[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.
[Show abstract][Hide abstract] ABSTRACT: Background
Diarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease.
We use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age.
Our findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.
[Show abstract][Hide abstract] ABSTRACT: Phenotypic and genetic characteristics of Shigella species isolated from diarrhoeal and asymptomatic children aged up to five years were analysed in this study. In this study, 91 and 17 isolates were identified from diarrhoeal (case) and asymptomatic (control) children, respectively. All the isolates were tested for antimicrobial resistance, presence of integrons, plasmid-mediated quinolone resistance (PMQR), virulence associated genes and Shigella-pathogenicity island (SH-PAI). Majority of the Shigella spp. from cases (68.1%) and controls (82.3%) were found to be resistant to fluoroquinolones. Integron carriage was detected more in cases (76.9%) than controls (35.5%). Atypical class 1 integron was exclusively detected in S. flexneri from cases but not from the controls. PMQR genes such as aac(6')-Ib-cr and qnrS1 were detected in 82.4% and 14.3% of the isolates from cases and 53% and 17.6% in controls, respectively. Shigella isolates from cases as well as controls were positive for the invasive plasmid antigen H encoding gene ipaH. The other virulence genes such as virF, sat, setA, setB, sen and ial were detected in Shigella isolates at the proportion of 80.2, 49.4, 27.4, 27.4, 80.2, 79.1% from cases and 64.7, 52.9, 17.6, 17.6, 64.7, 64.7% from controls, respectively. The entire SH-PAI was detected in S. flexneri serotype 2a from cases and controls. In an isolate from a control child, the SH-PAI was truncated. Integrons, PMQR and virulence encoding genes have been detected mostly in cases than controls. In diarrhoea endemic areas, the asymptomatic carriers may play a crucial role in the transmission of multidrug resistant Shigella spp. with all the putative virulence genes.
Journal of Medical Microbiology 05/2014; · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have reported that transcription of a hypothetical small open reading frame (orf60) in enteroaggregative E. coli (EAEC) strain 042 is impaired after mutation of aggR, which encodes a global virulence activator. We have also reported that the cryptic orf60 locus was linked to protection against EAEC diarrhea in two epidemiologic studies. Here, we report that the orf60 product acts as a negative regulator of aggR itself. The orf60 protein product lacks homology to known repressors, but displays 44-100% similarity to at least fifty previously undescribed small (<10 kDa) hypothetical proteins found in many gram negative pathogen genomes. Expression of orf60 homologs from enterotoxigenic E. coli (ETEC) repressed the expression of the AraC-transcriptional ETEC regulator CfaD/Rns and its regulon in ETEC strain H10407. Complementation in trans of EAEC 042orf60 by orf60 homologs from ETEC and the mouse pathogen Citrobacter rodentium resulted in dramatic suppression of aggR. A C. rodentium orf60 homolog mutant showed increased levels of activator RegA and increased colonization of the adult mouse. We propose the name Aar (AggR-activated regulator) for the clinically and epidemiologically important orf60 product in EAEC, and postulate the existence of a large family of homologs among pathogenic Enterobacteriaceae and Pasteurellaceae. We propose the name ANR (AraC Negative Regulators) for this family.
[Show abstract][Hide abstract] ABSTRACT: Handwashing practices among caretakers of case and control children < 5 years of age enrolled in the Global Enteric Multicenter Study in Mirzapur, Bangladesh were characterized and analyzed for association with moderate-to-severe diarrhea. Soap or detergent ownership was common, yet 48% of case and 47.7% of control caretakers also kept ashes for handwashing, including 36.8% of the wealthiest households. Soap, detergent, and ash were used for multiple hygiene purposes and were kept together at handwashing areas. Caretakers preferred soap for handwashing, but frequently relied on ash, or a detergent/ash mixture, as a low-cost alternative. The moderate-to-severe diarrhea was equally likely for children of caretakers who kept soap versus those who kept ash (median odds ratio [mOR] = 0.91; 0.62-1.32). Contact with ash and water reduced concentrations of bacterial enteropathogens, without mechanical scrubbing. Thus, washing hands with ash is a prevalent behavior in Mirzapur and may help diminish transmission of diarrheal pathogens to children.
The American journal of tropical medicine and hygiene 04/2014; · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A growing family of virulence factors called serine protease autotransporters of Enterobacteriaceae (SPATEs), are secreted by Shigella, Salmonella and E. coli pathotypes. SPATEs are subdivided into class 1 or class 2 based on structural features and phylogenetics. Class 1 SPATEs induce cytopathic effects in numerous epithelial cell lines, and several have been shown to cleave the cytoskeletal protein spectrin in vitro. But to date the in vivo role of Class 1 SPATEs in enteric pathogenesis is unknown. Citrobacter rodentium, a natural mouse pathogen, has recently been shown to harbor class 1 and class 2 SPATEs. To better understand the contribution of class 1 SPATEs in enteric infection, we constructed a class 1 SPATE null mutant (Δcrc1) in C. rodentium. Upon infection of C57BL/6 mice, Δcrc1exhibited a hyper-virulent, hyper-inflammatory phenotype compared with its parent, accompanied by greater weight loss and trend towards increased mortality in young mice; the effect was reversed when the crc1 gene was restored. Using flow cytometry, we observed increased infiltration of T cells, B cells and neutrophils into the lamina propria of the distal colon in mice fed Δcrc1, starting as early as 5 days after infection. No significant difference in epithelial cytotoxicity was observed. RT-PCR analysis of distal colonic tissue on day 10 post infection. showed significant increases in mRNA encoding cytokines- IL-6, TNFα, IFNγ, IL-1β and iNOS, but not IL-17, IL-4 or IL-10 in the Δcrc1- infected mice. Our data suggest a previously unsuspected role for class 1 SPATEs in enteric infection.
Infection and immunity 04/2014; · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A long-standing challenge in developing vaccines against enterotoxigenic Escherichia coli (ETEC), the most common bacteria causing diarrhea in children of developing countries and travelers to these countries, is to protect against heat-stable toxin type Ib (STa or hSTa). STa and heat-labile toxin (LT) are virulence determinants in ETEC diarrhea. LT antigens are often used in vaccine development, but STa has not been included because of its poor immunogenicity and potent toxicity. Toxic STa is not safe for vaccines, but only STa possessing toxicity is believed to be able to induce neutralizing antibodies. However, recent studies demonstrated that nontoxic STa derivatives (toxoids), after being fused to an LT protein, induced neutralizing antibodies, and suggested that different STa toxoids fused to an LT protein might exhibit different STa antigenic propensity. In this study, we selected 14 STa toxoids from a mini STa toxoid library based on toxicity reduction and reactivity to anti-native STa antibodies, and genetically fused each toxoid to a monomeric double mutant LT (dmLT) peptide for 14 STa-toxoid-dmLT toxoid fusions. These toxoid fusions were used to immunize mice, and were characterized for induction of anti-STa antibody response. Results showed different STa toxoids (in fusions) varied greatly in anti-STa antigenicity. Among them, STaN12S, STaN12T and STaA14H were the top toxoids in inducing anti-STa antibodies. In vitro neutralization assays indicated antibodies induced by fusion 3xSTaN12S-dmLT exhibited the greatest neutralizing activity against STa toxin. These results suggested 3xSTaN12S-dmLT is a preferred fusion antigen to induce anti-STa antibody response, and provided long-awaited information for effective ETEC vaccine development.
Infection and immunity 02/2014; · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fimbria-mediated adherence to the intestinal epithelia is a key step in EAEC pathogenesis. To date, four fimbriae have been described for EAEC; the aggregative adherence fimbriae II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of bound cellular proteins to AafA-dsc identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein and 042 to recombinant CK8. Moreover, HEp-2 cells transfected with CK8-siRNA showed reduced 042 adherence when compared with cells transfected with scrambled siRNA as control. Adherence of 042 to HEp-2 cells pre-incubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the role of CK8 as a potential receptor for EAEC.
Infection and immunity 02/2014; · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rotavirus is the leading cause of diarrhea in children <5 years of age. In light of the implementation of rotavirus vaccines of limited valency, it is important to characterize the genotypic diversity of circulating rotavirus in sub-Saharan Africa.
We collected stool samples from children 0-59 months of age who presented at the health centres as cases with moderate-to-severe diarrhea in the Upper River Region of The Gambia. Stool samples were also collected from age, sex and area-matched healthy controls. All stool samples were assayed for rotavirus antigens by enzyme-linked immunosorbent assay and genotyping was done using reverse transcriptase polymerase chain reaction.
We enrolled 1029 cases and 1569 controls during the 3-year study period (2008-2010). The detection rate of rotavirus among the cases was 20% (204/1029) and 3% (42/1569) among controls. At least 18 genotypes were found and the predominant genotypes were G2P (28%), G1P (26%) and G1P (10%). The rare identified genotypes (<1%) were G2P, G8P, G9P and G4P. There was also a strong positive association between rotavirus infection and the dry season (odds ratio: 9.83, 95% confidence interval: 6.18-15.63, P < 0.001). A significant increase in the odds of rotavirus and G1P detection with the use of untreated water and the presence of cats, rodents and cows in the child's residence was also found.
This study provides important baseline data for the genotypes circulating before vaccine implementation. The wide diversity of genotypes circulating in The Gambia implies the need for vigilant effectiveness surveillance following the implementation of RotaTeq in August 2013.
[Show abstract][Hide abstract] ABSTRACT: Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin α5β1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin α5β1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin α5β1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin α5β1 to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin α5 shRNA. Altogether, our data support the involvement of integrin α5β1 in the fibronectin-mediated EAEC binding to intestinal cells.
BioMed Research International 01/2014; 2014:781246. · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent evidence suggests that the abundance of enteric pathogens in the stool correlates with the presence of clinical diarrhea. We quantified the fecal pathogen after feeding enterotoxigenic E. coli (ETEC) strain H10407 to 30 adult volunteers. Stools were collected daily and examined using qualitative and quantitative (Q) culture. DNA was isolated and quantitative (Q)PCR targeting the heat-labile toxin (LT) gene was performed. Nine volunteers developed diarrhea. Among 131 stool specimens with complete data, pathogen abundance by QPCR was strongly correlated with Qculture, rho=0.61, p<0.0001. Receiver operating characteristic curve analysis comparing quantitative data against diarrhea status suggested cut-points, based on a maximum Youden Index, of 2.8x10(4) LT gene copies and 1.8x10(7) CFU. Based on these cut-points, QPCR had a sensitivity and specificity compared to diarrheal status of 0.75 and 0.87, respectively, and an OR of 20.0 (95% CI 5.7-70.2), whereas Qculture had a sensitivity and specificity of 0.73 and 0.91, respectively, and an OR of 28.6 (95% CI 7.7-106.6). Qculture had a sensitivity and specificity of 0.82 and 0.48, respectively and an OR of 4.4 (95% CI 1.2-16.0).The correlation between Qculture and QPCR was highest in diarrheal specimens and both quantitative methods demonstrated stronger association with diarrhea than qualitative culture. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Changes in small bowel function early in infancy in developing countries are increasingly being demonstrated, probably accompanied by altered mucosal architecture in most individuals, including reduced enterocyte mass and evidence of immune activation and inflammation in the mucosa. These alterations appear to be the result of factors of uncertain nature in the environment, and may be a cause of growth faltering and stunting in young children. For these reasons, this constellation of findings is being referred to as environmental enteropathy, or as we propose herein, environmental enteric dysfunction. If the causes were known and effective interventions were available, strategies and policies to intervene at--or possibly before--birth could be developed and promoted in order to prevent subsequent malnutrition and recurrent infection, which are known to interact in a cyclical and synergistic manner in a downward clinical course often ending in death. Resources would be mobilized and applied differently, and the emphasis would change from treatment to prevention. In order to move in this highly desired direction, investments in research will be required to establish the criteria to assess environmental enteric dysfunction, determine its predictive value for growth faltering and stunting, identify the causes, and propose and test potential interventions. The concepts and tools are available. What is required is the decision to move forward along this pathway to better health for infants and children in low-income countries.
Food and nutrition bulletin 09/2013; 34(3):357-64. · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enteroaggregative Escherichia coli (EAEC) causes diarrhoea. The antibiotic of choice for treating EAEC infections is ciprofloxacin. EAEC differs from other subgroups of pathogenic E. coli by having a surface protein, dispersin, which has previously been shown to play an important role in ciprofloxacin susceptibility for EAEC model strain 042. To investigate further the role of dispersin in ciprofloxacin susceptibility, minimum inhibitory concentrations (MICs) were determined for 25 clinical isolates, including 15 with dispersin and 10 without. Dispersin-positive strains had a lower MIC than dispersin-negative strains. The mechanism of action behind this observation may be caused by dispersin (i) increasing the bacteria-antibiotic interaction or (ii) facilitating ciprofloxacin access to the intracellular target, DNA gyrase/topoisomerase. To test the role of dispersin in ciprofloxacin sensitivity, EAEC 042 as well as its isogenic mutants, dispersin mutant (042aap) and a mutant in the transporter apparatus gene aatA, believed to be involved in dispersin transport to the bacterial surface (042aatA), were utilised. As predicted, 042 had a higher sensitivity to ciprofloxacin than 042aap, but it was also found that the MIC of 042aatA was similar to 042aap. To address the question of the role of dispersin in ciprofloxacin susceptibility, the concentration of ciprofloxacin bound in biofilms of 042 and 042aap was quantified by treating bacteria with radiolabelled 2-(14)C-ciprofloxacin. The results showed that dispersin did not increase the amount of bound ciprofloxacin as a function of biomass, indicating instead that dispersin facilitates ciprofloxacin access to the intracellular target leading to increased antibiotic susceptibility.
International journal of antimicrobial agents 08/2013; · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cultivation based assays combined with PCR or ELISA based methods to detect virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya and Bangladesh. The children were identified as either controls or cases with moderate to severe diarrhea using currently accepted clinical protocols. 3,610 stool samples were tested by established clinical culture techniques; 3,179 DNA samples by the Universal Biosensor® (Ibis Biosciences, Inc.); 1,466 DNA samples by GoldenGate® (Illumina, Inc.); and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected differing proportions of samples testing positive for each of seven enteric pathogens- enteroaggregative Escherichia coli (EAEC), enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), Shigella, Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. Comparisons among detection methods included the frequency of positive stools and kappa values to make pair wise comparisons. Overall, standard culture methods detected Shigella, EPEC, ETEC and EAEC in a smaller proportion of the samples than either of the methods based on detection of the virulence genes from DNA in whole stool. The GoldenGate® method revealed the greatest agreement with the most other methods. Agreement among methods was higher in cases than controls. New molecular technologies have high potential for highly sensitive identification of bacterial diarrhea pathogens.
Journal of clinical microbiology 07/2013; · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Water, sanitation, and hygiene information was collected during a matched case-control study of moderate and severe diarrhea (MSD) among 4,096 children < 5 years of age in Bamako, Mali. Primary use of piped water (conditional odds ratio [cOR] = 0.45; 0.34-0.62), continuous water access (cOR = 0.30; 0.20-0.43), fetching water daily (cOR = 0.77; 0.63-0.96), and breastfeeding (cOR = 0.65; 0.49-0.88) significantly reduced the likelihood of MSD. Fetching water in > 30 minutes (cOR = 2.56; 1.55-4.23) was associated with MSD. Piped tap water and courier-delivered water contained high (> 2 mg/L) concentrations of free residual chlorine and no detectable Escherichia coli. However, many households stored water overnight, resulting in inadequate free residual chlorine (< 0.2 mg/L) for preventing microbial contamination. Coliforms and E. coli were detected in 48% and 8% of stored household water samples, respectively. Although most of Bamako's population enjoys access to an improved water source, water quality is often compromised during household storage.
The American journal of tropical medicine and hygiene 07/2013; · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Shigella infections are a public health problem in developing and transitional countries because of high transmissibility, severity of clinical disease, widespread antibiotic resistance and lack of a licensed vaccine. Whereas Shigellae are known to be transmitted primarily by direct fecal-oral contact and less commonly by contaminated food and water, the role of the housefly Musca domestica as a mechanical vector of transmission is less appreciated. We sought to assess the contribution of houseflies to Shigella-associated moderate-to-severe diarrhea (MSD) among children less than five years old in Mirzapur, Bangladesh, a site where shigellosis is hyperendemic, and to model the potential impact of a housefly control intervention.