detecting positive selection at amino 56. Ruiz-Garbajosa P, Top J, Coque TM, Cantón R, Bonten MJ, Baquero F, acid sites. Genetics 164:1229 –1236. Willems RJ. 2008. Abstr. 18th Eur. Cong. Clin Microbiol. Infect. Dis., 42. Cohan FM. 2001. Bacterial species and speciation. Syst. Biol. 50:513–524. abstr. P2043. 43. Fraser C, Hanage WP, Spratt BG. 2007. Recombination and the nature of 57. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, bacterial speciation. Science 315:476 – 480. Tiedje JM. 2007. DNA-DNA hybridization values and their relationship 44. Wiedenbeck J, Cohan FM. 2011. Origins of bacterial diversity through to whole-genome sequence similarities. Int. J. Syst. Evol. Microbiol. 57: horizontal genetic transfer and adaptation to new ecological niches. FEMS 81–91. Microbiol. Rev. 35:957–976. 58. Konstantinidis KT, Tiedje JM. 2005. Genomic insights that advance the 45. Petroutsa EI, Manolis SK. 2010. Reconstructing Late Bronze Age diet in species definition for prokaryotes. Proc. Natl. Acad. Sci. U. S. A. 102: mainland Greece using stable isotope analysis. J. Archaeol. Sci. 37: 2567–2572. 614 – 620. 59. Tettelin H, Riley D, Cattuto C, Medini D. 2008. Comparative genomics: 46. Jay M, Richards MP. 2006. Diet in the Iron Age cemetery population at the bacterial pan-genome. Curr. Opin. Microbiol. 11:472– 477. Wetwang Slack, East Yorkshire, UK: carbon and nitrogen stable isotope 60. Chang D, Zhu Y, Zou Y, Fang X, Li T, Wang J, Guo Y, Su L, Xia J, Yang evidence. J. Archaeol. Sci. 33:653– 662. R, Fang C, Liu C. 2012. Draft genome sequence of Enterococcus faecium 47. McEvedy C, Jones R. 1978. Atlas of world population history. Penguin strain LCT-EF90. J. Bacteriol. 194:3556 –3557. Books, Middlesex, United Kingdom. 61. Delaney NF, Balenger S, Bonneaud C, Marx CJ, Hill GE, Ferguson-Noel 48. Osborne R, Cunliffe B. 2005. Mediterranean urbanization 800 – 600 BC. N, Tsai P, Rodrigo A, Edwards SV. 2012. Ultrafast evolution and loss of Oxford University Press, Oxford, United Kingdom. CRISPRs following a host shift in a novel wildlife pathogen, Mycoplasma 49. Galloway-Peña JR, Nallapareddy SR, Arias CA, Eliopoulos GM, Murray gallisepticum. PLoS Genet. 8:e1002511. 10 mbio.asm.org July/August 2013 Volume 4 Issue 4 e00534-13

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