Comparative genomic analysis and molecular examination of the diversity of enterotoxigenic Escherichia coli isolates from Chile

Enterotoxigenic Escherichia coli (ETEC) is one of the most common diarrheal pathogens in the low- and middle-income regions of the world, however a systematic examination of the genomic content of isolates from Chile has not yet been undertaken. Whole genome sequencing and comparative analysis of a collection of 125 ETEC isolates from three geographic locations in Chile, allowed the interrogation of phylogenomic groups, sequence types and genes specific to isolates from the different geographic locations. A total of 80.8% (101/125) of the ETEC isolates were identified in E. coli phylogroup A, 15.2% (19/125) in phylogroup B, and 4.0% (5/125) in phylogroup E. The over-representation of genomes in phylogroup A was significantly different from other global ETEC genomic studies. The Chilean ETEC isolates could be further subdivided into sub-clades similar to previously defined global ETEC reference lineages that had conserved multi-locus sequence types and toxin profiles. Comparison of the gene content of the Chilean ETEC identified genes that were unique based on geographic location within Chile, phylogenomic classifications or sequence type. Completion of a limited number of genomes provided insight into the ETEC plasmid content, which is conserved in some phylogenomic groups and not conserved in others. These findings suggest that the Chilean ETEC isolates contain unique virulence factor combinations and genomic content compared to global reference ETEC isolates

Dynamic interactions of a conserved enterotoxigenic Escherichia coli adhesin with intestinal mucins govern epithelium engagement and toxin delivery

At present, there is no vaccine for enterotoxigenic Escherichia coli (ETEC), an important cause of diarrheal illness. Nevertheless, recent microbial pathogenesis studies have identified a number of molecules produced by ETEC that contribute to its virulence and are novel antigenic targets to complement canonical vaccine approaches. EtpA is a secreted two-partner adhesin that is conserved within the ETEC pathovar. EtpA interacts with the tips of ETEC flagella to promote bacterial adhesion, toxin delivery, and intestinal colonization by forming molecular bridges between the bacteria and the epithelial surface. However, the nature of EtpA interactions with the intestinal epithelium remains poorly defined. Here, we demonstrate that EtpA interacts with glycans presented by transmembrane and secreted intestinal mucins at epithelial surfaces to facilitate pathogen-host interactions that culminate in toxin delivery. Moreover, we found that a major effector molecule of ETEC, the heat-labile enterotoxin (LT), may enhance these interactions by stimulating the production of the gel-forming mucin MUC2. Our studies suggest, however, that EtpA participates in complex and dynamic interactions between ETEC and the gastrointestinal mucosae in which host glycoproteins promote bacterial attachment while simultaneously limiting the epithelial engagement required for effective toxin delivery. Collectively, these data provide additional insight into the intricate nature of ETEC interactions with the intestinal epithelium that have potential implications for rational approaches to vaccine design

Comparative genomics and transcriptomics of Escherichia coli isolates carrying virulence factors of both enteropathogenic and enterotoxigenic E. coli

AbstractEscherichia coli that are capable of causing human disease are often classified into pathogenic variants (pathovars) based on their virulence gene content. However, disease-associated hybrid E. coli, containing unique combinations of multiple canonical virulence factors have also been described. Such was the case of the E. coli O104:H4 outbreak in 2011, which caused significant morbidity and mortality. Among the pathovars of diarrheagenic E. coli that cause significant human disease are the enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). In the current study we use comparative genomics, transcriptomics, and functional studies to characterize isolates that contain virulence factors of both EPEC and ETEC. Based on phylogenomic analysis, these hybrid isolates are more genomically-related to EPEC, but appear to have acquired ETEC virulence genes. Global transcriptional analysis using RNA sequencing, demonstrated that the EPEC and ETEC virulence genes of these hybrid isolates were differentially-expressed under virulence-inducing laboratory conditions, similar to reference isolates. Immunoblot assays further verified that the virulence gene products were produced and that the T3SS effector EspB of EPEC, and heat-labile toxin of ETEC were secreted. These findings document the existence and virulence potential of an E. coli pathovar hybrid that blurs the distinction between E. coli pathovars.</jats:p

Highly conserved type 1 pili promote enterotoxigenic E. coli pathogen-host interactions

Enterotoxigenic Escherichia coli (ETEC), defined by their elaboration of heat-labile (LT) and/or heat-stable (ST) enterotoxins, are a common cause of diarrheal illness in developing countries. Efficient delivery of these toxins requires ETEC to engage target host enterocytes. This engagement is accomplished using a variety of pathovar-specific and conserved E. coli adhesin molecules as well as plasmid encoded colonization factors. Some of these adhesins undergo significant transcriptional modulation as ETEC encounter intestinal epithelia, perhaps suggesting that they cooperatively facilitate interaction with the host. Among genes significantly upregulated on cell contact are those encoding type 1 pili. We therefore investigated the role played by these pili in facilitating ETEC adhesion, and toxin delivery to model intestinal epithelia. We demonstrate that type 1 pili, encoded in the E. coli core genome, play an essential role in ETEC virulence, acting in concert with plasmid-encoded pathovar specific colonization factor (CF) fimbriae to promote optimal bacterial adhesion to cultured intestinal epithelium (CIE) and to epithelial monolayers differentiated from human small intestinal stem cells. Type 1 pili are tipped with the FimH adhesin which recognizes mannose with stereochemical specificity. Thus, enhanced production of highly mannosylated proteins on intestinal epithelia promoted FimH-mediated ETEC adhesion, while conversely, interruption of FimH lectin-epithelial interactions with soluble mannose, anti-FimH antibodies or mutagenesis of fimH effectively blocked ETEC adhesion. Moreover, fimH mutants were significantly impaired in delivery of both heat-stable and heat-labile toxins to the target epithelial cells in vitro, and these mutants were substantially less virulent in rabbit ileal loop assays, a classical model of ETEC pathogenesis. Collectively, our data suggest that these highly conserved pili play an essential role in virulence of these diverse pathogens

Evaluation of Pavement Edge Drains and the Effect on Pavement Performance

This report documents the performance of highway edge drains in Kentucky. Approximately eighteen high edge drain installations were inspected. The report also documents the laboratory testing, construction, short-term and long-term performance of these systems. Construction inspection and maintenance are addressed. It is apparent that the panels are distressed more under the old method of installation using excavated trench material and dynamic type compaction. It is apparent that using the sand slurry reduces the chances of installation damage. Proper density needs to be achieved during installation of the sand backfill or damage will occur due to trench settlement. In most cases, increasing the density of the sand increased the performance of the panel drain. It is apparent through the field analysis that the maintenance and construction of the edge drain systems need to be improved. Field inspections of the headwalls and outlets indicates that approximately 25 percent of the outlets are not properly installed, and that the headwalls are not properly maintained. Inspection data indicate approximately 45 percent of the outlets are partially covered to completely plugged. The center of mass is also located in the rear of the headwall causing the headwall to fill backwards over time. Soil moisture and thermography data indicate that edge drains help move water laterally across the pavement structure, and that the shoulder acts as a restraining dam for pavements without edge drains. The gradation analysis performed on the sand backfill from the current edge drain installation specification showed that the sand backfill effectively filters out some of the minus 200 material. Blinding of the sand at this time does not appear to be a problem. Although further testing is needed, preliminary data indicates the sand acts as a filter by not allowing the lines from the broken concrete to flush into the filter fabric immediately after construction. FWD data indicate that edge drains significantly increase the strength of the subgrade by removing water. Preliminary analyses of Ride index data indicates that the edge drains may add significant life to the pavement structure. These conclusions are based on data from edge drain systems that are not fully functional

Evaluation of Headwalls and Outlets for Geocomposite Edge Drains on I-75 and I-71

Longitudinal, round, perforated pipe edge drains have been used along Kentucky roadways for approximately two decades. Panel (fin) edge drains were first used in Kentucky in 1984. Most of these edge drains were installed on the Interstate and Parkway systems. Several problems related to the drains have been noted in the last seven years. A number of these problems have been observed to be related to flexible outlet pipes and headwalls. A recent study was initiated to evaluate headwalls and outlets on I-75 from Lexington, Kentucky to Cincinnati, Ohio (approximately 70 miles) and on I-71 from Louisville, Kentucky to I-75 in Northern Kentucky (approximately 68 miles). This study was initiated as a first phase of a much more intensive study which will evaluate all edge drains and outlets on Interstates and Parkways in Kentucky. This report documents findings of the investigation of 234 edge drain outlets. Of the 234 outlets investigated, approximately 43 percent of the edge drain outlets inspected were out of service. Approximately 50 percent of the outlet pipes had been damaged during installation. More significant problems were found at the headwall and outlet pipe connection than any other location in the drainage system. It appears that a maintenance program should be established to clean the troughs of the headwalls and to check the screens for clogging and rust. The metal screens should be replaced with galvanized screens. Positive flow should also be maintained from the headwalls. The buildup of grass and silt can eventually detour some of the flow. Headwalls located in cuts are more prone to become covered or ponded. Edge drains and outlets should be inspected after they are installed. Rigid outlet pipe should be precast into the headwalls. This should help eliminate problems occurring at the headwall connection. Currently, a rigid outlet pipe is connected to an approximately 2-foot long pigtail (4-inch flexible pipe) which is precast into the headwall