Extraintestinal pathogenic Escherichia coli O1:K1:H7/NM from human and avian origin: detection of clonal groups B2 ST95 and D ST59 with different host distribution

<p>Abstract</p> <p>Background</p> <p>Extraintestinal pathogenic <it>Escherichia coli </it>(ExPEC) strains of serotype O1:K1:H7/NM are frequently implicated in neonatal meningitis, urinary tract infections and septicemia in humans. They are also commonly isolated from colibacillosis in poultry. Studies to determine the similarities of ExPEC from different origins have indicated that avian strains potentially have zoonotic properties.</p> <p>Results</p> <p>A total of 59 ExPEC O1:K1:H7/NM isolates (21 from avian colibacillosis, 15 from human meningitis, and 23 from human urinary tract infection and septicemia) originated from four countries were characterized by phylogenetic PCR grouping, Multilocus Sequence Typing (MLST), Pulsed Field Gel Electrophoresis (PFGE) and genotyping based on several genes known for their association with ExPEC or avian pathogenic <it>Escherichia coli </it>(APEC) virulence.</p> <p>APEC and human ExPEC isolates differed significantly in their assignments to phylogenetic groups, being phylogroup B2 more prevalent among APEC than among human ExPEC (95% vs. 53%, <it>P </it>= 0.001), whereas phylogroup D was almost exclusively associated with human ExPEC (47% vs. 5%, <it>P </it>= 0.0000). Seven virulence genes showed significant differences, being <it>fimAv</it>_MT78and <it>sat </it>genes linked to human isolates, while <it>papGII</it>, <it>tsh</it>, <it>iron</it>, <it>cvaC </it>and <it>iss </it>were significantly associated to APEC. By MLST, 39 of 40 ExPEC belonging to phylogroup B2, and 17 of 19 belonging to phylogroup D exhibited the Sequence Types (STs) ST95 and ST59, respectively. Additionally, two novel STs (ST1013 and ST1006) were established. Considering strains sharing the same ST, phylogenetic group, virulence genotype and PFGE cluster to belong to the same subclone, five subclones were detected; one of those grouped six strains of human and animal origin from two countries.</p> <p>Conclusion</p> <p>Present results reveal that the clonal group B2 O1:K1:H7/NM ST95, detected in strains of animal and human origin, recovered from different dates and geographic sources, provides evidence that some APEC isolates may act as potential pathogens for humans and, consequently, poultry as a foodborne source, suggesting no host specificity for this type of isolates. A novel and important finding has been the detection of the clonal group D O1:K1:H7/NM ST59 almost exclusively in humans, carrying pathogenic genes linked to the phylogenetic group D. This finding would suggest D O1:K1:H7/NM ST59 as a host specific pathotype for humans.</p

Identification et caractérisation d'un îlot génomique d'une souche d'Escherichia coli pathogène aviaire

Certaines souches d'E. coli sont pathogènes et provoquent des infections intestinales ou extra-intestinales chez l'homme et les animaux. Chez les volailles, les APEC (Avian Pathogenic E. coli), provoquent une infection systémique à point de départ respiratoire pouvant conduire à la mort de l'animal. Plusieurs facteurs de virulence ont été identifiés pour ces souches, mais ne suffisent pas à expliquer tout le processus infectieux. L'identification de nouveaux facteurs de virulence devrait permettre de mieux comprendre le schéma infectieux de ces souches. Les facteurs de virulence bactériens sont fréquemment situés au sein d'éléments mobiles (plasmides, transposons ou îlots de pathogénicité (PAIs)). Les PAIs sont souvent associés à leurs extrémités au loci d'ARNt. Nous avons mis en évidence chez la souche APEC BEN2908, un nouvel îlot génomique nommé AGI-3 pour "APEC Genomic Island 3" inséré au locus selC et possédant la plupart des caractéristiques des PAIs. AGI-3 est encadré par des séquences directes répétées, possède des gènes codant pour des éléments de mobilité potentiels et est de taille importante (49 600 pb). Cet îlot est composé de 49 ORFs, dont 3, aec35 à aec37, semblent impliqués dans le métabolisme des sucres. Par comparaison du phénotype de la souche sauvage et de son mutant isogénique délété des ORFs aec35-37, nous avons mis en évidence l'implication de ce groupe de gènes dans l'assimilation des hydrates de carbone. Des reproductions expérimentales de la colibacillose chez le poulet ont permis de déterminer leur implication dans les étapes précoces de l'infection. La présence d'une intégrase de phage ainsi que des sites att indiquent qu'AGI-3 a probablement été acquis par transfert horizontal. En faveur de cette hypothèse, nous avons montré qu'AGI-3 pouvait exister sous forme épisomale dans le cytoplasme bactérien et être transféré à une souche d'E. coli K-12 par conjugaison. Ces données montrent qu'AGI-3 est un vecteur potentiel de dissémination de gènes de virulence entre souches bactériennes.Escherichia coli is also of causing a variety of intestinal or extraintestinal infections in humans and animals. In poultry flocks APEC (Avian pathogenic E. coli) are involved in a systemic infection initiated in the respiratory tract that can be lethal for the animals. Several bacterial factors have been associated with their virulence, however the mechanisms underlying pathogenicity are not fully understood yet. Identification of new factors involved in the pathogenesis of APEC should lead to a better understanding of the infectious process. In bacteria, virulence factors are often located in mobile genetic elements (transposons or pathogenicity islands (PAIs)). PAIs are often associated with tRNA loci. We identified a new PAI in the APEC strain BEN2908, that we named AGI-3 for "APEC genomic island 3".AGI-3 is located at the 3'-end of the selC tRNA and possesses several characteristics of PAIs (flanked by direct repeats, presence of genes coding for putative mobile elements and large size 49 600 pb). AGI-3 shows a mosaic structure. It is composed of 49 ORFs three of them, aec35 to aec37, seem to be implicated in sugar metabolism. Comparing the abilities of the wild type strain BEN2908 and of the isogenic mutant, deleted for the three genes, to assimilate carbohydrates and to induce colibacillosis in a chicken experimental model, we demonstrated the implication of the cluster in the uptake of seven carbohydrates and in pathogenesis at the early steps of the infection. The presence of a putative active integrase gene as well as att sites suggested that AGI-3 could have been acquired by horizontal gene transfer. We demonstrated that AGI-3 is able to excise from the chromosome and to persist in the bacterial cytoplasm as a circular form and be transferred to an E. coli K-12 strain by conjugation. These data show that AGI-3 could be a potential vector for dissemination of virulence genes between bacterial strains.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Rôle des protéines IbeA et IbeT dans les propriétés d'adhésion de la souche d'Escherichia coli pathogène aviaire BEN2908

Escherichia coli est une espèce bactérienne à multiples facettes. En effet, certaines souches sont présentes à l état commensal au niveau intestinal, ou sont utilisées comme probiotiques. À l inverse, d autres souches sont responsables d infections intestinales ou extra-intestinales chez l Homme et les animaux à sang chaud. Les souches d E. coli pathogènes extra-intestinales (ExPEC) sont responsables de nombreuses maladies infectieuses (méningites néo-natales, infections urinaires, septicémies ou infections respiratoires). Plusieurs facteurs de virulence ont été identifiés chez les souches ExPEC (adhésines, invasines ) et notamment la protéine IbeA, mise en évidence dans une souche isolée d un cas de méningite néo-natale humaine. Le gène ibeA est retrouvé chez différentes souches ExPEC, dont certaines d origine aviaire. Il est localisé au sein de l îlot génomique GimA, sur un des quatre opérons de cet îlot, entre ibeR codant un potentiel régulateur et ibeT codant un potentiel antiporteur. La protéine IbeA a été décrite comme jouant un rôle dans l invasion bactérienne des cellules endothéliales microvasculaires de cerveau humain (HBMEC). Afin de mieux comprendre le rôle d IbeA dans le processus infectieux et l invasion cellulaire, nous avons étudié l implication d IbeA dans l adhésion de la souche ExPEC d origine aviaire BEN2908 puis tenté de déterminer la localisation de cette protéine et son lien avec la protéine IbeT. L étude phénotypique comparative de la souche BEN2908 et de son mutant ibeA nous a montré qu IbeA intervenait dès le stade de l adhésion aux HBMEC. Des tests d adhésion en absence des fimbriae de type 1 (adhésine majeure de notre souche) nous ont montré que dans ce contexte, IbeA n avait pas d action sur l adhésion. Ce résultat nous a suggéré qu il pouvait y avoir une baisse d expression des fimbriae de type 1 à la surface bactérienne dans un mutant ibeA, ce que nous avons montré par dots blots. Pour comprendre comment IbeA entraînait une modification de l expression des fimbriae de type 1, nous nous sommes intéressés au contrôle de l expression des gènes de l opéron fim. Nous avons ainsi montré que le promoteur de ces gènes, localisé sur un élément invertible, était préférentiellement dans une orientation ne permettant pas la transcription des gènes fim dans un mutant ibeA. Nous avons ensuite mis en évidence chez le mutant ibeA une baisse de l expression des gènes fimB et fimE qui codent pour deux recombinases participant au contrôle de l orientation de l élément invertible. Ces baisses d expression de fimB et fimE pourraient expliquer la diminution d expression des fimbriae de type 1 dans le mutant ibeA. Enfin, des phénotypes similaires à ceux du mutant ibeA ont été observés chez un mutant ibeT. La localisation d IbeA est indispensable pour comprendre comment cette protéine peut agir sur l expression des recombinases FimB et FimE. Nous avons localisé IbeA dans le compartiment cytoplasmique, mais l incertitude sur la fonctionnalité d IbeA dans les constructions génétiques utilisées nécessite de confirmer ces premiers résultats. Enfin, nous avons recherché un rôle métabolique pour IbeA et IbeT étant données les homologies d IbeT avec des transporteurs de composés carbonés. Nous avons observé qu un mutant ibeT présentait un retard de croissance par rapport à la souche sauvage et au mutant ibeA dans des cultures en milieu minimum avec du fumarate, du succinate, du malate ou de l aspartate comme seule source de carbone. Ces résultats suggèrent un lien entre le métabolisme de certains dicarboxylates, l expression des fimbriae de type 1 et les protéines IbeA et IbeT. Ils ouvrent de nombreuses perspectives pour la compréhension du mécanisme d action d IbeA et IbeT.Escherichia coli is bacterial species with multiple facets. Indeed, some strains are present at a commensal state in the intestinal tract of humans and warm-blooded animals, or are used as probiotics. Conversely, other strains are responsible for intestinal or extra-intestinal infections in Humans and warm-blooded animals. Extra-intestinal pathogenic E. coli (ExPEC) strains are responsible for multiple infectious diseases (neonatal meningitis, urinary tract infections, septicaemias or respiratory infections). Several virulence factors have been identified in ExPEC strains (adhesins, invasins, ) and notably the IbeA protein, originally identified in a strain isolated from a case of human neonatal meningitis. The ibeA gene is found in different ExPEC strains, of including strains avian origin. It is located on one of the four operon of the GimA genomic island, between ibeR coding a putative regulator and ibeT coding a putative antiporter. The IbeA protein is known for its role in bacterial invasion of human brain microvascular endothelial cells (HBMEC). In order to better understand the role of IbeA in the infectious process and cellular invasion, we have studied the involvement of IbeA in adhesion of the avian pathogenic E. coli strain BEN2908 and attempted to determine the localisation of this protein and its link with the IbeT protein. The comparative henotypic study of strain BEN2908 and its ibeA mutant showed that IbeA was involved in the adhesion to HBMEC. Adhesion tests in the absence of type 1 fimbriae ( the major adhesin of our strain) showed that IbeA did not have a direct role in adhesion in this context. This result suggested there could be a decrease in type 1 fimbriae expression at the bacterial surface in the ibeA mutant. This was demonstrated by dot blots. To understand how IbeA led to a modification of type 1 fimbriae, we investigated the role of IbeA in the control of the expression of genes that belong to the fim operon. Thus we showed that the promoter of these genes, located on an invertible element, was preferentially in an orientation preventing transcription of the fim genes in the ibeA mutant. Then, we highlighted in the ibeA mutant, a decrease of expression of the fimB and fimE genes encoding two recombinases involved in the orientational control of invertible element. These decreases of fimB and fimE expression could explain the reduction of type 1 fimbriae expression in the ibeA mutant. Lastly, phenotypes similar to that of the ibeA mutant were observed in a ibeT mutant. The localisation of IbeA is necessary to understand how this protein can act on fimB and fimE expression. We localised IbeA in the bacterial cytoplasm, but the doubt on the functionality of IbeA in the genetic constructions used demands that these results be confirmed. Finally, we have looked for a metabolic role for IbeA and IbeT, given the IbeT homology with carbon compound transporters. We have observed that, in minimal broth with fumarate, succinate, malate or aspartate as sole carbon sources, the ibeT mutant presented a lower growth rate than the wild type strain and ibeA mutant. Altogether, these results suggest a link between metabolism of dicarboxylates, type 1 fimbriae expression and IbeA and IbeT proteins. They open numerous perspectives for the comprehension of IbeA and IbeT mechanisms.TOURS-Bibl.électronique (372610011) / SudocSudocFranceF

A Genomic Island of an Extraintestinal Pathogenic Escherichia coli Strain Enables the Metabolism of Fructooligosaccharides, Which Improves Intestinal Colonization▿ §

Prebiotics such as fructooligosaccharides (FOS) are increasingly being used in some countries for improving human and animal health and as an alternative to antibiotic growth promoters in animals, with various degrees of success. It has been observed that FOS stimulate the proliferation of probiotic bacteria and, at the same time, decrease the population of bacteria associated with disease. This observation assumes that pathogenic bacteria do not metabolize FOS and, therefore, lose their competitive advantage over beneficial bacteria. Here we present evidence that some pathogenic Escherichia coli strains can metabolize FOS and show that this property helps the bacterium colonize the intestine. These findings highlight the potential risk that a high level of prebiotic usage could lead to the emergence of well-adapted pathogenic strains that metabolize prebiotic substances

Escherichia coli Strains from Pregnant Women and Neonates: Intraspecies Genetic Distribution and Prevalence of Virulence Factors

To determine the extent to which the vagina, endocervix, and amniotic fluid screen the Escherichia coli strains responsible for neonatal infections, we studied the genetic relationships among 105 E. coli strains isolated from all of the ecosystems involved in this infectious process. Twenty-four strains were isolated from the intestinal flora, and 25 strains were isolated from the vaginas of pregnant women. Twenty-seven strains were isolated from the amniotic fluid, blood, and cerebrospinal fluid (CSF) of infected neonates. The intraspecies genetic characteristics of all of the isolates were determined by random amplified polymorphic DNA (RAPD) analysis, PCR ECOR (E. coli reference) grouping, and PCR virulence genotyping. A correlation was found between the intraspecies distributions of the strains in the A, B1, B2, and D ECOR groups and in the two major RAPD groups (I and II). Nevertheless, the distribution of the E. coli strains in the RAPD groups according to their anatomical origins was more significant than their distribution in the ECOR groups. This may be explained by the existence of an E. coli subpopulation, defined by the RAPD I group, within the ECOR B2 group. This RAPD I group presents a major risk for neonates: 75% of the strains isolated from patients with meningitis and 100% of the strains isolated from patients with bacteremia were in this group. The vagina and the amniotic fluid are two barriers that favor colonization by highly infectious strains. Indeed, only 17% of fecal strains belonged to the RAPD I group, whereas 52% of vaginal strains and 67% of amniotic fluid strains belonged to this subpopulation. The ibeA and iucC genes were significantly associated with CSF strains, whereas the hly and sfa/foc genes were more frequent in blood strains. These findings could serve as a basis for developing tools to recognize vaginal strains, which present a high risk for neonates, for use in prophylaxis programs

Extraintestinal Pathogenic Escherichia coli Strains of Avian and Human Origin: Link between Phylogenetic Relationships and Common Virulence Patterns▿

Extraintestinal pathogenic Escherichia coli (ExPEC) strains of human and avian origin show similarities that suggest that the avian strains potentially have zoonotic properties. However, the phylogenetic relationships between avian and human ExPEC strains are poorly documented, so this possibility is difficult to assess. We used PCR-based phylotyping and multilocus sequence typing (MLST) to determine the phylogenetic relationships between 39 avian pathogenic E. coli (APEC) strains of serogroups O1, O2, O18, and O78 and 51 human ExPEC strains. We also compared the virulence genotype and pathogenicity for chickens of APEC strains and human ExPEC strains. Twenty-eight of the 30 APEC strains of serogroups O1, O2, and O18 were classified by MLST into the same subcluster (B2-1) of phylogenetic group B2, whereas the 9 APEC strains of serogroup O78 were in phylogenetic groups D (3 strains) and B1 (6 strains). Human ExPEC strains were closely related to APEC strains in each of these three subclusters. The 28 avian and 25 human strains belonging to phylogenetic subcluster B2-1 all expressed the K1 antigen and presented no significant differences concerning the presence of other virulence factors. Moreover, human strains of this phylogenetic subcluster were highly virulent for chicks, so no host specificity was identified. Thus, APEC strains of serotypes O1:K1, O2:K1, and O18:K1 belong to the same highly pathogenic clonal group as human E. coli strains of the same serotypes isolated from cases of neonatal meningitis, urinary tract infections, and septicemia. These APEC strains constitute a potential zoonotic risk

The extra-intestinal avian pathogenic Escherichia coli strain BEN2908 invades avian and human epithelial cells and survives intracellularly

 Extra-intestinal pathogenic Escherichia coli (ExPEC) strains are responsible for a wide range of diseases in humans and animals. Using in vitro invasion assays and transmission electron microscopy, we showed that BEN2908, an ExPEC strain of avian origin (also termed APEC for Avian Pathogenic E. coli), is able to usurp cellular endocytic pathways to invade A549 human type II pneumocytes and LMH avian hepatocytes where it is able to survive over several days. Although type 1 fimbriae are the major adhesin of BEN2908, proportions of adherent fimbriated or afimbriated bacteria that entered cells were comparable. Internalization of BEN2908 into human pneumocytes reinforces previous studies indicating that APEC strains could represent a zoonotic risk

Contribution of the SitABCD, MntH, and FeoB Metal Transporters to the Virulence of Avian Pathogenic Escherichia coli O78 Strain χ7122▿

The roles of SitABCD, MntH, and FeoB metal transporters in the virulence of avian pathogenic Escherichia coli (APEC) O78 strain χ7122 were assessed using isogenic mutants in chicken infection models. In a single-strain infection model, compared to χ7122, the Δsit strain demonstrated reduced colonization of the lungs, liver, and spleen. Complementation of the Δsit strain restored virulence. In a coinfection model, compared to the virulent APEC strain, the Δsit strain demonstrated mean 50-fold, 126-fold, and 25-fold decreases in colonization of the lungs, liver, and spleen, respectively. A ΔmntH Δsit strain was further attenuated, demonstrating reduced persistence in blood and mean 1,400-fold, 954-fold, and 83-fold reduced colonization in the lungs, liver, and spleen, respectively. In coinfections, the ΔfeoB Δsit strain demonstrated reduced persistence in blood but increased colonization of the liver. The ΔmntH, ΔfeoB, and ΔfeoB ΔmntH strains were as virulent as the wild type in either of the infection models. Strains were also tested for sensitivity to oxidative stress-generating agents. The ΔmntH Δsit strain was the most sensitive strain and was significantly more sensitive than the other strains to hydrogen peroxide, plumbagin, and paraquat. sit sequences were highly associated with APEC and human extraintestinal pathogenic E. coli compared to commensal isolates and diarrheagenic E. coli. Comparative genomic analyses also demonstrated that sit sequences are carried on conjugative plasmids or associated with phage elements and were likely acquired by distinct genetic events among pathogenic E. coli and Shigella sp. strains. Overall, the results demonstrate that SitABCD contributes to virulence and, together with MntH, to increased resistance to oxidative stress