Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells

Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria. They not only represent a potential platform for development of novel vaccines but also provide a tool for efficient adjuvant and antigen delivery system. In the present study, we investigated the interaction between BGs of Escherichia coli (E. coli) and bovine monocyte-derived dendritic cells (MoDCs). MoDCs are highly potent antigen-presenting cells and have the potential to act as a powerful tool for manipulating the immune system. We generated bovine MoDCs in vitro from blood monocytes using E. coli expressed bovine GM-CSF and IL-4 cytokines. These MoDCs displayed typical morphology and functions similar to DCs. We further investigated the E. coli BGs to induce maturation of bovine MoDCs in comparison to E. coli lipopolysaccharide (LPS). We observed the maturation marker molecules such as MHC-II, CD80 and CD86 were induced early and at higher levels in BG stimulated MoDCs as compared to the LPS stimulated MoDCs. BG mediated stimulation induced significantly higher levels of cytokine expression in bovine MoDCs than LPS. Both pro-inflammatory (IL-12 and TNF-α) and anti-inflammatory (IL-10) cytokines were induced in MoDCs after BGs stimulation. We further analysed the effects of BGs on the bovine MoDCs in an allogenic mixed lymphocyte reaction (MLR). We found the BG-treated bovine MoDCs had significantly (p<0.05) higher capacity to stimulate allogenic T cell proliferation in MLR as compared to the LPS. Taken together, these findings demonstrate the E. coli BGs induce a strong activation and maturation of bovine MoDCs.     

Identification and Actions of a Novel Third Maresin Conjugate in Tissue Regeneration: MCTR3

Maresin conjugates in tissue regeneration (MCTR) are a new family of evolutionarily conserved chemical signals that orchestrate host responses to promote tissue regeneration and resolution of infections. Herein, we identified the novel MCTR3 and established rank order potencies and matched the stereochemistries of MCTR1, MCTR2 and MCTR3 using material prepared by total organic synthesis and mediators isolated from both mouse and human systems. MCTR3 was produced from endogenous substrate by E. coli activated human macrophages and identified in sepsis patients. Each of the three synthetic MCTR dose-dependently (1–100nM) accelerated tissue regeneration in planaria by 0.6–0.9 days. When administered at the onset or peak of inflammation, each of the MCTR promoted resolution of E. coli infections in mice. They increased bacterial phagocytosis by exudate leukocytes (~15–50%), limited neutrophil infiltration (~20–50%), promoted efferocytosis (~30%) and reduced eicosanoids. MCTR1 and MCTR2 upregulated human neutrophil and macrophage phagocytic responses where MCTR3 also proved to possess potent actions. These results establish the complete stereochemistry and rank order potencies for MCTR1, MCTR2 and MCTR3 that provide novel resolution moduli in regulating host responses to clear infections and promote tissue regeneration.     

Differential Expression of Inflammatory Cytokines and Stress Genes in Male and Female Mice in Response to a Lipopolysaccharide Challenge

BackgroundSex plays a key role in an individual’s immune response against pathogenic challenges such that females fare better when infected with certain pathogens. It is thought that sex hormones impact gene expression in immune cells and lead to sexually dimorphic responses to pathogens. We predicted that, in the presence of E. coli gram-negative lipopolysaccharide (LPS), there would be a sexually dimorphic response in proinflammatory cytokine production and acute phase stress gene expression and that these responses might vary among different mouse strains and times in a pattern opposite to that of body temperature associated with LPS-induced shock.ResultsStatistical analysis using analyses of variance (ANOVAs) showed that the levels of the all six traits changed over time, generally peaking at 2 hours after LPS injection. Mt-1, Fgb, and IL-6 showed differences among strains, although these were time-specific. Sexual dimorphism was seen for Fgb and IL6, and was most pronounced at the latest time period (7 hours) where male levels exceeded those for females. Trends for all six cytokine/gene expression traits were negatively correlated with those for body temperatures.DiscussionThe higher levels of expression of Fgb and IL6 in males compared with females are consistent with the greater vulnerability of males to infection and subsequent inflammation. Temperature appears to be a useful proxy for mortality in endotoxic shock, but sexual dimorphism in cytokine and stress gene expression levels may persist after an LPS challenge even if temperatures in the two sexes are similar and have begun to stabilize.     

Proteomic Analysis of Duodenal Tissue from Escherichia coli F18-Resistant and -Susceptible Weaned Piglets

Diarrhea and edema disease in weaned piglets due to infection by Escherichia coli F18 is a leading cause of economic loss in the pig industry. Resistance to E. coli F18 depends on expression of receptors on intestinal epithelial cells, and individual immunity. This study was conducted in Sutai pig E. coli F18-resistant and -susceptible full sib-pair individuals, identified on the basis of resource populations and verification of adhesion assays. The molecular mechanism underlying E. coli F18 resistance was investigated through analysis of the expression of E. coli F18 receptor associated and innate immunity proteins, using proteomics and bioinformatics techniques. Two-dimensional electrophoresis analysis revealed a total of 20 differentially expressed proteins in E. coli F18-resistant and -susceptible groups (10 upregulated and 10 downregulated). A total of 16 differentially expressed proteins were identified by MALDI TOF/TOF mass spectral analysis. According to gene ontology and pathway analysis, differentially expressed proteins were mainly involved in cell adhesion, immune response and other biologically relevant functions. Network analysis of interactions between differentially expressed proteins indicated a likelihood of their involvement in E. coli F18 infection. The expression levels of several important proteins including actin beta (ACTB), vinculin (VCL), heat stress proteins (HSPs) and transferrin (TF) in E. coli F18-resistant and -susceptible individuals were verified by Western blotting, supporting the identification of ACTB, VCL, HSPs and TF as promising candidate proteins for association with E. coli F18 susceptibility.     

Infiltration of myeloid cells into decidua is a critical early event in the labour cascade and post‐partum uterine remodelling

Leucocyte infiltration in the decidua (maternal–foetal interface) before, during and after term (TL) and preterm labour (PTL) was studied in mouse. We also investigated the mechanism of peripheral leucocyte recruitment into decidua by analysing the tissue cytokine profiles. Decidual tissues were collected during late gestation, TL and post‐partum (PP). PTL was initiated on gestational day 15 by intrauterine injection of Lipopolysaccharide (LPS, 125 μg) or progesterone signalling antagonism by RU486. Animals were killed during PTL or PP. Decidua basalis was analysed using FACS and immunohistochemistry. Markers of myeloid cell differentiation (Gr1, Ly6G, Neu7/4, F4/80) were assessed to define tissue monocytes (M), neutrophils (N) and macrophages (Macs). Flow cytometry revealed a significant (P < 0.05) increase in decidual Macs prior to TL; M and N numbers increased during TL and further increased during PP, which correlated with immunohistochemistry data. Massive influx of N, but not Macs and M, was detected by FACS during LPS‐PTL (P < 0.05) but not RU486‐PTL. Highest levels of N infiltration into the decidua occurred PP in both LPS and RU486 groups. Decidual infiltration during TL and RU486‐PTL was accompanied by an increase in pro‐inflammatory cytokines (IL1b and IL6) and CCL2 chemokine; LPS‐PTL showed increases in multiple cytokines. PP period following TL and PTL was associated with further up‐regulation of multiple cytokines/chemokines (P < 0.05). Our data suggest a programme of myeloid cells involvement in parturition with the pre‐partum influx of Macs into the decidua contributing to the progression of labour, whereas the later influx of M and N contribute to PP decidual involution.     

Response of the insect immune system to three different immune challenges

Insects rely on an innate immune system to effectively respond to pathogenic challenges. Most studies on the insect immune system describe changes in only one or two immune parameters following a single immune challenge. In addition, a variety of insect models, often at different developmental stages, have been used, making it difficult to compare results across studies. In this study, we used adult male Acheta domesticus crickets to characterize the response of the insect innate immune system to three different immune challenges: injection of bacterial lipopolysaccharides (LPS); injection of live Serratia marcescens bacteria; or insertion of a nylon filament into the abdomen. For each challenge, we measured and compared hemolymph phenoloxidase (PO) and lysozyme-like enzyme activities; the number of circulating hemocytes; and the nodulation responses of challenged and un-challenged crickets. We found that injection of an LD₅₀ dose of LPS from Escherichia coli elicited a more rapid response than an LD₅₀ dose of LPS from S. marcescens. LPS injection could cause a rapid decrease 2 hpi, followed by an increase by 7 dpi, in the number of circulating hemocytes. In contrast, injection of live S. marcescens produced a rapid increase and then decrease in hemocyte number. This was followed by an increase in the number of hemocytes at 7 dpi, similar to that observed following LPS injection. Both LPS and live bacteria decreased hemolymph PO activity, but the timing of this effect was dependent on the challenge. Live bacteria, but not LPS, induced an increase in lysozyme-like activity in the hemolymph. Insertion of a nylon filament induced a decrease in hemolymph PO activity 2 h after insertion of the filament, but had no effect on hemocyte number or lytic activity. Our results indicate that the innate immune system’s response to each type of challenge can vary greatly in both magnitude and timing, so it is important to assess multiple parameters at multiple time points in order to obtain a comprehensive view of such responses.     

Epidemiology of Antimicrobial Resistance in Escherichia coli Isolates from Raccoons (Procyon lotor) and the Environment on Swine Farms and Conservation Areas in Southern Ontario

Antimicrobial resistance is a global threat to livestock, human and environmental health. Although resistant bacteria have been detected in wildlife, their role in the epidemiology of antimicrobial resistance is not clear. Our objective was to investigate demographic, temporal and climatic factors associated with carriage of antimicrobial resistant Escherichia coli in raccoons and the environment. We collected samples from raccoon paws and feces and from soil, manure pit and dumpsters on five swine farms and five conservation areas in Ontario, Canada once every five weeks from May to November, 2011–2013 and tested them for E. coli and susceptibility to 15 antimicrobials. Of samples testing positive for E. coli, resistance to ≥ 1 antimicrobials was detected in 7.4% (77/1044; 95% CI, 5.9–9.1) of raccoon fecal samples, 6.3% (23/365; 95% CI, 4.0–9.3) of paw samples, 9.6% (121/1260; 8.0–11.4) of soil samples, 57.4% (31/54; 95% CI, 43.2–70.8) of manure pit samples, and 13.8% (4/29; 95% CI, 3.9–31.7) of dumpster samples. Using univariable logistic regression, there was no significant difference in the occurrence of resistant E. coli in raccoon feces on conservation areas versus farms; however, E. coli isolates resistant to ≥ 1 antimicrobials were significantly less likely to be detected from raccoon paw samples on swine farms than conservation areas and significantly more likely to be detected in soil samples from swine farms than conservation areas. Resistant phenotypes and genotypes that were absent from the swine farm environment were detected in raccoons from conservation areas, suggesting that conservation areas and swine farms may have different exposures to resistant bacteria. However, the similar resistance patterns and genes in E. coli from raccoon fecal and environmental samples from the same location types suggest that resistant bacteria may be exchanged between raccoons and their environment.     

Moderate Exercise Attenuates Lipopolysaccharide-Induced Inflammation and Associated Maternal and Fetal Morbidities in Pregnant Rats

Fetal growth restriction (FGR) and coagulopathies are often associated with aberrant maternal inflammation. Moderate-intensity exercise during pregnancy has been shown to increase utero-placental blood flow and to enhance fetal nutrition as well as fetal and placental growth. Furthermore, exercise is known to reduce inflammation. To evaluate the effect of moderate-intensity exercise on inflammation associated with the development of maternal coagulopathies and FGR, Wistar rats were subjected to an exercise regime before and during pregnancy. To model inflammation-induced FGR, pregnant rats were administered daily intraperitoneal injections of E. coli lipopolysaccharide (LPS) on gestational days (GD) 13.5–16.5 and sacrificed at GD 17.5. Control rats were injected with saline. Maternal hemostasis was assessed by thromboelastography. Moderate-intensity exercise prevented LPS-mediated increases in white blood cell counts measured on GD 17.5 and improved maternal hemostasis profiles. Importantly, our data reveal that exercise prevented LPS-induced FGR. Moderate-intensity exercise initiated before and maintained during pregnancy may decrease the severity of maternal and perinatal complications associated with abnormal maternal inflammation.     

Protozoan Predation of Escherichia coli O157:H7 Is Unaffected by the Carriage of Shiga Toxin-Encoding Bacteriophages

Escherichia coli O157:H7 is a food-borne bacterium that causes hemorrhagic diarrhea and hemolytic uremic syndrome in humans. While cattle are a known source of E. coli O157:H7 exposure resulting in human infection, environmental reservoirs may also be important sources of infection for both cattle and humans. Bacteriophage-encoded Shiga toxins (Stx) carried by E. coli O157:H7 may provide a selective advantage for survival of these bacteria in the environment, possibly through their toxic effects on grazing protozoa. To determine Stx effects on protozoan grazing, we co-cultured Paramecium caudatum, a common ciliate protozoon in cattle water sources, with multiple strains of Shiga-toxigenic E. coli O157:H7 and non-Shiga toxigenic cattle commensal E. coli. Over three days at ambient laboratory temperature, P. caudatum consistently reduced both E. coli O157:H7 and non-Shiga toxigenic E. coli populations by 1–3 log cfu. Furthermore, a wild-type strain of Shiga-toxigenic E. coli O157:H7 (EDL933) and isogenic mutants lacking the A subunit of Stx 2a, the entire Stx 2a-encoding bacteriophage, and/or the entire Stx 1-encoding bacteriophage were grazed with similar efficacy by both P. caudatum and Tetrahymena pyriformis (another ciliate protozoon). Therefore, our data provided no evidence of a protective effect of either Stx or the products of other bacteriophage genes on protozoan predation of E. coli. Further research is necessary to determine if the grazing activity of naturally-occurring protozoa in cattle water troughs can serve to decrease cattle exposure to E. coli O157:H7 and other Shiga-toxigenic E. coli.     

Induction of cyclooxygenase 2 by Escherichia coli but not Helicobacter pylori lipopolysaccharide in gastric epithelial cells in vitro

Background. Cyclooxygenase 2 (COX‐2) is an inducible enzyme that plays a key role in the synthesis of prostaglandins in response to inflammatory stimuli. It is expressed in the gastric mucosa as part of the response to infection with Helicobacter pylori. The specific interaction between H. pylori and the gastric epithelium that results in COX‐2 expression has not been identified. Methods. In order to investigate the hypothesis that lipopolysaccharide (LPS) from H. pylori plays a role in the induction of cyclooxygenase 2 in the stomach, gastric cell lines MKN‐7 and MKN‐45 were incubated with LPS from either H. pylori NCTC 11637 or Escherichia coli 055:B5. Incubation of cells with live H. pylori NCTC 11637 was also carried out as a positive control. Cells were then analysed for COX‐2 protein and mRNA and prostaglandin E2 synthesis. Results. Cyclooxygenase 2 protein and mRNA expression was induced by E. coli LPS and live H. pylori, but not by H. pylori LPS. Prostaglandin E₂ synthesis increased in a dose‐dependent manner in both cell lines with E. coli but not H. pylori LPS. Conclusions. H. pylori LPS is of low biological activity when compared with E. coli LPS in its ability to induce the expression of cyclooxygenase 2 and synthesis of prostaglandin E₂. This may provide one mechanism by which H. pylori minimizes the inflammatory response in the gastric mucosa, allowing chronic infection.     

D-Alanine-Controlled Transient Intestinal Mono-Colonization with Non-Laboratory-Adapted Commensal E. coli Strain HS

Soon after birth the mammalian gut microbiota forms a permanent and collectively highly resilient consortium. There is currently no robust method for re-deriving an already microbially colonized individual again-germ-free. We previously developed the in vivo growth-incompetent E. coli K-12 strain HA107 that is auxotrophic for the peptidoglycan components D-alanine (D-Ala) and meso-diaminopimelic acid (Dap) and can be used to transiently associate germ-free animals with live bacteria, without permanent loss of germ-free status. Here we describe the translation of this experimental model from the laboratory-adapted E. coli K-12 prototype to the better gut-adapted commensal strain E. coli HS. In this genetic background it was necessary to complete the D-Ala auxotrophy phenotype by additional knockout of the hypothetical third alanine racemase metC. Cells of the resulting fully auxotrophic strain assembled a peptidoglycan cell wall of normal composition, as long as provided with D-Ala and Dap in the medium, but could not proliferate a single time after D-Ala/Dap removal. Yet, unsupplemented bacteria remained active and were able to complete their cell cycle with fully sustained motility until immediately before autolytic death. Also in vivo, the transiently colonizing bacteria retained their ability to stimulate a live-bacteria-specific intestinal Immunoglobulin (Ig)A response. Full D-Ala auxotrophy enabled rapid recovery to again-germ-free status. E. coli HS has emerged from human studies and genomic analyses as a paradigm of benign intestinal commensal E. coli strains. Its reversibly colonizing derivative may provide a versatile research tool for mucosal bacterial conditioning or compound delivery without permanent colonization.     

Isolation of an Aptamer that Binds Specifically to E. coli

Escherichia coli is a bacterial species found ubiquitously in the intestinal flora of animals, although pathogenic variants cause major public health problems. Aptamers are short oligonucleotides that bind to targets with high affinity and specificity, and have great potential for use in diagnostics and therapy. We used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to isolate four single stranded DNA (ssDNA) aptamers that bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the nanomolar range. Fluorescently labeled aptamers label the surface of E. coli cells, as viewed by fluorescent microscopy. Specificity tests with twelve different bacterial species showed that one of the aptamers–called P12-31—is highly specific for E. coli. Importantly, this aptamer binds to Meningitis/sepsis associated E. coli (MNEC) clinical isolates, and is the first aptamer described with potential for use in the diagnosis of MNEC-borne pathologies.     

Genomic Comparison of Translocating and Non-Translocating Escherichia coli

Translocation of E. coli across the gut epithelium can result in fatal sepsis in post-surgical patients. In vitro and in vivo experiments have identified the existence of a novel pathotype of translocating E. coli (TEC) that employs an unknown mechanism for translocating across epithelial cells to the mesenteric lymph nodes and the blood stream in both humans and animal models. In this study the genomes of four TEC strains isolated from the mesenteric lymph nodes of a fatal case of hospitalised patient (HMLN-1), blood of pigs after experimental shock (PC-1) and after non-lethal haemorrhage in rats (KIC-1 and KIC-2) were sequenced in order to identify the genes associated with their adhesion and/or translocation. To facilitate the comparison, the genomes of a non-adhering, non-translocating E. coli (46–4) and adhering but non-translocating E. coli (73–89) were also sequenced and compared. Whole genome comparison revealed that three (HMLN-1, PC-1 and KIC-2) of the four TEC strains carried a genomic island that encodes a Type 6 Secretion System that may contribute to adhesion of the bacteria to gut epithelial cells. The human TEC strain HMLN-1 also carried the invasion ibeA gene, which was absent in the animal TEC strains and is likely to be associated with host-specific translocation. Phylogenetic analysis revealed that the four TEC strains were distributed amongst three distinct E. coli phylogroups, which was supported by the presence of phylogroup specific fimbriae gene clusters. The genomic comparison has identified potential genes that can be targeted with knock-out experiments to further characterise the mechanisms of E. coli translocation.     

Vaginal versus Obstetric Infection Escherichia coli Isolates among Pregnant Women: Antimicrobial Resistance and Genetic Virulence Profile

Vaginal Escherichia coli colonization is related to obstetric infections and the consequent development of infections in newborns. Ampicillin resistance among E. coli strains is increasing, which is the main choice for treating empirically many obstetric and neonatal infections. Vaginal E. coli strains are very similar to extraintestinal pathogenic E. coli with regards to the virulence factors and the belonging to phylogroup B2. We studied the antimicrobial resistance and the genetic virulence profile of 82 E. coli isolates from 638 vaginal samples and 63 isolated from endometrial aspirate, placental and amniotic fluid samples from pregnant women with obstetric infections. The prevalence of E. coli in the vaginal samples was 13%, which was significant among women with associated risk factors during pregnancy, especially premature preterm rupture of membranes (p<0.0001). Sixty-five percent of the strains were ampicillin-resistant. The E. coli isolates causing obstetric infections showed higher resistance levels than vaginal isolates, particularly for gentamicin (p = 0.001). The most prevalent virulence factor genes were those related to the iron uptake systems revealing clear targets for interventions. More than 50% of the isolates belonged to the virulent B2 group possessing the highest number of virulence factor genes. The ampicillin-resistant isolates had high number of virulence factors primarily related to pathogenicity islands, and the remarkable gentamicin resistance in E. coli isolates from women presenting obstetric infections, the choice of the most appropriate empiric treatment and clinical management of pregnant women and neonates should be carefully made. Taking into account host-susceptibility, the heterogeneity of E. coli due to evolution over time and the geographical area, characterization of E. coli isolates colonizing the vagina and causing obstetric infections in different regions may help to develop interventions and avoid the aetiological link between maternal carriage and obstetric and subsequent puerperal infections.     

Genomic Comparative Study of Bovine Mastitis Escherichia coli

Escherichia coli, one of the main causative agents of bovine mastitis, is responsible for significant losses on dairy farms. In order to better understand the pathogenicity of E. coli mastitis, an accurate characterization of E. coli strains isolated from mastitis cases is required. By using phylogenetic analyses and whole genome comparison of 5 currently available mastitis E. coli genome sequences, we searched for genotypic traits specific for mastitis isolates. Our data confirm that there is a bias in the distribution of mastitis isolates in the different phylogenetic groups of the E. coli species, with the majority of strains belonging to phylogenetic groups A and B1. An interesting feature is that clustering of strains based on their accessory genome is very similar to that obtained using the core genome. This finding illustrates the fact that phenotypic properties of strains from different phylogroups are likely to be different. As a consequence, it is possible that different strategies could be used by mastitis isolates of different phylogroups to trigger mastitis. Our results indicate that mastitis E. coli isolates analyzed in this study carry very few of the virulence genes described in other pathogenic E. coli strains. A more detailed analysis of the presence/absence of genes involved in LPS synthesis, iron acquisition and type 6 secretion systems did not uncover specific properties of mastitis isolates. Altogether, these results indicate that mastitis E. coli isolates are rather characterized by a lack of bona fide currently described virulence genes.     

Myometrial immune cells contribute to term parturition,preterm labour and post‐partum involution in mice

This study aimed to determine the mechanism of uterine activation during labour, both term (TL) and preterm (PTL). We hypothesized that the peripheral leucocytes are recruited to uterine tissues by locally produced cytokines where they contribute to the initiation of parturition. Mouse uteri were collected (i) during gestation, TL and post‐partum (PP), (ii) during PTL initiated by intrauterine infusion of LPS (125 μg) or (iii) injection of the progesterone receptor antagonist RU486 and analysed for multiple cytokine expression levels by real‐time polymerase chain reaction (RT‐PCR) and 23‐plex Cytokine assay or enzymatically dispersed for assessment of immune cell populations. Markers of myeloid cell differentiation (Gr1, Neu7/4 and F4/80) were evaluated by FACS to define tissue macrophages (Macs), monocytes (M) and neutrophils (N) and by immunohistochemistry to detect tissue Macs and N. Our results indicate that: (1) Macs were elevated in mouse myometrium before TL (P < 0.05) followed by an increase in M and N; these changes were accompanied by an increase in multiple pro‐inflammatory cytokines/chemokines genes. The expression of corresponding proteins increased PP. (2) TL and RU486‐PTL models showed similar gene/protein expression profiles, (3) LPS‐PTL was characterized by strong pro‐inflammatory response and massive influx of N in myometrial tissues showing a pattern different from TL and RU486‐PTL, (4) The PP period appears similar in all three models, with elevated myometrial cytokine levels and high infiltration of immune cells. We concluded that leucocytes infiltrate myometrium around the time of parturition implicating their potential role in labour activation (both term and preterm) and major role in PP uterine involution.     

Genome Based Phylogeny and Comparative Genomic Analysis of Intra-Mammary Pathogenic Escherichia coli

Escherichia coli is an important cause of bovine mastitis and can cause both severe inflammation with a short-term transient infection, as well as less severe, but more chronic inflammation and infection persistence. E. coli is a highly diverse organism that has been classified into a number of different pathotypes or pathovars, and mammary pathogenic E. coli (MPEC) has been proposed as a new such pathotype. The purpose of this study was to use genome sequence data derived from both transient and persistent MPEC isolates (two isolates of each phenotype) to construct a genome-based phylogeny that places MPEC in its phylogenetic context with other E. coli pathovars. A subsidiary goal was to conduct comparative genomic analyses of these MPEC isolates with other E. coli pathovars to provide a preliminary perspective on loci that might be correlated with the MPEC phenotype. Both concatenated and consensus tree phylogenies did not support MPEC monophyly or the monophyly of either transient or persistent phenotypes. Three of the MPEC isolates (ECA-727, ECC-Z, and ECA-O157) originated from within the predominately commensal clade of E. coli, referred to as phylogroup A. The fourth MPEC isolate, of the persistent phenotype (ECC-1470), was sister group to an isolate of ETEC, falling within the E. coli B1 clade. This suggests that the MPEC phenotype has arisen on numerous independent occasions and that this has often, although not invariably, occurred from commensal ancestry. Examination of the genes present in the MPEC strains relative to the commensal strains identified a consistent presence of the type VI secretion system (T6SS) in the MPEC strains, with only occasional representation in commensal strains, suggesting that T6SS may be associated with MPEC pathogenesis and/or as an inter-bacterial competitive attribute and therefore could represent a useful target to explore for the development of MPEC specific inhibitors.     

Small Intestine Early Innate Immunity Response during Intestinal Colonization by Escherichia coli Depends on Its Extra-Intestinal Virulence Status

Uropathogenic Escherichia coli (UPEC) strains live as commensals in the digestive tract of the host, but they can also initiate urinary tract infections. The aim of this work was to determine how a host detects the presence of a new UPEC strain in the digestive tract. Mice were orally challenged with UPEC strains 536 and CFT073, non-pathogenic strain K12 MG1655, and ΔPAI-536, an isogenic mutant of strain 536 lacking all 7 pathogenicity islands whose virulence is drastically attenuated. Intestinal colonization was measured, and cytokine expression was determined in various organs recovered from mice after oral challenge. UPEC strain 536 efficiently colonized the mouse digestive tract, and prior Enterobacteriaceae colonization was found to impact strain 536 colonization efficiency. An innate immune response, detected as the production of TNFα, IL-6 and IL-10 cytokines, was activated in the ileum 48 hours after oral challenge with strain 536, and returned to baseline within 8 days, without a drop in fecal pathogen load. Although inflammation was detected in the ileum, histology was normal at the time of cytokine peak. Comparison of cytokine secretion 48h after oral gavage with E. coli strain 536, CFT073, MG1655 or ΔPAI-536 showed that inflammation was more pronounced with UPECs than with non-pathogenic or attenuated strains. Pathogenicity islands also seemed to be involved in host detection, as IL-6 intestinal secretion was increased after administration of E. coli strain 536, but not after administration of ΔPAI-536. In conclusion, UPEC colonization of the mouse digestive tract activates acute phase inflammatory cytokine secretion but does not trigger any pathological changes, illustrating the opportunistic nature of UPECs. This digestive tract colonization model will be useful for studying the factors controlling the switch from commensalism to pathogenicity.     

FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Unfortunately, overexpression of fabI cannot be used to select medium –copy number plasmids, typically used for the expression of heterologous proteins in E. coli. Here we report that Vibrio cholera FabV, a functional homologue of E. coli FabI, can be used as a suitable marker for the selection and maintenance of both high and medium -copy number plasmid vectors in E. coli.