Olivier Clermont

Identification and characterization of a spinal muscular atrophy-determining gene

Spinal muscular atrophy (SMA) is a common fatal autosomal recessive disorder characterized by degeneration of lower motor neurons, leading to progressive paralysis with muscular atrophy. The gene for SMA has been mapped to chromosome 5q13, where large-scale deletions have been reported. We describe here the inverted duplication of a 500 kb element in normal chromosomes and narrow the critical region to 140 kb within the telomeric region. This interval contains a 20 kb gene encoding a novel protein of 294 amino acids. An highly homologous gene is present in the centromeric element of 95% of controls. The telomeric gene is either lacking or interrupted in 226 of 229 patients, and patients retaining this gene (3 of 229) carry either a point mutation (Y272C) or short deletions in the consensus splice sites of introns 6 and 7. These data suggest that this gene, termed the survival motor neuron (SMN) gene, is an SMA-determining gene.

Rapid and simple determination of the Escherichia coli phylogenetic group.

ABSTRACT Phylogenetic analysis has shown that Escherichia coliis composed of four main phylogenetic groups (A, B1, B2, and D) and that virulent extra-intestinal strains mainly belong to groups B2 and D. Actually, phylogenetic groups can be determined by multilocus enzyme electrophoresis or ribotyping, both of which are complex, time-consuming techniques. We describe a simple and rapid phylogenetic grouping technique based on triplex PCR. The method, which uses a combination of two genes (chuA and yjaA) and an anonymous DNA fragment, was tested with 230 strains and showed excellent correlation with reference methods.

The Clermont Escherichia coli phylo‐typing method revisited: improvement of specificity and detection of new phylo‐groups

There is extensive genetic substructure within the species Escherichia coli. In 2000 a simple triplex PCR method was described by Clermont and colleagues that enables an E. coli isolate to be assigned to one of the phylo-groups A, B1, B2 or D. The growing body of multi-locus sequence data and genome data for E. coli has refined our understanding of E. colis phylo-group structure and eight phylo-groups are now recognized: seven (A, B1, B2, C, D, E, F) belong to E. coli sensu stricto, whereas the eighth is the Escherichia cryptic clade I. Here a new PCR-based method is developed that enables an E. coli isolate to be assigned to one of the eight phylo-groups and which allows isolates that are members of the other cryptic clades (II to V) of Escherichia to be identified. The development of the method is described and the method is validated. Over 95% of E. coli isolates can be correctly assigned to a phylo-group. Two collections of human faecal isolates were screened using the new phylo-group assignment method demonstrating that about 13% of E. coli isolates belong to the newly described phylo-groups C, E, F and clade I.

Commensal Escherichia coli isolates are phylogenetically distributed among geographically distinct human populations.

An intraspecies phylogenetic grouping of 168 human commensal Escherichia coli strains isolated from the stools of three geographically distinct human populations (France, Croatia, Mali) was generated by triplex PCR. The distributions of seven known extraintestinal virulence determinants (ibeA, pap, sfa/foc, afa, hly, cnf1, aer) were also determined by PCR. The data from the three populations were compiled, which showed that strains from phylogenetic groups A (40%) and B1 (34%) were the most common, followed by phylogenetic group D strains (15%). Strains of the phylogenetic group B2 were rare (11%). However, a significant specific distribution for strains of groups A, B1 and B2 within each population was observed, which may indicate the influence of (i) geographic/climatic conditions, (ii) dietary factors and/or the use of antibiotics or (iii) host genetic factors on the commensal flora. Virulence determinants were rarely detected, with only 25.6% of the strains harbouring at least one of the virulence genes tested. The strains with virulence factors most frequently belonged to phylogenetic group B2. The commensal strains of phylogenetic groups A, B1 and D had fewer virulence determinants than pathogenic strains of the corresponding groups when these data were compared with those for previous collections of virulent extraintestinal infection strains studied using the same approach. However, the virulence patterns of commensal and pathogenic B2 phylogenetic group strains were the same. The data thus suggest that strains of the A, B1 and D phylogenetic groups predominate in the gut flora and that these strains must acquire virulence factors to become pathogenic. In contrast, commensal phylogenetic group B2 strains are rare but appear to be potentially virulent.

Assigning Escherichia coli strains to phylogenetic groups: multi-locus sequence typing versus the PCR triplex method.

It is well recognized that Escherichia coli consists of a number of distinct phylo-groups and that strains of the different phylo-groups vary in their ecological niches, life-history characteristics and propensity to cause disease. Consequently, much can be learnt by assigning a strain of E. coli to one of the recognized phylo-groups. A triplex PCR-based method that enables strains of E. coli to be assigned to a phylo-group using a dichotomous key approach based on the presence or absence of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2) has been developed. However, the accuracy with which this method assigns strains to their correct phylo-group has not been adequately evaluated. Consequently, 662 strains of E. coli were characterized using a multi-locus sequence typing approach. Unsupervised population assignment algorithms were used to assign strains to phylo-groups based on the multi-locus sequence typing data. The analyses revealed that 85-90% of E. coli strains can be assigned to a phylo-group and that 80-85% of the phylo-group memberships assigned using the Clermont method are correct. However, the accuracy with which strains are assigned to the correct phylo-group depends on their Clermont genotype. For example, strains yielding a Clermont genotype consistent with phylo-groups B1 and B2 are assigned correctly 95% of the time. Strains failing to yield any PCR products using the Clermont method are seldom members of phylo-group A and strains with such a genotype should not be assigned to a phylo-group.

The CTX-M-15-producing Escherichia coli diffusing clone belongs to a highly virulent B2 phylogenetic subgroup

OBJECTIVES A clone of CTX-M-15-producing Escherichia coli has recently been reported to be spreading through Europe and Africa. The aim of this work was to thoroughly characterize this clone. MATERIALS AND METHODS Representative isolates of this clone were subjected to multilocus sequence typing, O typing, virulence gene detection, adhesion assay on human cells, biofilm production assay and mouse lethality assay. RESULTS The clone: (i) belongs to a unique B2 phylogenetic subgroup encompassing the pyelonephritogenic diffusely adhering EC7372 strain; (ii) exhibits a specific O25b molecular subtype; (iii) is identical to the E. coli clone O25:H4-ST131 producing CTX-M-15; (iv) produces biofilm; and (v) is highly virulent in mice despite lacking classical extraintestinal pathogenicity islands (except for high pathogenicity island) and the afa/dra gene. CONCLUSIONS The CTX-M-15-producing E. coli diffusing clone is associated with a high level of antibiotic resistance and with high virulence, showing that, under certain selective pressures, the previously observed trade-off between resistance and virulence may not apply.

Phylogenetic and genomic diversity of human bacteremic Escherichia coli strains

BackgroundExtraintestinal pathogenic Escherichia coli (ExPEC) strains represent a huge public health burden. Knowledge of their clonal diversity and of the association of clones with genomic content and clinical features is a prerequisite to recognize strains with a high invasive potential. In order to provide an unbiased view of the diversity of E. coli strains responsible for bacteremia, we studied 161 consecutive isolates from patients with positive blood culture obtained during one year in two French university hospitals. We collected precise clinical information, multilocus sequence typing (MLST) data and virulence gene content for all isolates. A subset representative of the clonal diversity was subjected to comparative genomic hybridization (CGH) using 2,324 amplicons from the flexible gene pool of E. coli.ResultsRecombination-insensitive phylogenetic analysis of MLST data in combination with the ECOR collection revealed that bacteremic E. coli isolates were highly diverse and distributed into five major lineages, corresponding to the classical E. coli phylogroups (A+B1, B2, D and E) and group F, which comprises strains previously assigned to D. Compared to other strains of phylogenetic group B2, strains belonging to MLST-derived clonal complexes (CCs) CC1 and CC4 were associated (P < 0.05) with a urinary origin. In contrast, no CC appeared associated with severe sepsis or unfavorable outcome of the bacteremia. CGH analysis revealed genomic characteristics of the distinct CCs and identified genomic regions associated with CC1 and/or CC4.ConclusionOur results demonstrate that human bacteremia strains distribute over the entire span of E. coli phylogenetic diversity and that CCs represent important phylogenetic units for pathogenesis and comparative genomics.

Phylogenetic analysis and prevalence of urosepsis strains of Escherichia coli bearing pathogenicity island-like domains

ABSTRACT We characterized 100 Escherichia coli urosepsis isolates from adult patients according to host compromise status by means of ribotyping, PCR phylogenetic grouping, and PCR detection of papG alleles and the virulence-related genes sfa/foc, fyuA, irp-2, aer, hly, cnf-1 and hra. We also tested these strains for copies of pap and hly and their direct physical linkage with other virulence genes in an attempt to look for pathogenicity islands (PAIs) described for the archetypal uropathogenic strains J96, CFT073, and 536. Most of the isolates belonged to E. coli phylogenetic groups B2 and D and bore papG allele II, aer, and fyuA/irp-2. papG allele II-bearing strains were more common in noncompromised patients, while papG allele-negative strains were significantly more frequent in compromised patients. Fifteen ribotypes were identified. The three archetypal strains harbored different ribotypes, and only one-third of our urosepsis strains were genetically related to one of the archetypal strains. Three and 18 strains harbored three and two copies of pap, respectively, and 5 strains harbored two copies of hly. papGIII was physically linked to hly, cnf-1, and hra (reported to be PAI IIJ96-like genetic elements) in 14% of the strains. The PAI IIJ96-like domain was inserted within pheR tRNA in 11 strains and near leuX tRNA in 3 strains. Moreover, the colocalized genes cnf-1, hra, and hly were physically linked to papGII in four strains and to no pap gene in three strains. papGII and hly (reported to be PAI ICFT073-like genetic elements) were physically linked in 16 strains, pointing to a PAI ICFT073-like domain. Three strains contained both a PAI IIJ96-like domain and a PAI ICFTO73-like domain. Forty-two strains harbored papGII but not hly, in keeping with the presence of a PAI IICFT073-like domain. Only one strain harbored a PAI I536-like domain (hly only), and none harbored a PAI IJ96-like domain (papGI plus hly) or a PAI II536-like domain (papGIII plus hly). This study provides new data on the prevalence and variability of physical genetic linkage between pap and certain virulence-associated genes that are consistent with their colocalization on archetypal PAIs.

Identification of regions of the Escherichia coli chromosome specific for neonatal meningitis-associated strains.

ABSTRACT Specific virulence factors associated with the pathogenesis ofEscherichia coli strains causing neonatal meningitis (ECNM), such as the K1 capsular polysaccharide, the S fimbriae, and the Ibe10 protein, have been previously identified. However, some other yet unidentified factors are likely to be involved in the pathogenesis of ECNM. To identify specialized unique DNA regions associated with ECNM virulence, we used the representational difference analysis technique. The genomes of two strains belonging to nonpathogenic phylogenetic group A of the ECOR reference collection were subtracted from E. coli strain C5, isolated from a case of neonatal meningitis. Strain C5 belongs to the phylogenetic group B2 as do the majority of ECNM. We have isolated and mapped 64 DNA fragments which are specific for strain C5 and not found in nonpathogenic strains. Of these clones, 44 were clustered in six distinct regions on the chromosome. The sfa and ibe10 genes were located in regions 2 and 6, respectively. A group of genes (cnf1, hra, hly, andprs) known to be present in a pathogenicity island of the uropathogenic strain E. coli J96 colocalized with region 6. The occurrence of these DNA regions was tested in a set of meningitis-associated strains and in a control group composed of non-meningitis-associated strains belonging to the same B2 group. Regions 1, 3, and 4 were present in 91, 82, and 81%, respectively, of the meningitis strains and in 40, 13, and 47% of the control strains. Together, these data suggest that regions 1, 3, and 4 code for factors associated with the ability of E. coli to invade the meninges of neonates.

Animal and human pathogenic Escherichia coli strains share common genetic backgrounds.

Escherichia coli is a versatile species encompassing both commensals of the digestive tracts of many vertebrates, including humans, and pathogenic strains causing various intra- and extraintestinal infections. Despite extensive gene flow between strains, the E. coli species has a globally clonal population structure, consisting of distinct phylogenetic groups. Little is known about the relationships between phylogenetic groups and host specificity. We therefore used multilocus sequence typing (MLST) to investigate phylogenetic relationships and evaluated the virulence gene content of 35 E. coli strains representative of the diverse diseases encountered in domestic animals. We compared these strains with a panel of 101 human pathogenic and 98 non-human and human commensal strains representative of the phylogenetic and pathovar diversity of this species. A global factorial analysis of correspondence indicated that extraintestinal infections were caused mostly by phylogenetic group B2 strains, whereas intraintestinal infections were caused mostly by phylogenetic group A/B1/E strains, with strains responsible from extraintestinal or intraintestinal infections having specific virulence factors. It was not possible to distinguish between strains of human and animal origin. A detailed phylogenetic analysis of the MLST data showed that numerous pathogenic animal and human strains are very closely related, and had a number of virulence genes in common. However, a set of specific adhesins was identified in animal non-B2 group strains of all pathotypes. In conclusion, human and animal pathogenic strains share common genetic backgrounds, but non-B2 strains of different origins seem to have different sets of adhesins that could be involved in host specificity.