Previous studies suggesting a link between Escherichia coli phylogenetic groups and extraintestinal virulence have been hampered by the difficulty in establishing the intrinsic virulence of a bacterial strain. Indeed, unidentified virulence factors do exist, and the susceptibility of the host to infection is highly variable. To overcome these difficulties, we have developed a mouse model of extraintestinal virulence to test the virulence of the strains under normalized conditions. We then assessed the phylogenetic relationships compared to the E. coli reference (ECOR) collection, the presence of several known virulence determinants, and the lethality to mice of 82 human adult E. coli strains isolated from normal feces and during the course of extraintestinal infections. Commensal strains belong mainly to phylogenetic groups A and B1, are devoid of virulence determinants, and do not kill the mice. Strains exhibiting the same characteristics as the commensal strains can be isolated under pathogenic conditions, thus indicating the role of host-dependent factors, such as susceptibility linked to underlying disease, in the development of infection. Some strains of phylogenetic groups A, B1, and D are able to kill the mice, their virulence being most often correlated with the presence of virulence determinants. Lastly, strains of the B2 phylogenetic group represent a divergent lineage of highly virulent strains which kill the mice at high frequency and possess the highest level of virulence determinants. The observed link between virulence and phylogeny could correspond to the necessity of virulence determinants in a genetic background that is adequate for the emergence of a virulent clone, an expression of the interdependency of pathogenicity and metabolic activities in pathogenic bacteria.

The selective pressures leading to the evolution and maintenance of virulence in the case of facultative pathogens are quite unclear. For example, Escherichia coli, a commensal of the gut of warm-blooded animals and humans, can cause severe extraintestinal diseases, such as septicemia and meningitis, which represent evolutionary dead ends for the pathogen as they are associated to rapid host death and poor interhost transmission. Such infectious process has been linked to the presence of so-called "virulence genes." To understand the evolutionary forces that select and maintain these genes, we focused our study on E. coli B2 phylogenetic group strains that encompass both commensal and pathogenic (extra- and intraintestinal) strains. Multilocus sequence typing (MLST), comparative genomic hybridization of the B2 flexible gene pool, and quantification of extraintestinal virulence using a mouse model of septicemia were performed on a panel of 60 B2 strains chosen for their genetic and ecologic diversity. The phylogenetic history of the strains reconstructed from the MLST data indicates the emergence of at least 9 subgroups of strains. A high polymorphism is observed in the B2 flexible gene pool among the strains with a good correlation between the MLST-inferred phylogenetic history of the strains and the presence/absence of specific genomic regions, indicating coevolution between the chromosomal background and the flexible gene pool. Virulence in the mouse model is a highly prevalent and widespread character present in all subgroups except one. Association studies reveal that extraintestinal virulence is a multigenic process with a common set of "virulence determinants" encompassing genes involved in transcriptional regulation, iron metabolism, adhesion, lipopolysaccharide (LPS) biosynthesis, and the recently reported peptide polyketide hybrid synthesis system. Interestingly, these determinants can also be viewed as intestinal colonization and survival factors linked to commensalism as they can increase the fitness of the strains within the normal gut environment. Altogether, these data argue for an ancestral emergence of the extraintestinal virulence character that is a coincidental by-product of commensalism. Furthermore, the phenotypic and genotypic markers identified in this work will allow further epidemiological studies devoted to test the niche specialization hypothesis for the B2 phylogenetic subgroups.

The study of several Escherichia coli intestinal commensal isolates per individual in 265 healthy human subjects belonging to seven populations distributed worldwide showed that the E. coli population is highly structured, with major differences between the tropical and temperate populations.Escherichia coli is a commensal inhabitant of the intestinal tracts of healthy humans and many animal species, but it can also cause a wide range of diseases, ranging from diarrhea to extraintestinal infections (8). As it has been proposed that pathogenic E. coli strains are derived from commensal strains by the acquisition of chromosomal or extrachromosomal virulence operons (19), identifying the factors that shape the genetic structure of commensal strains might help us understand the emergence of virulence. E. coli can be considered to have a clonal genetic structure with a low level of recombination (7, 10). Four main phylogenetic groups, A, B1, B2, and D, constitute the bulk of the species (15). A few authors have examined commensal strains from humans (3-5, 13) using population genetics molecular tools (for a pioneer review, see reference 14). A significant locale-specific distribution among groups A, B1, B2, and D has been observed in human commensal strains in three geographically distinct human populations (French, Croatian, and Malian) (9).To gain insight into the composition of the E. coli human commensal microbiota, we characterized the relative abundance of E. coli phylogenetic groups in a large collection of 1,740 isolates from 265 subjects belonging to seven populations spread over three continents and compared the results to those of previous studies using the same approach.Bacterial isolates. Isolates were collected between 1999 and 2001 from seven human populations composed of healthy adult subjects of both sexes from 15 to 65 years of age, except when otherwise stated. The populations were the following: (i) 27 subjects living in the Paris area (mainland France, Europe), (ii) 21 university students living in Brest (Brittany, mainland France), (iii) 25 bank and insurance workers (BIW) living in seven distinct areas of Brittany (mainland France), (iv) 25 pig farmers (PF) living in the same seven areas as the BIW, (v) 93 ethnically homogeneous Wayampi Amerindians living in three villages of southern French Guyana (South America) with no modern sanitary or hygienic facilities, (vi) 46 women living in Cotonou (Benin, Africa), and (vii) 28 subjects living in Bogotá (Colombia, South America). The individuals in the BIW and PF populations in Brittany were matched for county of residence, age (20 to 60 years), and sex (13 men and 12 women). A subset of 25 Amerindians was also matched for age and sex with the BIW and PF populations. These matched populations had neither been hospitalized nor had taken antibiotics for at least 1 month before stool sampling. In all, 1,740 E. coli isolates were obtained after plating fresh fecal samples on Drigalski agar, with 5 or 10 randomly chosen E. coli isolates per individual.The...

To identify forces shaping the Escherichia coli intraspecies ecological structure, we have characterized in terms of phylogenetic group (A, B1, D and B2) belonging, presence/absence of extraintestinal virulence genes (pap, sfa, hly and aer) and intra-host phylotype diversity a collection of 1898 commensal isolates originating from 387 animals (birds and mammals) sampled in the 1980s and the 2000s. These data have been compared with 760 human commensal isolates, sampled from 152 healthy subjects in the 2000s, and analysed with the same approach. The prevalence of the E. coli phylogenetic groups in birds, non-human mammals and humans is clearly different with a predominance of D/B1, A/B1 and A/B2 strains respectively. A major force shaping the ecological structure is the environment with a strong effect of domestication and the year of sampling followed by the climate. Host characteristics, as the diet and body mass, also influence the ecological structure. Human microbiota are characterized by a higher prevalence of virulence genes and a lower intra-host diversity than the non-human mammal ones. This work identifies for the first time a group of strains specific to the animals, the B1 phylogenetic group strains exhibiting the hly gene. In conclusion, a complex network of factors seems to shape the ecological structure of commensal E. coli, with anthropogenic factors playing a major role and perturbing natural niche equilibrium.

The Yersinia high-pathogenicity island (HPI) encodes an iron uptake system mediated by the siderophore yersiniabactin (Ybt) and confers the virulence of highly pathogenic Yersinia species. This HPI is also widely distributed in human pathogenic members of the family of Enterobacteriaceae, above all in extraintestinal pathogenic Escherichia coli (ExPEC). In the present study we demonstrate a highly significant correlation of a functional HPI and extraintestinal virulence in E. coli. Moreover, using a mouse infection model, we show for the first time that the HPI contributes to the virulence of ExPEC.