Inflammation, the kynurenines, and mucosal injury during human experimental enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in children and travelers, especially in low- and middle-income countries. ETEC is a non-invasive gut pathogen colonizing the small intestinal wall before secreting diarrhea-inducing enterotoxins. We sought to investigate the impact of ETEC infection on local and systemic host defenses by examining plasma markers of inflammation and mucosal injury as well as kynurenine pathway metabolites. Plasma samples from 21 volunteers experimentally infected with ETEC were collected before and 1, 2, 3, and 7 days after ingesting the ETEC dose, and grouped based on the level of intestinal ETEC proliferation: 14 volunteers experienced substantial proliferation (SP) and 7 had low proliferation (LP). Plasma markers of inflammation, kynurenine pathway metabolites, and related cofactors (vitamins B2 and B6) were quantified using targeted mass spectrometry, whereas ELISA was used to quantify the mucosal injury markers, regenerating islet-derived protein 3A (Reg3a), and intestinal fatty acid-binding protein 2 (iFABP). We observed increased concentrations of plasma C-reactive protein (CRP), serum amyloid A (SAA), neopterin, kynurenine/tryptophan ratio (KTR), and Reg3a in the SP group following dose ingestion. Vitamin B6 forms, pyridoxal 5'-phosphate and pyridoxal, decreased over time in the SP group. CRP, SAA, and pyridoxic acid ratio correlated with ETEC proliferation levels. The changes following experimental ETEC infection indicate that ETEC, despite causing a non-invasive infection, induces systemic inflammation and mucosal injury when proliferating substantially, even in cases without diarrhea. It is conceivable that ETEC infections, especially when repeated, contribute to negative health impacts on children in ETEC endemic areas.

Colicins and T6SS-based competition systems enhance enterotoxigenic E. coli (ETEC) competitiveness.

Diarrheal diseases are still a significant problem for humankind, causing approximately half a million deaths annually. To cause diarrhea, enteric bacterial pathogens must first colonize the gut, which is a niche occupied by the normal bacterial microbiota. Therefore, the ability of pathogenic bacteria to inhibit the growth of other bacteria can facilitate the colonization process. Although enterotoxigenic Escherichia coli (ETEC) is one of the major causative agents of diarrheal diseases, little is known about the competition systems found in and used by ETEC and how they contribute to the ability of ETEC to colonize a host. Here, we collected a set of 94 fully assembled ETEC genomes by performing whole-genome sequencing and mining the NCBI RefSeq database. Using this set, we performed a comprehensive search for delivered bacterial toxins and investigated how these toxins contribute to ETEC competitiveness in vitro. We found that type VI secretion systems (T6SS) were widespread among ETEC (n = 47). In addition, several closely related ETEC strains were found to encode Colicin Ia and T6SS (n = 8). These toxins provide ETEC competitive advantages during in vitro competition against other E. coli, suggesting that the role of T6SS as well as colicins in ETEC biology has until now been underappreciated.

Reduced Plasma Guanylin Levels Following Enterotoxigenic Escherichia coli-Induced Diarrhea.

The intestinal peptide hormones guanylin (GN) and uroguanylin (UGN) interact with the epithelial cell receptor guanylate cyclase C to regulate fluid homeostasis. Some enterotoxigenic Escherichia coli (ETEC) produce heat-stable enterotoxin (ST), which induces diarrhea by mimicking GN and UGN. Plasma concentrations of prohormones of GN (proGN) and UGN (proUGN) are reportedly decreased during chronic diarrheal diseases. Here we investigate whether prohormone concentrations also drop during acute diarrhea caused by ST-producing ETEC strains TW10722 and TW11681. Twenty-one volunteers were experimentally infected with ETEC. Blood (n = 21) and urine (n = 9) specimens were obtained immediately before and 1, 2, 3, and 7 days after ETEC ingestion. Concentrations of proGN and proUGN were measured by ELISA. Urine electrolyte concentrations were measured by photometry and mass spectrometry. Ten volunteers developed diarrhea (D group), and eleven did not (ND group). In the D group, plasma proGN, but not proUGN, concentrations were substantially reduced on days 2 and 3, coinciding with one day after diarrhea onset. No changes were seen in the ND group. ETEC diarrhea also seemed to affect diuresis, the zinc/creatinine ratio, and sodium and chloride secretion levels in urine. ETEC-induced diarrhea causes a reduction in plasma proGN and could potentially be a useful marker for intestinal isotonic fluid loss.

Dynamics of circulating lymphocytes responding to human experimental enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of children's and travelers' diarrhea, with no licensed vaccine. This study aimed to explore the role of cellular immunity in protection against human ETEC infection. Nine volunteers were experimentally infected with ETEC, of which six developed diarrhea. Lymphocytes were collected from peripheral blood buffy coats, before and 3, 5, 6, 7, 10, and 28 days after dose ingestion, and 34 phenotypic and functional markers were examined by mass cytometry. Thirty-three cell populations, derived by manually merging 139 cell clusters from the X-shift unsupervised clustering algorithm, were analyzed. Initially, the diarrhea group responded with increased CD56dim CD16+ natural killer cells, dendritic cells tended to rise, and mucosal-associated invariant T cells decreased. On day 5-7, an increase in plasmablasts was paralleled by a consistent rise in CD4+ Th17-like effector memory and regulatory cell subsets. CD4+ Th17-like central memory cells peaked on day 10. All Th17-like cell populations showed increased expression of activation, gut-homing, and proliferation markers. Interestingly, in the nondiarrhea group, these same CD4+ Th17-like cell populations expanded earlier, normalizing around day 7. Earlier development of these CD4+ Th17-like cell populations in the nondiarrhea group may suggest a recall response and a potential role in controlling ETEC infections.

Strong Association between Diarrhea and Concentration of Enterotoxigenic Escherichia coli Strain TW10722 in Stools of Experimentally Infected Volunteers.

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal illness in children and travelers in low- and middle-income countries. When volunteers are infected with ETEC strains, as part of experimental infection studies, some do not develop diarrhea. To improve our understanding of how these volunteers are protected, we investigated the association between stool ETEC DNA concentration, as determined by quantitative PCR, and the development and severity of disease in 21 volunteers who had been experimentally infected with ETEC strain TW10722. We found a strong association between maximum stool ETEC DNA concentration and the development of diarrhea: all of the 11 volunteers who did not develop diarrhea had <0.99% TW10722-specific DNA in their stools throughout the follow-up period of up to 9 days, while all of the 10 volunteers who did develop diarrhea had maximum DNA concentrations of ≥0.99%. Most likely, these maximum stool TW10722 DNA concentrations reflect the level of intestinal colonization and the risk of experiencing diarrhea, thereby, seems to be directly dependent on the level of colonization. Thus, the development and availability of vaccines and other prophylactic measures, even if they only partially reduce colonization, could be important in the effort to reduce the burden of ETEC diarrhea.

Umbilical Cord Stump Infections in Central Uganda: Incidence, Bacteriological Profile, and Risk Factors.

Umbilical cord stump infection (omphalitis) is a risk factor for neonatal sepsis and death. We assessed the incidence of omphalitis, described the bacteriological and antibiotic-resistance profile of potentially pathogenic bacteria isolated from the umbilical cord stump of omphalitis cases, and evaluated whether bacteria present in the birth canal during birth predicted omphalitis. We enrolled 769 neonates at birth at three primary healthcare facilities and followed them for 28 days with scheduled visits on days 3, 7, 14, and 28. Cox regression models were used to estimate the rates of omphalitis associated with potential risk factors. Sixty-five (8.5%) neonates developed omphalitis, with an estimated incidence of 0.095 cases per 28 child-days (95% CI 0.073, 0.12). Potentially pathogenic bacteria were isolated from the cord stump area of 41 (63.1%) of the 65 neonates with omphalitis, and the most commonly isolated species were Escherichia coli (n = 18), Klebsiella pneumoniae (n = 10), Citrobacter freundii (n = 5), and Enterobacter spp. (n = 4). The Enterobacteriaceace isolates were resistant to gentamicin (10.5%, 4/38), ampicillin (86.8%, 33/38), and ceftriaxone (13.2%, 5/38). Delayed initiation of breastfeeding was associated with an increased risk of omphalitis (aHR 3.1; 95% CI 1.3, 7.3); however, vaginal colonization with potentially pathogenic bacteria did not predict omphalitis.

Vaginal colonization with antimicrobial-resistant bacteria among women in labor in central Uganda: prevalence and associated factors.

BACKGROUND: According to WHO ( CISMAC. Centre for Intervention Science in Maternal and Child health), the antimicrobial resistant bacteria considered to be clinically most important for human health and earmarked for surveillance include extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, carbapenem-resistant bacteria, methicillin-resistant (MRSA) and, macrolide-lincosamide-streptogramin B -resistant vancomycin-resistant (VRSA) Staphylococcus aureus and vancomycin-resistant Enterococcus (VRE). If these bacteria are carried in the female genital tract, they may be transmitted to the neonate causing local or systemic neonatal infections that can be difficult to treat with conventionally available antimicrobials. In order to develop effective treatment strategies, there is need for updated information about the prevalence of colonization with important antimicrobial-resistant pathogens. OBJECTIVE: We sought to estimate the prevalence of vaginal colonization with potentially pathogenic and clinically important AMR bacteria among women in labour in Uganda and to identify factors associated with colonization. METHODS: We conducted a cross-sectional study among HIV-1 and HIV-2 negative women in labour at three primary health care facilities in Uganda. Drug susceptibility testing was done using the disk diffusion method on bacterial isolates cultured from vaginal swabs. We calculated the prevalence of colonization with potentially pathogenic and clinically important AMR bacteria, in addition to multidrug-resistant (MDR) bacteria, defined as bacteria resistant to antibiotics from ≥ 3 antibiotic classes. RESULTS: We found that 57 of the 1472 enrolled women (3.9% prevalence; 95% Confidence interval [CI] 3.0%, 5.1%) were colonized with ESBL-producing Enterobacteriaceace, 27 (1.8%; 95% CI 1.2%, 2.6%) were colonized with carbapenem-resistant Enterobacteriaceae, and 85 (5.8%; 95% CI 4.6%, 7.1%) were colonized with MRSA. The prevalence of colonization with MDR bacteria was high (750/1472; 50.9%; 95% CI 48.4%, 53.5%). Women who were ≥ 30 years of age had higher odds of being colonized with MDR bacteria compared to women aged 20-24 years (OR 1.6; 95% CI 1.1, 2.2). CONCLUSION: Most of the women included in our study were vaginally colonized with potentially pathogenic MDR and other clinically important AMR bacteria. The high prevalence of colonization with these bacteria is likely to further increase the incidence of difficult-to-treat neonatal sepsis.

Characterization of Glycosylation-Specific Systemic and Mucosal IgA Antibody Responses to Escherichia coli Mucinase YghJ (SslE).

Efforts to develop broadly protective vaccines against pathogenic Escherichia coli are ongoing. A potential antigen candidate for vaccine development is the metalloprotease YghJ, or SslE. YghJ is a conserved mucinase that is immunogenic, heavily glycosylated, and produced by most pathogenic E. coli. To develop efficacious YghJ-based vaccines, there is a need to investigate to what extent potentially protective antibody responses target glycosylated epitopes in YghJ and to describe variations in the quality of YghJ glycosylation in the E. coli population. In this study we estimated the proportion of anti-YghJ IgA antibodies that targeted glycosylated epitopes in serum and intestinal lavage samples from 21 volunteers experimentally infected with wild-type enterotoxigenic E. coli (ETEC) strain TW10722. Glycosylated and non-glycosylated YghJ was expressed, purified, and then gycosylation pattern was verified by BEMAP analysis. Then we used a multiplex bead flow cytometric assay to analyse samples from before and 10 days after TW10722 was ingested. We found that 20 (95%) of the 21 volunteers had IgA antibody responses to homologous, glycosylated YghJ, with a median fold increase in IgA levels of 7.9 (interquartile range [IQR]: 7.1, 11.1) in serum and 3.7 (IQR: 2.1, 10.7) in lavage. The median proportion of anti-YghJ IgA response that specifically targeted glycosylated epitopes was 0.45 (IQR: 0.30, 0.59) in serum and 0.07 (IQR: 0.01, 0.22) in lavage. Our findings suggest that a substantial, but variable, proportion of the IgA antibody response to YghJ in serum during ETEC infection is targeted against glycosylated epitopes, but that gut IgA responses largely target non-glycosylated epitopes. Further research into IgA targeting glycosylated YghJ epitopes is of interest to the vaccine development efforts.

Distinct Metabolic Features of Pathogenic Escherichia coli and Shigella spp. Determined by Label-Free Quantitative Proteomics.

Escherichia coli and Shigella spp. causing illnesses in humans represent a genotypically and phenotypically diverse group of pathogens. Although E. coli diversity has been studied by comparative genomics, the intra-species variation at the proteome level is currently unknown. The proteomes of 16 pathogenic E. coli, 2 non-pathogenic E. coli, and 5 Shigella strains originating from 18 phylogenetic lineages are investigated. By applying label-free quantitative proteomics on trypsin-digested cell extracts from bacteria grown on blood agar, 4018 proteins are detected, 3285 of which arequantified, and 261 represented virulence factors. Of 753 proteins quantified in all strains, the levels of 153 vary substantially between strains and are functionally associated mostly with stress response and peripheral metabolism. The levels of proteins associated with the central metabolism vary considerably less than the levels of proteins from other metabolic pathways. Hierarchical clustering analysis based on the protein levels results in strains grouping that differ from that obtained by gene-based phylogenetic analysis. Finally, strains of some E. coli pathotypes have more similar protein profiles even when the strains are not genetically closely related. The results suggest that the degree of genetic relatedness may not necessarily be a good predictor of E. coli phenotypic characteristics.

Human Mucosal IgA Immune Responses against Enterotoxigenic Escherichia coli.

Infection with enterotoxigenic Escherichia coli (ETEC) is a major contributor to diarrheal illness in children in low- and middle-income countries and travelers to these areas. There is an ongoing effort to develop vaccines against ETEC, and the most reliable immune correlate of protection against ETEC is considered to be the small intestinal secretory IgA response that targets ETEC-specific virulence factors. Since isolating IgA from small intestinal mucosa is technically and ethically challenging, requiring the use of invasive medical procedures, several other indirect methods are used as a proxy for gauging the small intestinal IgA responses. In this review, we summarize the literature reporting on anti-ETEC human IgA responses observed in blood, activated lymphocyte assayss, intestinal lavage/duodenal aspirates, and saliva from human volunteers being experimentally infected with ETEC. We describe the IgA response kinetics and responder ratios against classical and noncanonical ETEC antigens in the different sample types and discuss the implications that the results may have on vaccine development and testing.

Vaginal colonisation of women in labour with potentially pathogenic bacteria: a cross sectional study at three primary health care facilities in Central Uganda.

BACKGROUND: Potentially pathogenic bacteria that colonise the lower genital tract of women in labour can be passed to the baby during birth. While many babies become colonised with these bacteria after delivery, a few develop neonatal infections. The lower genital tract is a reservoir for potential pathogens and a source of infection for neonates. We determined the prevalence of vaginal colonisation of potentially pathogenic bacteria among women in labour in Central Uganda and identified potential risk factors associated with this colonisation. METHODS: We conducted a cross sectional study at three primary health care facilities and collected vaginal swabs from HIV-1 negative women in labour. Specimens were cultured on different selective microbiological media, and biochemical tests were used to classify bacterial isolates on the species level. Multivariable logistic regression analyses were used to estimate the association between relevant exposures and colonisation with potentially pathogenic bacteria. RESULTS: We recruited 1472 women in labour whose mean age was 24.6 years (standard deviation [SD] 4.9). Of these, 955 (64.9%; 95% Confidence Interval [CI] 62.4, 67%) were vaginally colonised with at least one potentially pathogenic bacterial species. The most commonly isolated species were Escherichia coli (n = 508; 34.5%), Klebsiella pneumoniae (n = 144; 9.8%) and Staphylococcus aureus (n = 121; 8.2%). Results from exploratory multivariable regression analyses indicated that having had ≥5 previous pregnancies (adjusted odds ratio [aOR] 0.59; 95% CI 0.35, 0.97) or being ≥30 years old (aOR 1.52; 95% CI 1.03, 2.23) could be associated with vaginal colonisation with any potentially pathogenic bacteria, as well as with vaginal colonisation with S. aureus (aOR 0.33; 95% CI 0.12, 0.88, and aOR 2.17; 95% CI 1.17, 4.00, respectively). Possession of domestic animals in a household (aOR 0.57; 95% CI 0.35, 0.92) could be associated with vaginal colonisation with E. coli. CONCLUSIONS: Two-thirds of HIV-1 negative women in labour were vaginally colonised by potentially pathogenic bacteria, mainly E. coli, K. pneumoniae, and S. aureus.

A new human challenge model for testing heat-stable toxin-based vaccine candidates for enterotoxigenic Escherichia coli diarrhea - dose optimization, clinical outcomes, and CD4+ T cell responses.

TRIAL REGISTRATION: ClinicalTrials.gov ClinicalTrials.gov, Project ID: NCT02870751.

Experimental Infection of Human Volunteers with the Heat-Stable Enterotoxin-Producing Enterotoxigenic Escherichia coli Strain TW11681.

Infection with enterotoxigenic Escherichia coli (ETEC) producing the heat-stable enterotoxin (ST) is one of the most important causes of childhood diarrhoea in low- and middle-income countries. Here, we undertook a controlled human infection model (CHIM) study to investigate whether ST-producing ETEC strain TW11681 would be suitable for testing the protective efficacy of new ST-based vaccine candidates in vaccine challenge models. In groups of three, nine volunteers ingested 1 × 106, 1 × 107, or 1 × 108 colony-forming units (CFU) of TW11681. Flow cytometry-based assays were used to measure CD4+ T cell responses and antibody levels targeting virulence factors expressed by the strain. We found that infection with TW11681 elicited few and mild symptoms, including mild diarrhoea in two volunteers, both of whom ingested 1 × 106 CFU. Averaged across all volunteers, the CD4+ T cell responses specific for E. coli YghJ mucinase peaked 10 days after infection (3.2-fold (p = 0.016)), while the CD4+ T cell responses specific for Colonization Factor Antigen I (CFA/I) major fimbrial subunit (CfaB) peaked after 28 days (3.6-fold (p = 0.063)). The serum CfaB-specific anti-IgA and anti-IgG/IgM levels were significantly increased and peaked 3 months after infection. Both remained elevated for the duration of the 12-month follow-up. The corresponding anti-YghJ serological response was strongest after 10 days, although a significant increase was seen only for IgA levels (3.2-fold (p = 0.008)). In conclusion, due to its low diarrhoea attack risk, TW11681 is probably not suitable for testing the efficacy of new vaccines in human challenge studies at doses 1 × 106 to 1 × 108. However, the strain may still be useful in CHIMs for studying ETEC host-pathogen interactions.

Immunizations with Enterotoxigenic Escherichia coli Heat-Stable Toxin Conjugates Engender Toxin-Neutralizing Antibodies in Mice That Also Cross-React with Guanylin and Uroguanylin.

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of childhood diarrhea in low- and middle-income countries, as well as of diarrhea among travelers to these countries. In children, ETEC strains secreting the heat-stable toxin (ST) are the most pathogenic, and there are ongoing efforts to develop vaccines that target ST. One important challenge for ST vaccine development is to construct immunogens that do not elicit antibodies that cross-react with guanylin and uroguanylin, which are endogenous peptides involved in regulating the activity of the guanylate cyclase-C (GC-C) receptor. We immunized mice with both human ST (STh) and porcine ST (STp) chemically coupled to bovine serum albumin, and the resulting sera neutralized the toxic activities of both STh and STp. This suggests that a vaccine based on either ST variant can confer cross-protection. However, several anti-STh and anti-STp sera cross-reacted with the endogenous peptides, suggesting that the ST sequence must be altered to reduce the risk of unwanted cross-reactivity. Epitope mapping of four monoclonal anti-STh and six anti-STp antibodies, all of which neutralized both STh and STp, revealed that most epitopes appear to have at least one amino acid residue shared with guanylin or uroguanylin. Despite this, only one monoclonal antibody displayed demonstrable cross-reactivity to the endogenous peptides, suggesting that targeted mutations of a limited number of ST residues may be sufficient to obtain a safe ST-based vaccine.

Proliferation of enterotoxigenic Escherichia coli strain TW11681 in stools of experimentally infected human volunteers.

BACKGROUND: As part of the effort to develop an enterotoxigenic Escherichia coli (ETEC) human challenge model for testing new heat-stable toxin (ST)-based vaccine candidates, a controlled human infection model study based on the ST-producing ETEC strain TW11681 was undertaken. Here, we estimate stool TW11681 DNA concentration and evaluate its association with dose, clinical symptoms, and with levels of antibodies targeting the CfaB subunit of the ETEC Colonization Factor Antigen I and the E. coli mucinase YghJ. Nine volunteers ingested different doses of the strain and were subsequently followed for 9 days with daily stool specimen collection and clinical examination. Stool DNA was purified by using a newly developed microplate-based method, and DNA originating from TW11681 was quantified by using a probe-based quantitative PCR assay. Antibody levels against CfaB and YghJ were measured in serum collected before and 10 and 28 days after TW11681 was ingested by using a bead-based flow cytometry immunoassay. RESULTS: For 6 of the 9 volunteers, the stool TW11681 DNA concentration increased sharply a median 3.5 (range 2-5) days after dose ingestion, peaking at a median of 5.4% (range 3.3-8.2%) of the total DNA in the specimen. The concentration then fell sharply during the subsequent days, sometimes even before the onset of antibiotic treatment. The size or timing of these proliferation peaks did not seem to be associated with the number of TW11681 bacteria ingested, but the 2 volunteers who developed diarrhea and all five who experienced abdominal pains or cramps had these peaks. The 3 volunteers who did not have the proliferation peaks experienced fewer symptoms and they generally had relatively low CfaB- and YghJ-specific antibody levels before ingesting the strain and subsequently weaker responses than the other volunteers afterwards. CONCLUSIONS: Since the lack of proliferation peaks appears to be associated with fewer clinical symptoms and lower serum antibody responses to virulence factors of the infecting strain, it may be important to account for proliferation peaks when explaining results from controlled human infection model studies and for improving the accuracy of protective efficacy estimates when testing new ETEC diarrhea vaccine candidates.

Longitudinal cohort study of serum antibody responses towards Giardia lamblia variant-specific surface proteins in a non-endemic area.

INTRODUCTION/AIMS: The long-term humoral immune response after a natural giardiasis infection is not well understood. The aim of this study was to evaluate longitudinal serum IgA and IgG/M responses towards conserved regions of two Giardia variant-specific surface proteins (VSP) and whether these responses differ between Giardia assemblages and durations of infection. METHODS: We recruited thirty Giardia-positive patients, mainly returning travellers, and eighteen healthy adults presumed to be Giardia unexposed. Blood samples were collected before treatment, and at 6 weeks, 6 months and 12 months after the infection cleared. We used a multiplex bead-based flow cytometry immunoassay to measure Giardia specific IgA and IgG/M responses targeting two recombinant antigens from G. lamblia VSP proteins 3 and 5 (VSP3 and VSP5). RESULTS: Serum levels of anti-VSP5 and anti-VSP3 IgA decreased rapidly to low levels after treatment but continued to be substantially higher than that of presumed unexposed controls even after 6 and 12 months. The IgG/M response decreased more gradually but remained significantly higher than presumed unexposed controls at all time points, except for anti-VSP3 at 12 months. There were no significant difference in responses for infections with assemblage A and assemblage B Giardia lamblia. Chronic infections (>8 weeks) were associated with a significantly lower anti-VSP5 IgG/M response. CONCLUSION: This study describes the kinetics of the humoral immune response against two Giardia VSP proteins over one year, and the considerable cross reactivity between the two human infective Giardia assemblages. Persons with chronic Giardia infection seem to have lower levels of VSP antibodies.

Comparative Proteomics of Enterotoxigenic Escherichia coli Reveals Differences in Surface Protein Production and Similarities in Metabolism.

Enterotoxigenic Escherichia coli (ETEC) infections are an important cause of diarrhea among young children living in low- and middle-income countries and visiting travelers. The development of effective vaccines is complicated by substantial genomic diversity that exists among ETEC isolates. To investigate how ETEC genomic variation is reflected at expressed proteome level, we applied label-free quantitative proteomics to seven human ETEC strains representing five epidemiologically important lineages. We further determined the proteome profile of the nonpathogenic E. coli B strain BL21(DE3) to discriminate features specific for ETEC. The analysis yielded a data set of 2893 proteins, of which 1729 were present in all strains. Each ETEC strain produced on average 27 plasmid- or chromosomally-encoded proteins with known or putative connections to virulence, and a number of strain-specific proteins associated with the biosynthesis of surface antigens. Statistical comparison of protein levels between the ETEC strains and BL21(DE3) revealed several proteins with considerably increased levels only in BL21(DE3) including enzymes of arginine biosynthesis and metabolism of melibiose, galactitol, and gluconate. ETEC strains displayed consistently increased levels of proteins that were functional in iron acquisition, maltose metabolism, and acid resistance. The latter results suggest that specific metabolic functions might be shared among ETEC isolates.

An Evidenced-Based Scale of Disease Severity following Human Challenge with Enteroxigenic Escherichia coli.

BACKGROUND: Experimental human challenge models have played a major role in enhancing our understanding of infectious diseases. Primary outcomes have typically utilized overly simplistic outcomes that fail to entirely account for complex illness syndromes. We sought to characterize clinical outcomes associated with experimental infection with enterotoxigenic Escherichia coli (ETEC) and to develop a disease score. METHODS: Data were obtained from prior controlled human ETEC infection studies. Correlation and univariate regression across sign and symptom severity was performed. A multiple correspondence analysis was conducted. A 3-parameter disease score with construct validity was developed in an iterative fashion, compared to standard outcome definitions and applied to prior vaccine challenge trials. RESULTS: Data on 264 subjects receiving seven ETEC strains at doses from 1x105 to 1x1010 cfu were used to construct a standardized dataset. The strongest observed correlation was between vomiting and nausea (r = 0.65); however, stool output was poorly correlated with subjective activity-impacting outcomes. Multiple correspondence analyses showed covariability in multiple signs and symptoms, with severity being the strongest factor corresponding across outcomes. The developed disease score performed well compared to standard outcome definitions and differentiated disease in vaccinated and unvaccinated subjects. CONCLUSION: Frequency and volumetric definitions of diarrhea severity poorly characterize ETEC disease. These data support a disease severity score accounting for stool output and other clinical signs and symptoms. Such a score could serve as the basis for better field trial outcomes and gives an additional outcome measure to help select future vaccines that warrant expanded testing in pivotal pre-licensure trials.

Improving genome annotation of enterotoxigenic Escherichia coli TW10598 by a label-free quantitative MS/MS approach.

The most commonly used genome annotation processes are to a great extent based on computational methods. However, those can only predict genes that have been described earlier or that have sequence signatures indicative of a gene function. Here, we report a synonymous proteogenomic approach for experimentally improving microbial genome annotation based on label-free quantitative MS/MS. The approach is exemplified by analysis of cell extracts from in vitro cultured enterotoxigenic Escherichia coli (ETEC) strain TW10598, as part of an effort to create a new reference ETEC genome sequence. The proteomic analysis yielded identification of 2060 proteins, out of which 312 proteins were originally described as hypothetical. For 84% of the identified proteins we have provided description of their relative quantitative levels, among others, for 20 abundantly expressed ETEC virulence factors. Proteogenomic mapping supported the existence of four protein-coding genes that had not been annotated, and led to correction of translation start positions of another nine. The addition of the proteomic analysis into TW10598 genome re-annotation project improved quality of the annotation, and provided experimental evidence for a significant portion of ETEC expressed proteome. Data are available via ProteomeXchange with identifier PXD002473 (http://proteomecentral.proteomexchange.org/dataset/PXD002473).

Experimental infection of healthy volunteers with enterotoxigenic Escherichia coli wild-type strain TW10598 in a hospital ward.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood diarrhea in resource-limited regions. It is also an important cause of diarrhea in travellers to these areas.To evaluate the protective efficacy of new ETEC vaccines that are under development, there is a need to increase the capacity to undertake Phase IIB (human challenge) clinical trials and to develop suitable challenge models. METHODS: An in-hospital study was performed where fasting adult volunteers were experimentally infected with 1 × 106 to 1 × 109 colony forming units (CFUs) of the wild-type ETEC strain TW10598, which had been isolated from a child with diarrhea in West Africa in 1997. We recorded symptoms and physical signs and measured serum immune response to the TW10598 bacterium. RESULTS: We included 30 volunteers with mean age 22.8 (range 19.8, 27.4) years. The most common symptoms were diarrhea (77%), abdominal pain (67%), nausea (63%), and abdominal cramping (53%). Seven subjects (23%) experienced fever, none were hypotensive. Most of the volunteers responded with a substantial rise in the level of serum IgA antibodies against the challenge strain. CONCLUSIONS: We established the capacity and methods for safely undertaking challenge studies to measure the efficacy of ETEC vaccine candidates in a hospital ward. Strain TW10598 elicited both clinical symptoms and an immune response across the doses given.