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Global spread of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex.

Vancomycin-resistant enterococci (VRE) have caused hospital outbreaks worldwide, and the vancomycin-resistance gene (vanA) has crossed genus boundaries to methicillin-resistant Staphylococcus aureus. Spread of VRE, therefore, represents an immediate threat for patient care and creates a reservoir of mobile resistance genes for other, more virulent pathogens. Evolutionary genetics, population structure, and geographic distribution of 411 VRE and vancomycin-susceptible Enterococcus faecium isolates, recovered from human and nonhuman sources and community and hospital reservoirs in 5 continents, identified a genetic lineage of E. faecium (complex-17) that has spread globally. This lineage is characterized by 1) ampicillin resistance, 2) a pathogenicity island, and 3) an association with hospital outbreaks. Complex-17 is an example of cumulative evolutionary processes that improved the relative fitness of bacteria in hospital environments. Preventing further spread of this epidemic E. faecium subpopulation is critical, and efforts should focus on the early disclosure of ampicillin-resistant complex-17 strains.

Variant esp gene as a marker of a distinct genetic lineage of vancomycin-resistant Enterococcus faecium spreading in hospitals

In the USA, vancomycin-resistant Enterococcus faecium (VREF) is endemic in hospitals, despite lack of carriage among healthy individuals. In Europe, however, hospital outbreaks are rare, but VREF carriage among healthy individuals and livestock is common. We used amplified fragment-length polymorphism analysis to genotype 120 VREF isolates associated with hospital outbreaks and 45 non-epidemic isolates from the USA, Europe, and Australia. We also looked for the esp virulence gene in these isolates and in 98 VREF from animals. A specific E. faecium subpopulation genetically distinct from non-epidemic VREF isolates was found to be the cause of the hospital epidemics in all three continents. This subpopulation contained a variant of the esp gene that was absent in all non-epidemic and animal isolates. Identification of the variant esp gene will be important in guiding infection-control strategies, and the Esp protein could be a new target for antibacterial therapy.

Multilocus Sequence Typing Scheme for Enterococcus faecalis Reveals Hospital-Adapted Genetic Complexes in a Background of High Rates of Recombination

ABSTRACT A multilocus sequence typing (MLST) scheme based on seven housekeeping genes was used to investigate the epidemiology and population structure of Enterococcus faecalis. MLST of 110 isolates from different sources and geographic locations revealed 55 different sequence types that grouped into four major clonal complexes (CC2, CC9, CC10, and CC21) by use of eBURST. Two of these clonal complexes, CC2 and CC9, are particularly fit in the hospital environment, as CC2 includes the previously described BVE clonal complex identified by an alternative MLST scheme and CC9 includes exclusively isolates from hospitalized patients. Identical alleles were found in genetically diverse isolates with no linkage disequilibrium, while the different MLST loci gave incongruent phylogenetic trees. This demonstrates that recombination is an important mechanism driving genetic variation in E. faecalis and suggests an epidemic population structure for E. faecalis. Our novel MLST scheme provides an excellent tool for investigating local and short-term epidemiology as well as global epidemiology, population structure, and genetic evolution of E. faecalis.

A Novel Putative Enterococcal Pathogenicity Island Linked to the esp Virulence Gene of Enterococcus faecium and Associated with Epidemicity

Enterococcus faecalis harbors a virulence-associated surface protein encoded by the esp gene. This gene has been shown to be part of a 150-kb putative pathogenicity island. A gene similar to esp has recently been found in Enterococcus faecium isolates recovered from hospitalized patients. In the present study we analyzed the polymorphism in the esp gene of E. faecium, and we investigated the association of esp with neighboring chromosomal genes. The esp gene showed considerable sequence heterogeneity in the regions encoding the nonrepeat N- and C-terminal domains of the Esp protein as well as differences in the number of repeats. DNA sequencing of chromosomal regions flanking the esp gene of E. faecium revealed seven open reading frames, representing putative genes implicated in virulence, regulation of transcription, and antibiotic resistance. These flanking regions were invariably associated with the presence or absence of the esp gene in E. faecium, indicating that esp in E. faecium is part of a distinct genetic element. Because of the presence of virulence genes in this gene cluster, the lower G+C content relative to that of the genome, and the presence of esp in E. faecium isolates associated with nosocomial outbreaks and clinically documented infections, we conclude that this genetic element constitutes a putative pathogenicity island, the first one described in E. faecium. Except for the presence of esp and araC, this pathogenicity island is completely different from the esp-containing pathogenicity island previously disclosed in E. faecalis.

Emergence of CC17 Enterococcus faecium: from commensal to hospital‐adapted pathogen

For many years, Enterococcus faecium was considered to be a commensal of the digestive tract, which only sporadically caused opportunistic infections in severely ill patients. Over the last two decades, vancomycin-resistant E. faecium (VREF) has emerged worldwide as an important cause of nosocomial infections, especially in immunocompromised patients. The global Vancomycin-resistant enterococci (VRE) epidemic was preceded by the emergence of ampicillin-resistant E. faecium (AREfm) in the United States in the early 1980s, followed by the rapid emergence of VRE in the 1990s. A similar increase of VRE may occur in countries with still low levels of VRE in hospitals (such as The Netherlands), but increasing incidence of AREfm infections. Molecular epidemiological studies of both human- and animal-derived E. faecium isolates using multilocus sequence typing revealed the existence of host-specific genogroups, including a specific genetic lineage designated CC17, associated with hospital-related isolates. These strains were characterized by ampicillin and quinolone resistance. In addition, the majority of these CC17 isolates contain over hundred hospital-clade-specific genes, including mobile elements, phage genes and plasmid sequences, hypothetical and membrane proteins and antibiotic and regulatory genes and a putative pathogenicity island including the esp gene.

Host Specificity of Vancomycin-Resistant Enterococcus faecium

Amplified-fragment length polymorphism (AFLP) analysis was used to investigate the genetic relationships among 255 vancomycin-resistant Enterococcus faecium (VREF) strains isolated from hospitalized patients, nonhospitalized persons, and various animal sources. Four major AFLP genogroups (A-D) were discriminated. The strains of each taxon shared >/=65% of the restriction fragments. Most isolates recovered from nonhospitalized persons (75%) were grouped together with all pig isolates in genogroup A. Most isolates from hospitalized patients (84%), a subset of veal calf isolates (25%), and all isolates from cats and dogs clustered in genogroup C. Most isolates from chickens (97%) and turkeys (86%) were grouped in genogroup B, whereas most veal calf isolates (70%) clustered in genogroup D. Therefore, VREF strains are predominantly host-specific, and strains isolated from hospitalized patients are genetically different from the prevailing VREF strains present in the fecal flora of nonhospitalized persons.

Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island

BackgroundThe Gram-positive bacterium Enterococcus faecium is an important cause of nosocomial infections in immunocompromized patients.ResultsWe present a pyrosequencing-based comparative genome analysis of seven E. faecium strains that were isolated from various sources. In the genomes of clinical isolates several antibiotic resistance genes were identified, including the vanA transposon that confers resistance to vancomycin in two strains. A functional comparison between E. faecium and the related opportunistic pathogen E. faecalis based on differences in the presence of protein families, revealed divergence in plant carbohydrate metabolic pathways and oxidative stress defense mechanisms. The E. faecium pan-genome was estimated to be essentially unlimited in size, indicating that E. faecium can efficiently acquire and incorporate exogenous DNA in its gene pool. One of the most prominent sources of genomic diversity consists of bacteriophages that have integrated in the genome. The CRISPR-Cas system, which contributes to immunity against bacteriophage infection in prokaryotes, is not present in the sequenced strains. Three sequenced isolates carry the esp gene, which is involved in urinary tract infections and biofilm formation. The esp gene is located on a large pathogenicity island (PAI), which is between 64 and 104 kb in size. Conjugation experiments showed that the entire esp PAI can be transferred horizontally and inserts in a site-specific manner.ConclusionsGenes involved in environmental persistence, colonization and virulence can easily be aquired by E. faecium. This will make the development of successful treatment strategies targeted against this organism a challenge for years to come.

Restricted Gene Flow among Hospital Subpopulations of Enterococcus faecium

ABSTRACT Enterococcus faecium has recently emerged as an important multiresistant nosocomial pathogen. Defining population structure in this species is required to provide insight into the existence, distribution, and dynamics of specific multiresistant or pathogenic lineages in particular environments, like the hospital. Here, we probe the population structure of E. faecium using Bayesian-based population genetic modeling implemented in Bayesian Analysis of Population Structure (BAPS) software. The analysis involved 1,720 isolates belonging to 519 sequence types (STs) (491 for E. faecium and 28 for Enterococcus faecalis). E. faecium isolates grouped into 13 BAPS (sub)groups, but the large majority (80%) of nosocomial isolates clustered in two subgroups (2-1 and 3-3). Phylogenetic and eBURST analysis of BAPS groups 2 and 3 confirmed the existence of three separate hospital lineages (17, 18, and 78), highlighting different evolutionary trajectories for BAPS 2-1 (lineage 78) and 3-3 (lineage 17 and lineage 18) isolates. Phylogenomic analysis of 29 E. faecium isolates showed agreement between BAPS assignment of STs and their relative positions in the phylogenetic tree. Odds ratio calculation confirmed the significant association between hospital isolates with BAPS 3-3 and lineages 17, 18, and 78. Admixture analysis showed a scarce number of recombination events between the different BAPS groups. For the E. faecium hospital population, we propose an evolutionary model in which strains with a high propensity to colonize and infect hospitalized patients arise through horizontal gene transfer. Once adapted to the distinct hospital niche, this subpopulation becomes isolated, and recombination with other populations declines. IMPORTANCE Multiresistant Enterococcus faecium has become one of the most important nosocomial pathogens, causing increasing numbers of nosocomial infections worldwide. Here, we used Bayesian population genetic analysis to identify groups of related E. faecium strains and show a significant association of hospital and farm animal isolates to different genetic groups. We also found that hospital isolates could be divided into three lineages originating from sequence types (STs) 17, 18, and 78. We propose that, driven by the selective pressure in hospitals, the three hospital lineages have arisen through horizontal gene transfer, but once adapted to the distinct pathogenic niche, this population has become isolated and recombination with other populations declines. Elucidation of the population structure is a prerequisite for effective control of multiresistant E. faecium since it provides insight into the processes that have led to the progressive change of E. faecium from an innocent commensal to a multiresistant hospital-adapted pathogen. Multiresistant Enterococcus faecium has become one of the most important nosocomial pathogens, causing increasing numbers of nosocomial infections worldwide. Here, we used Bayesian population genetic analysis to identify groups of related E. faecium strains and show a significant association of hospital and farm animal isolates to different genetic groups. We also found that hospital isolates could be divided into three lineages originating from sequence types (STs) 17, 18, and 78. We propose that, driven by the selective pressure in hospitals, the three hospital lineages have arisen through horizontal gene transfer, but once adapted to the distinct pathogenic niche, this population has become isolated and recombination with other populations declines. Elucidation of the population structure is a prerequisite for effective control of multiresistant E. faecium since it provides insight into the processes that have led to the progressive change of E. faecium from an innocent commensal to a multiresistant hospital-adapted pathogen.

Multiple-Locus Variable-Number Tandem Repeat Analysis, a Novel Typing Scheme To Study the Genetic Relatedness and Epidemiology of Enterococcus faecium Isolates

ABSTRACT Multiresistant Enterococcus faecium is a major cause of hospital acquired infections and outbreaks. Here, we describe the development of multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) as a novel typing method to assess the genetic relatedness of E. faecium isolates. Six VNTR loci were used to genotype 392 isolates recovered from different animals and human community, hospital survey, and clinical isolates. From 3 to 13 alleles were found per locus, resulting in 127 different MLVA profiles. Clustering of MLVA profiles confirmed the host-specific genogroups found by multilocus sequence typing (MLST) and showed the grouping of clinical and epidemic isolates that belonged to the MLST-C1 cluster in a distinct MLVA-C1 cluster (sensitivity of 97% and specificity of 90%). Furthermore, the discriminatory power of MLVA is comparable to MLST. MLVA profiles appeared to be relatively stable, since isolates from a single outbreak shared the same MLVA profile, which is a prerequisite when MLVA is used to study hospital outbreaks. Our data show that MLVA is a highly reproducible and portable typing method; in contrast to MLST, it is fast, relatively cheap, and easy to perform. Furthermore, it has the abilities of MLST to recognize genetically related and potential epidemic isolates. Submission of MLVA profiles is possible via a Web-based database for international comparison.

Epidemic and Nonepidemic Multidrug-Resistant Enterococcus faecium

The epidemiology of vancomycin-resistant Enterococcus faecium (VREF) in Europe is characterized by a large community reservoir. In contrast, nosocomial outbreaks and infections (without a community reservoir) characterize VREF in the United States. Previous studies demonstrated host-specific genogroups and a distinct genetic lineage of VREF associated with hospital outbreaks, characterized by the variant esp-gene and a specific allele-type of the purK housekeeping gene (purK1). We investigated the genetic relatedness of vanA VREF (n=108) and vancomycin-susceptible E. faecium (VSEF) (n=92) from different epidemiologic sources by genotyping, susceptibility testing for ampicillin, sequencing of purK1, and testing for presence of esp. Clusters of VSEF fit well into previously described VREF genogroups, and strong associations were found between VSEF and VREF isolates with resistance to ampicillin, presence of esp, and purK1. Genotypes characterized by presence of esp, purK1, and ampicillin resistance were most frequent among outbreak-associated isolates and almost absent among community surveillance isolates. Vancomycin-resistance was not specifically linked to genogroups. VREF and VSEF from different epidemiologic sources are genetically related; evidence exists for nosocomial selection of a subtype of E. faecium, which has acquired vancomycin-resistance through horizontal transfer.