Characterization of unconventional pathogenic Escherichia coli isolated from bloodstream infection: virulence beyond the opportunism.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of urinary tract infection worldwide and a critical bloodstream infection agent. There are more than 50 virulence factors (VFs) related to ExPEC pathogenesis; however, many strains isolated from extraintestinal infections are devoid of these factors. Since opportunistic infections may occur in immunocompromised patients, E. coli strains that lack recognized VFs are considered opportunist, and their virulence potential is neglected. We assessed eleven E. coli strains isolated from bloodstream infections and devoid of the most common ExPEC VFs to understand their pathogenic potential. The strains were evaluated according to their capacity to interact in vitro with human eukaryotic cell lineages (Caco-2, T24, HEK293T, and A549 cells), produce type 1 fimbriae and biofilm in diverse media, resist to human sera, and be lethal to Galleria mellonella. One strain displaying all phenotypic traits was sequenced and evaluated. Ten strains adhered to Caco-2 (colon), eight to T24 (bladder), five to HEK-293 T (kidney), and four to A549 (lung) cells. Eight strains produced type 1 fimbriae, ten adhered to abiotic surfaces, nine were serum resistant, and seven were virulent in the G. mellonella model. Six of the eleven E. coli strains displayed traits compatible with pathogens, five of which were isolated from an immune-competent host. The genome of the EC175 strain, isolated from a patient with urosepsis, reveals that the strain belonged to ST504-A, and serotype O11:H11; harbors thirteen VFs genes, including genes encoding UpaG and yersiniabactin as the only ExPEC VFs identified. Together, our results suggest that the ExPEC pathotype includes pathogens from phylogroups A and B1, which harbor VFs that remain to be uncovered.

Surface Protein Dispersin of Enteroaggregative Escherichia coli Binds Plasminogen That Is Converted Into Active Plasmin.

Dispersin is a 10.2 kDa-immunogenic protein secreted by enteroaggregative Escherichia coli (EAEC). In the prototypical EAEC strain 042, dispersin is non-covalently bound to the outer membrane, assisting dispersion across the intestinal mucosa by overcoming electrostatic attraction between the AAF/II fimbriae and the bacterial surface. Also, dispersin facilitates penetration of the intestinal mucus layer. Initially characterized in EAEC, dispersin has been detected in other E. coli pathotypes, including those isolated from extraintestinal sites. In this study we investigated the binding capacity of purified dispersin to extracellular matrix (ECM), since dispersin is exposed on the bacterial surface and is involved in intestinal colonization. Binding to plasminogen was also investigated due to the presence of conserved carboxy-terminal lysine residues in dispersin sequences, which are involved in plasminogen binding in several bacterial proteins. Moreover, some E. coli components can interact with this host protease, as well as with tissue plasminogen activator, leading to plasmin production. Recombinant dispersin was produced and used in binding assays with ECM molecules and coagulation cascade compounds. Purified dispersin bound specifically to laminin and plasminogen. Interaction with plasminogen occurred in a dose-dependent and saturable manner. In the presence of plasminogen activator, bound plasminogen was converted into plasmin, its active form, leading to fibrinogen and vitronectin cleavage. A collection of E. coli strains isolated from human bacteremia was screened for the presence of aap, the dispersin-encoding gene. Eight aap-positive strains were detected and dispersin production could be observed in four of them. Our data describe new attributes for dispersin and points out to possible roles in mechanisms of tissue adhesion and dissemination, considering the binding capacity to laminin, and the generation of dispersin-bound plasmin(ogen), which may facilitate E. coli spread from the colonization site to other tissues and organs. The cleavage of fibrinogen in the bloodstream, may also contribute to the pathogenesis of sepsis caused by dispersin-producing E. coli.

Virulence Potential of a Multidrug-Resistant Escherichia coli Strain Belonging to the Emerging Clonal Group ST101-B1 Isolated from Bloodstream Infection.

Escherichia coli EC121 is a multidrug-resistant (MDR) strain isolated from a bloodstream infection of an inpatient with persistent gastroenteritis and T-zone lymphoma that died due to septic shock. Despite causing an extraintestinal infection, previous studies showed that it did not have the usual characteristics of an extraintestinal pathogenic E. coli. Instead, it belonged to phylogenetic group B1 and harbored few known virulence genes. To evaluate the pathogenic potential of strain EC121, an extensive genome sequencing and in vitro characterization of various pathogenicity-associated properties were performed. The genomic analysis showed that strain EC121 harbors more than 50 complete virulence genetic clusters. It also displays the capacity to adhere to a variety of epithelial cell lineages and invade T24 bladder cells, as well as the ability to form biofilms on abiotic surfaces, and survive the bactericidal serum complement activity. Additionally, EC121 was shown to be virulent in the Galleria mellonella model. Furthermore, EC121 is an MDR strain harboring 14 antimicrobial resistance genes, including blaCTX-M-2. Completing the scenario, it belongs to serotype O154:H25 and to sequence type 101-B1, which has been epidemiologically linked to extraintestinal infections as well as to antimicrobial resistance spread. This study with E. coli strain EC121 shows that clinical isolates considered opportunistic might be true pathogens that go underestimated.

Pic-Producing Escherichia coli Induces High Production of Proinflammatory Mediators by the Host Leading to Death by Sepsis.

Escherichia coli is an important pathogen responsible for a variety of diseases. We have recently shown that Pic, a serine protease secreted by E. coli, mediates immune evasion by the direct cleavage of complement molecules. The aim of this study was to investigate the action of a Pic-producing bacteria in a murine model of sepsis. Mice were infected with Pic-producing E. coli (F5) or F5∆pic mutant. Animal survival was monitored for five days, and a subset of mice was euthanized after 12 h for sample acquisition. The inoculation of Pic-producing bacteria induced 100% death within 24 h. The colony forming units count in the organs was significantly higher in F5. Hematological analysis showed a decrease of total leukocytes. Nitric oxide and cytokines were detected in serum, as well as on peritoneal lavage of the F5 group in higher levels than those detected in the other groups. In addition, immunophenotyping showed a decrease of activated lymphocytes and macrophages in the F5 group. Therefore, Pic represents an important virulence factor, allowing the survival of the bacterium in the bloodstream and several organs, as well as inducing a high production of proinflammatory mediators by the host, and concomitantly a cellular immunosuppression, leading to sepsis and death.

Serine protease autotransporters of Enterobacteriaceae (SPATEs) are largely distributed among Escherichia coli isolated from the bloodstream.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the major cause of Gram-negative-related sepsis. Bacterial survival in the bloodstream is mediated by a variety of virulence traits, including those mediating immune system evasion. Serine protease autotransporters of Enterobacteriaceae (SPATE) constitute a superfamily of virulence factors that can cause tissue damage and cleavage of molecules of the complement system, which is a key feature for the establishment of infection in the bloodstream. In this study, we analyzed 278 E. coli strains isolated from human bacteremia from inpatients of both genders, different ages, and clinical conditions. These strains were screened for the presence of SPATE-encoding genes as well as for phylogenetic classification and intrinsic virulence of ExPEC. SPATE-encoding genes were detected in 61.2% of the strains and most of these strains (44.6%) presented distinct SPATE-encoding gene profiles. sat was the most frequent gene among the entire collection, found in 34.2%, followed by vat (28.4%), pic (8.3%), and tsh (4.7%). Although in low frequencies, espC (0.7%), eatA (1.1%), and espI (1.1%) were detected and are being reported for the first time in extraintestinal isolates. The presence of SPATE-encoding genes was positively associated to phylogroup B2 and intrinsic virulent strains. These findings suggest that SPATEs are highly prevalent and involved in diverse steps of the pathogenesis of bacteremia caused by E. coli.

Virulence potential of a multidrug-resistant escherichia coli strain belonging to the emerging clonal group ST101-B1 isolated from bloodstream infection

Escherichia coli EC121 is a multidrug-resistant (MDR) strain isolated from a bloodstream infection of an inpatient with persistent gastroenteritis and T-zone lymphoma that died due to septic shock. Despite causing an extraintestinal infection, previous studies showed that it did not have the usual characteristics of an extraintestinal pathogenic E. coli. Instead, it belonged to phylogenetic group B1 and harbored few known virulence genes. To evaluate the pathogenic potential of strain EC121, an extensive genome sequencing and in vitro characterization of various pathogenicity-associated properties were performed. The genomic analysis showed that strain EC121 harbors more than 50 complete virulence genetic clusters. It also displays the capacity to adhere to a variety of epithelial cell lineages and invade T24 bladder cells, as well as the ability to form biofilms on abiotic surfaces, and survive the bactericidal serum complement activity. Additionally, EC121 was shown to be virulent in the Galleria mellonella model. Furthermore, EC121 is an MDR strain harboring 14 antimicrobial resistance genes, including blaCTX-M-2. Completing the scenario, it belongs to serotype O154:H25 and to sequence type 101-B1, which has been epidemiologically linked to extraintestinal infections as well as to antimicrobial resistance spread. This study with E. coli strain EC121 shows that clinical isolates considered opportunistic might be true pathogens that go underestimated.

Pic-producing escherichia coli induces high production of proinflammatory mediators by the host leading to death by sepsis

Escherichia coli is an important pathogen responsible for a variety of diseases. We have recently shown that Pic, a serine protease secreted by E. coli, mediates immune evasion by the direct cleavage of complement molecules. The aim of this study was to investigate the action of a Pic-producing bacteria in a murine model of sepsis. Mice were infected with Pic-producing E. coli (F5) or F5?pic mutant. Animal survival was monitored for five days, and a subset of mice was euthanized after 12 h for sample acquisition. The inoculation of Pic-producing bacteria induced 100% death within 24 h. The colony forming units count in the organs was significantly higher in F5. Hematological analysis showed a decrease of total leukocytes. Nitric oxide and cytokines were detected in serum, as well as on peritoneal lavage of the F5 group in higher levels than those detected in the other groups. In addition, immunophenotyping showed a decrease of activated lymphocytes and macrophages in the F5 group. Therefore, Pic represents an important virulence factor, allowing the survival of the bacterium in the bloodstream and several organs, as well as inducing a high production of proinflammatory mediators by the host, and concomitantly a cellular immunosuppression, leading to sepsis and death

Serine protease autotransporters of Enterobacteriaceae (SPATEs) are largely distributed among Escherichia coli isolated from the bloodstream

Extraintestinal pathogenic Escherichia coli (ExPEC) is the major cause of Gram-negative-related sepsis. Bacterial survival in the bloodstream is mediated by a variety of virulence traits, including those mediating immune system evasion. Serine protease autotransporters of Enterobacteriaceae (SPATE) constitute a superfamily of virulence factors that can cause tissue damage and cleavage of molecules of the complement system, which is a key feature for the establishment of infection in the bloodstream. In this study, we analyzed 278 E. coli strains isolated from human bacteremia from inpatients of both genders, different ages, and clinical conditions. These strains were screened for the presence of SPATE-encoding genes as well as for phylogenetic classification and intrinsic virulence of ExPEC. SPATE-encoding genes were detected in 61.2% of the strains and most of these strains (44.6%) presented distinct SPATE-encoding gene profiles. sat was the most frequent gene among the entire collection, found in 34.2%, followed by vat (28.4%), pic (8.3%), and tsh (4.7%). Although in low frequencies, espC (0.7%), eatA (1.1%), and espI (1.1%) were detected and are being reported for the first time in extraintestinal isolates. The presence of SPATE-encoding genes was positively associated to phylogroup B2 and intrinsic virulent strains. These findings suggest that SPATEs are highly prevalent and involved in diverse steps of the pathogenesis of bacteremia caused by E. coli.

Surface protein dispersin of enteroaggregative escherichia coli binds plasminogen that Is converted Into active plasmin

Dispersin is a 10.2 kDa-immunogenic protein secreted by enteroaggregative Escherichia coli (EAEC). In the prototypical EAEC strain 042, dispersin is non-covalently bound to the outer membrane, assisting dispersion across the intestinal mucosa by overcoming electrostatic attraction between the AAF/II fimbriae and the bacterial surface. Also, dispersin facilitates penetration of the intestinal mucus layer. Initially characterized in EAEC, dispersin has been detected in other E. coli pathotypes, including those isolated from extraintestinal sites. In this study we investigated the binding capacity of purified dispersin to extracellular matrix (ECM), since dispersin is exposed on the bacterial surface and is involved in intestinal colonization. Binding to plasminogen was also investigated due to the presence of conserved carboxy-terminal lysine residues in dispersin sequences, which are involved in plasminogen binding in several bacterial proteins. Moreover, some E. coli components can interact with this host protease, as well as with tissue plasminogen activator, leading to plasmin production. Recombinant dispersin was produced and used in binding assays with ECM molecules and coagulation cascade compounds. Purified dispersin bound specifically to laminin and plasminogen. Interaction with plasminogen occurred in a dose-dependent and saturable manner. In the presence of plasminogen activator, bound plasminogen was converted into plasmin, its active form, leading to fibrinogen and vitronectin cleavage. A collection of E. coli strains isolated from human bacteremia was screened for the presence of aap, the dispersin-encoding gene. Eight aap-positive strains were detected and dispersin production could be observed in four of them. Our data describe new attributes for dispersin and points out to possible roles in mechanisms of tissue adhesion and dissemination, considering the binding capacity to laminin, and the generation of dispersin-bound plasmin(ogen), which may facilitate E. coli spread from the colonization site to other tissues and organs. The cleavage of fibrinogen in the bloodstream, may also contribute to the pathogenesis of sepsis caused by dispersin-producing E. coli.

Virulence potential of a multidrug-resistant escherichia coli strain belonging to the emerging clonal group ST101-B1 isolated from bloodstream infection

Escherichia coli EC121 is a multidrug-resistant (MDR) strain isolated from a bloodstream infection of an inpatient with persistent gastroenteritis and T-zone lymphoma that died due to septic shock. Despite causing an extraintestinal infection, previous studies showed that it did not have the usual characteristics of an extraintestinal pathogenic E. coli. Instead, it belonged to phylogenetic group B1 and harbored few known virulence genes. To evaluate the pathogenic potential of strain EC121, an extensive genome sequencing and in vitro characterization of various pathogenicity-associated properties were performed. The genomic analysis showed that strain EC121 harbors more than 50 complete virulence genetic clusters. It also displays the capacity to adhere to a variety of epithelial cell lineages and invade T24 bladder cells, as well as the ability to form biofilms on abiotic surfaces, and survive the bactericidal serum complement activity. Additionally, EC121 was shown to be virulent in the Galleria mellonella model. Furthermore, EC121 is an MDR strain harboring 14 antimicrobial resistance genes, including blaCTX-M-2. Completing the scenario, it belongs to serotype O154:H25 and to sequence type 101-B1, which has been epidemiologically linked to extraintestinal infections as well as to antimicrobial resistance spread. This study with E. coli strain EC121 shows that clinical isolates considered opportunistic might be true pathogens that go underestimated.

Pic-producing escherichia coli induces high production of proinflammatory mediators by the host leading to death by sepsis

Escherichia coli is an important pathogen responsible for a variety of diseases. We have recently shown that Pic, a serine protease secreted by E. coli, mediates immune evasion by the direct cleavage of complement molecules. The aim of this study was to investigate the action of a Pic-producing bacteria in a murine model of sepsis. Mice were infected with Pic-producing E. coli (F5) or F5?pic mutant. Animal survival was monitored for five days, and a subset of mice was euthanized after 12 h for sample acquisition. The inoculation of Pic-producing bacteria induced 100% death within 24 h. The colony forming units count in the organs was significantly higher in F5. Hematological analysis showed a decrease of total leukocytes. Nitric oxide and cytokines were detected in serum, as well as on peritoneal lavage of the F5 group in higher levels than those detected in the other groups. In addition, immunophenotyping showed a decrease of activated lymphocytes and macrophages in the F5 group. Therefore, Pic represents an important virulence factor, allowing the survival of the bacterium in the bloodstream and several organs, as well as inducing a high production of proinflammatory mediators by the host, and concomitantly a cellular immunosuppression, leading to sepsis and death

Serine protease autotransporters of Enterobacteriaceae (SPATEs) are largely distributed among Escherichia coli isolated from the bloodstream

Extraintestinal pathogenic Escherichia coli (ExPEC) is the major cause of Gram-negative-related sepsis. Bacterial survival in the bloodstream is mediated by a variety of virulence traits, including those mediating immune system evasion. Serine protease autotransporters of Enterobacteriaceae (SPATE) constitute a superfamily of virulence factors that can cause tissue damage and cleavage of molecules of the complement system, which is a key feature for the establishment of infection in the bloodstream. In this study, we analyzed 278 E. coli strains isolated from human bacteremia from inpatients of both genders, different ages, and clinical conditions. These strains were screened for the presence of SPATE-encoding genes as well as for phylogenetic classification and intrinsic virulence of ExPEC. SPATE-encoding genes were detected in 61.2% of the strains and most of these strains (44.6%) presented distinct SPATE-encoding gene profiles. sat was the most frequent gene among the entire collection, found in 34.2%, followed by vat (28.4%), pic (8.3%), and tsh (4.7%). Although in low frequencies, espC (0.7%), eatA (1.1%), and espI (1.1%) were detected and are being reported for the first time in extraintestinal isolates. The presence of SPATE-encoding genes was positively associated to phylogroup B2 and intrinsic virulent strains. These findings suggest that SPATEs are highly prevalent and involved in diverse steps of the pathogenesis of bacteremia caused by E. coli.

Draft genome sequence of a multidrug-resistant Aeromonas hydrophila ST508 strain carrying rmtD and blaCTX-M-131 isolated from a bloodstream infection.

Here we report the draft genome sequence of a multidrug-resistant (MDR) Aeromonas hydrophila strain belonging to sequence type 508 (ST508) isolated from a human bloodstream infection. Assembly and annotation of this draft genome resulted in 5028498bp and revealed the presence of 16S rRNA methylase rmtD and blaCTX-M-131 genes encoding high-level resistance to aminoglycosides and cephalosporins, respectively, as well as multiple virulence genes. This draft genome can provide significant information for understanding mechanisms on the establishment and treatment of infections caused by this pathogen.

Cochlear epithelial of dog fetuses: a new source of multipotent stem cells.

Hearing loss caused by the damage of cochlea sensory cells or neurons is a common human disease, but also affects dogs and other animals. To test their progenitor nature as potential value for future therapies, we characterized cells derived from the cochlear epithelium in dog fetuses. In total, 8 fetuses of 35-40 days of gestation, derived from castration campaigns, were investigated. Cells were analysed by the MTT colorimetric assay and in regard to cell cycle, differentiation capacities, immunophenotypes and qPCR analysis. In culture, cells had a fibroblast-like morphology. Phenotypic immunocharacterization showed positive staining for mesenchymal stem cell and pluripotency markers and were negative for hematopoietic cell markers. Cells possessed differentiation capacity for the three main cell lineages: osteogenic, adipogenic and chondrogenic, altogether indicating their nature as mesenchymal stem cells. Thus, cells derived from fetal cochlear tissues indeed may provide valuable sources of progenitor cells for cell therapy of canine deafness and other diseases.