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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.Although classically considered a commensal of the gastrointestinal tracts of humans and animals rather than a specialized human pathogen, enterococci have become extremely relevant in hospital-acquired infections. Their ability to acquire specific genetic traits, such as virulence and antibiotic resistance determinants that could increase their fitness in such a complex ecosystem, has been recognized (18). The paradigm of this evolutionary development is the emergence and spread of vancomycin-resistant enterococci (VRE) (20).Among enterococcal species, Enterococcus faecalis is responsible for most human infections in both community and hospital settings. Though resistance to vancomycin and penicillins is very rare, E. faecalis seems to harbor a broader repertoire of potential virulence traits than E. faecium (34). However, little is known about the relationship between the population structure and global epidemiology of E. faecalis. Different molecular typing methods have been developed to analyze E. faecalis epidemiology (3,11,19,36,37,40). Pulsed-field gel electrophoresis (PFGE) is considered a practical "gold standard" due to its high discriminatory abilities (3, 37), but the most important limitation of PFGE is its low interlaboratory reproducibility and its unsuitability for both global and long-term epidemiology studies or for phylogenetic or population structure studies.For many different bacterial species, the most appropriate technique for global and long-term epidemiology studies is multilocus sequence typing (MLST) (38). MLST provides an unambiguous nomenclature for genotypes, and clones and data are easily stored in databases that can be exchanged between different laboratories via the Internet (1). For E. faecium, the development of an MLST scheme has been critical in the understanding of global epidemiology, genetic evolution, and population structure (14,41). A previous ML...

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In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacteriurn tuberculosis in the biochemical tests and in its 16s rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium afiicanurn, M. bovis, and Mycobacteriurn microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".The Mycobactenum tuberculosis complex constitutes a genetically closely related group, and its members, M. tuberculosis, Mycobactenum afncanum, Mycobactenurn bovis, and Mycobacterium microti, may be considered as subspecies of M. tuberculosis (39, 47). The close relatedness between M. tuberculosis complex bacteria has been established by DNA-DNA hybridization (>95%) (l), multiple-locus enzyme electrophoresis (15), and Sequencing of 16s ribosomal RNA and housekeeping genes (15). Furthermore, repetitive DNA elements, such as the insertion sequence IS6110 (43) and the direct repeat (DR) (18), have been found restricted to the M. tuberculosis complex, Nevertheless, the host range and pathogenicity of the M. tuberculosis complex species vary enormously. The natural reservoir of M. tuberculosis and M. afncanum is limited to humans (47) and that of M. microti is limited to voles (48). In contrast, the host range of M. bovis is very broad, and this species causes disease among a wide range of wild and domestic mammals as well as in humans (38).Colonies of primary M. tuberculasis cultures almost invariably have a characterist...

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.

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.

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.