Factors associated with typical enteropathogenic Escherichia coli infection among children <5 years old with moderate-to-severe diarrhoea in rural western Kenya, 2008-2012.

Typical enteropathogenic Escherichia coli (tEPEC) infection is a major cause of diarrhoea and contributor to mortality in children <5 years old in developing countries. Data were analysed from the Global Enteric Multicenter Study examining children <5 years old seeking care for moderate-to-severe diarrhoea (MSD) in Kenya. Stool specimens were tested for enteric pathogens, including by multiplex polymerase chain reaction for gene targets of tEPEC. Demographic, clinical and anthropometric data were collected at enrolment and ~60-days later; multivariable logistic regressions were constructed. Of 1778 MSD cases enrolled from 2008 to 2012, 135 (7.6%) children tested positive for tEPEC. In a case-to-case comparison among MSD cases, tEPEC was independently associated with presentation at enrolment with a loss of skin turgor (adjusted odds ratio (aOR) 2.08, 95% confidence interval (CI) 1.37-3.17), and convulsions (aOR 2.83, 95% CI 1.12-7.14). At follow-up, infants with tEPEC compared to those without were associated with being underweight (OR 2.2, 95% CI 1.3-3.6) and wasted (OR 2.5, 95% CI 1.3-4.6). Among MSD cases, tEPEC was associated with mortality (aOR 2.85, 95% CI 1.47-5.55). This study suggests that tEPEC contributes to morbidity and mortality in children. Interventions aimed at defining and reducing the burden of tEPEC and its sequelae should be urgently investigated, prioritised and implemented.

Critical Role of Zinc in a New Murine Model of Enterotoxigenic Escherichia coli Diarrhea.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveler's diarrhea as well as of endemic diarrhea and stunting in children in developing areas. However, a small-mammal model has been badly needed to better understand and assess mechanisms, vaccines, and interventions. We report a murine model of ETEC diarrhea, weight loss, and enteropathy and investigate the role of zinc in the outcomes. ETEC strains producing heat-labile toxins (LT) and heat-stable toxins (ST) that were given to weaned C57BL/6 mice after antibiotic disruption of normal microbiota caused growth impairment, watery diarrhea, heavy stool shedding, and mild to moderate intestinal inflammation, the latter being worse with zinc deficiency. Zinc treatment promoted growth in zinc-deficient infected mice, and subinhibitory levels of zinc reduced expression of ETEC virulence genes cfa1, cexE, sta2, and degP but not of eltA in vitro Zinc supplementation increased shedding and the ileal burden of wild-type (WT) ETEC but decreased shedding and the tissue burden of LT knockout (LTKO) ETEC. LTKO ETEC-infected mice had delayed disease onset and also had less inflammation by fecal myeloperoxidase (MPO) assessment. These findings provide a new murine model of ETEC infection that can help elucidate mechanisms of growth, diarrhea, and inflammatory responses as well as potential vaccines and interventions.

Diarrhoea, enteric pathogen detection and nutritional indicators among controls in the Global Enteric Multicenter Study, Kenya site: an opportunity to understand reference populations in case-control studies of diarrhoea.

Given the challenges in accurately identifying unexposed controls in case-control studies of diarrhoea, we examined diarrhoea incidence, subclinical enteric infections and growth stunting within a reference population in the Global Enteric Multicenter Study, Kenya site. Within 'control' children (0-59 months old without diarrhoea in the 7 days before enrolment, n = 2384), we examined surveys at enrolment and 60-day follow-up, stool at enrolment and a 14-day post-enrolment memory aid for diarrhoea incidence. At enrolment, 19% of controls had ⩾1 enteric pathogen associated with moderate-to-severe diarrhoea ('MSD pathogens') in stool; following enrolment, many reported diarrhoea (27% in 7 days, 39% in 14 days). Controls with and without reported diarrhoea had similar carriage of MSD pathogens at enrolment; however, controls reporting diarrhoea were more likely to report visiting a health facility for diarrhoea (27% vs. 7%) or fever (23% vs. 16%) at follow-up than controls without diarrhoea. Odds of stunting differed by both MSD and 'any' (including non-MSD pathogens) enteric pathogen carriage, but not diarrhoea, suggesting control classification may warrant modification when assessing long-term outcomes. High diarrhoea incidence following enrolment and prevalent carriage of enteric pathogens have implications for sequelae associated with subclinical enteric infections and for design and interpretation of case-control studies examining diarrhoea.

Characteristics of the enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli O104:H4 strain causing the outbreak of haemolytic uraemic syndrome in Germany, May to June 2011.

The Escherichia coli strain causing a large outbreak of haemolytic uraemic syndrome and bloody diarrhoea in Germany in May and June 2011 possesses an unusual combination of pathogenic features typical of enteroaggregative E. coli together with the capacity to produce Shiga toxin. Through rapid national and international exchange of information and strains the known occurrence in humans was quickly assessed.We describe simple diagnostic screening tools to detect the outbreak strain in clinical specimens and a novel real-time PCR for its detection in foods.

Enteroaggregative Escherichia coli expresses a novel flagellin that causes IL-8 release from intestinal epithelial cells.

Enteroaggregative Escherichia coli (EAEC) is an emerging cause of acute and persistent diarrhea worldwide. EAEC infections are associated with intestinal inflammation and growth impairment in infected children, even in the absence of diarrhea. We previously reported that prototype EAEC strains rapidly induce IL-8 production by Caco-2 intestinal epithelial cells, and that this effect is mediated by a soluble, heat-stable factor released by these bacteria in culture. We herein report the cloning, sequencing, and expression of this biologically active IL-8-releasing factor from EAEC, and its identification as a flagellin that is unique among known expressed proteins. Flagella purified from EAEC 042 and several other EAEC isolates potently release IL-8 from Caco-2 cells; an engineered aflagellar mutant of 042 does not release IL-8. Finally, cloned EAEC flagellin expressed in nonpathogenic E. coli as a polyhistidine-tagged fusion protein maintains its proinflammatory activity. These findings demonstrate a major new means by which EAEC may cause intestinal inflammation, persistent diarrhea, and growth impairment that characterize human infection with these organisms. Furthermore, they open new approaches for diagnosis and vaccine development. This novel pathogenic mechanism of EAEC extends an emerging paradigm of bacterial flagella as inflammatory stimuli.

Role of the eaeA gene in experimental enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infant diarrhea in developing countries. Recently eaeA, a gene necessary for the characteristic intimate attachment of EPEC to epithelial cells in tissue culture, was described. We conducted a randomized, double-blind study to determine the role of the eaeA gene in human EPEC infection. 11 adult volunteers ingested 2 x 10(10) colony-forming units of O127:H6 EPEC strain E2348/69, and an equal number received the same dose of an isogenic eaeA deletion mutant constructed from E2348/69. Volunteers were monitored for the development of diarrhea, fever, and systemic and gastrointestinal complaints. Diarrhea developed in all 11 volunteers who received E2348/69 and in 4 of 11 who received the mutant (P = 0.002). Fever was more common in recipients of the wild-type strain (P = 0.024). Stool volumes were lower in recipients of the mutant. All volunteers seroconverted to E2348/69 LPS, but the geometric mean peak titers of serum IgG and IgA in recipients of the mutant were lower than those of recipients of the wild-type strain. IgA against LPS was detected in the jejunal fluid of six of six recipients of E2348/69 and 5/6 recipients of the mutant. This study unambiguously assigns a role for eaeA as an EPEC virulence gene, but the residual diarrhea seen in recipients of the mutant indicates that other factors are involved.

Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM.

Enteroaggregative Escherichia coli (EAEC) is pathogenic and produces severe diarrhea in humans. A mutant of EAEC that does not produce dispersin, a cell surface protein, is not pathogenic. It has been proposed that dispersin imparts a positive charge to the bacterial cell surface allowing the bacteria to colonize on the negatively charged intestinal mucosa. However, physical properties of the bacterial cell surface, such as rigidity, may be influenced by the presence of dispersin and may contribute to pathogenicity. Using the system developed in our laboratory for mounting and imaging bacterial cells by atomic force microscopy (AFM), in liquid, on gelatin coated mica surfaces, studies were initiated to measure cell surface elasticity. This was carried out in both wild type EAEC, that produces dispersin, and the mutant that does not produce dispersin. This was accomplished using AFM force-distance (FD) spectroscopy on the wild type and mutant grown in liquid or on solid medium. Images in liquid and in air of both the wild-type and mutant grown in liquid and on solid media are presented. This work represents an initial step in efforts to understand the pathogenic role of the dispersin protein in the wild-type bacteria.

The great escape: structure and function of the autotransporter proteins.

The autotransporters, a family of secreted proteins from Gram-negative bacteria, possess an overall unifying structure comprising three functional domains: the amino-terminal leader sequence, the secreted mature protein (passenger domain) and a carboxy-terminal (beta-) domain that forms a beta-barrel pore to allow secretion of the passenger protein. Members of this family have been implicated as important or putative virulence factors in many Gram-negative pathogens.

Characterization of the AfaD-like family of invasins encoded by pathogenic Escherichia coli associated with intestinal and extra-intestinal infections.

The afimbrial adhesive sheath, encoded by the afa-3 gene cluster, is composed of two proteins with different roles in bacterium-HeLa cell interactions. AfaE is required for adhesion and AfaD for internalization. In this study, we found that the AfaD invasin was structurally and functionally conserved among human afa-expressing strains, independently of AfaE subtype and clinical origin of the Escherichia coli isolate. The AggB protein from enteroaggregative E. coli was also found to be an AfaD-related invasin. These data suggest that AfaD is the prototype of a family of invasins encoded by adhesion-associated operons in pathogenic E. coli.

Enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) are an increasingly important cause of diarrhoea. E. coli belonging to this category cause watery diarrhoea, which is often persistent and can be inflammatory. EAEC have been implicated in sporadic diarrhoea in children and adults, in both developing and developed countries, and have been identified as the cause of several outbreaks worldwide. EAEC are defined by their ability to adhere to epithelial cells in a characteristic "stacked-brick" pattern but are otherwise highly heterogeneous. Genes that could contribute to the pathogenicity of EAEC encode adhesins, toxins, and other factors, all of which are only partially conserved. Practicable tools are needed to improve diagnosis and identify risk factors. EAEC-infected individuals can be treated with fluoroquinolones but there is a need to examine alternative treatment protocols.

Endemic necrotizing enterocolitis: lack of association with a specific infectious agent.

We conducted a comprehensive analysis of bacterial, parasitic and viral agents present in stool samples of 23 necrotizing enterocolitis cases and 23 matched and 10 random controls. Enterococcus spp., Staphylococcus epidermidis, and Escherichia coli were the most common aerobic bacterial species isolated. Astrovirus was identified in a stool sample from one control. Eight infants were bacteremic; in 7 of 8 the same organism was also present in the stool. No one bacterial species or strain (as identified by plasmid profile analysis) was associated with occurrence of illness. Plasmid analysis further suggested that each infant was colonized with his or her own distinctive aerobic bacterial flora. With the exception of isolates from two control patients which hybridized with a probe for diffuse adherence, no diarrheagenic E. coli was identified. Five (45%) of 11 case infants were colonized with coagulase-negative staphylococci (all S. epidermidis) that produced delta-hemolysin in vitro, as compared with 13 (87%) of 15 control infants. Necrotizing enterocolitis was not associated with an increased ability to ferment carbohydrate, as measured by in vitro beta-galactosidase activity. Our data do not support the hypothesis that endemic necrotizing enterocolitis in our institution is caused by a single infectious agent, nor was there evidence that previously proposed virulence mechanisms such as production of delta-hemolysin or increased in vitro carbohydrate fermentation play a critical role in disease occurrence.

Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells.

A total of 516 Escherichia coli strains randomly isolated from coprocultures of 154 Chilean children with diarrhea and 66 controls were examined with DNA probes and tested for adherence to HEp-2 cells. Three adherence patterns were distinguished, localized, true diffuse and "aggregative." Enteropathogenic E. coli (EPEC) were detected by EPEC adherence factor probe among 86 of the 372 isolates (23%) from patients with diarrhea vs. 14 of 144 (10%) strains from controls (P less than 0.0002). Of 95 strains that manifested localized adherence, 97% were EPEC adherence factor probe-positive; thus the HEp-2 assay may serve as an alternative to the probe in identifying EPEC adherence factor-positive EPEC. True diffuse adherence was not associated with diarrhea. In contrast the aggregative pattern appears to signify a new, distinct class of diarrheagenic E. coli (enteroadherent-aggregative E. coli). The aggregative pattern was found in only 3 of 27 enterotoxigenic, 0 of 4 enteroinvasive, 0 of 2 enterohemorrhagic and 2 of 86 EPEC strains but in 84 of 253 probe-negative strains (P less than 0.00001) from patients with diarrhea; in comparison only 20 of 134 probe-negative strains from controls were aggregative E. coli (P less than 0.00001 vs. probe-negative strains from diarrhea patients).

Population-based study of the incidence of Shigella diarrhea and causative serotypes in Santiago, Chile.

BACKGROUND: Shigella is an important cause of diarrheal disease in children in developing countries. The increasing prevalence of antibiotic-resistant strains has stimulated interest in the use of multivalent Shigella vaccines. Because Shigella vaccines under development are based on eliciting immunity to O antigens, monitoring the distribution of serotypes in defined target populations is critical. We initiated health center-based surveillance in a poor semirural community in Colina, Santiago (7489 children <60 months of age) to determine the age-specific incidence of Shigella disease and the responsible serotypes. FINDINGS: Surveillance was maintained at the 2 health centers during warm seasons (November 1 through April 30) for 4 successive years (1994 to 1998). Shigella was recovered from 54 of 243 cases of dysentery (22%) and from 215 of 3966 cases of nondysenteric diarrhea (5.4%) (P < 0.001). The peak mean annual incidence of shigellosis occurred among children 12 to 47 months of age (9.0 to 12.6 cases/10(3) children), although the incidence in infants (5.2/10(3)) and children 48 to 59 months of age (6.2/10(3)) was also substantial. During the 1995 through 1996 season, an age-matched healthy control was cultured for every child <60 months of age with diarrhea. Shigella isolation from cases (34 of 576, 5.9%) was >8-fold higher than controls (4 of 576, 0.7%) (P < 0.01). Four serotypes, Shigella sonnei (45%), Shigella flexneri 2b (19%), S. flexneri 2a (14%) and S. flexneri 6 (11%), accounted for 89% of all cases. INTERPRETATION: Shigella remains an important pediatric pathogen in Santiago. The serotype distribution from Colina, which closely resembles data from a population-based surveillance study in Santiago in the mid-1980s, demonstrates a remarkable degree of serotype stability in Santiago during a 15-year period.

Association of torovirus with acute and persistent diarrhea in children.

OBJECTIVE: To study the etiologic role of toroviruses as a cause of gastroenteritis in humans. METHODS: The design was a case-control study. We compared the rate of torovirus detection in fecal specimens from a selection of children with acute or persistent diarrhea and controls without diarrhea from a study of childhood diarrhea in an urban Brazilian slum. Stool samples were coded and tested in a blinded fashion for the presence of torovirus antigen by enzyme-linked immunosorbent assay, other enteropathogens, toxins and fecal leukocytes. RESULTS: Thirty-three children with acute diarrhea, 41 children with persistent diarrhea and 17 controls were enlisted in the study. Torovirus antigen was detected in 9 (27%) samples from children with acute diarrhea, 11 (27%) samples from children with persistent diarrhea and none of the samples from controls (P < 0.05). In addition the presence of enteroaggregative E. coli was associated with persistent diarrhea and the presence of Cryptosporidium oocysts was common although not significant (P = 0.08); torovirus and Cryptosporidium occurred in different subsets of samples, whereas torovirus and enteroaggregative Escherichia coli were commonly found in combination. CONCLUSIONS: These data indicate that toroviruses, alone or in combination with enteroaggregative E. coli, may play a pathogenic role in acute and possibly persistent diarrhea. Further studies are warranted to determine the etiologic role of toroviruses in gastroenteritis.

Short report: characterization of enteroaggregative Escherichia coli isolates from Iranian children.

We have previously shown that enteroaggregative Escherichia coli (EAEC) is an important pathogen among Iranian infants and children. To better understand the characteristics of EAEC in Iran, we analyzed EAEC isolates for the presence of pAA plasmid-borne factors. Ninety-eight E. coli strains that displayed the aggregative adherence (AA) pattern on HeLa cells were hybridized with the CVD432 (AA) probe and with genes encoding enteroaggregative heat-stable enterotoxin-1 and aggregative adherence fimbriae (AAF) I and II. Our data suggest that AAF/II is common in this population and that AAF/I and AAF/II can sometimes be detected in the same E. coli isolate. Surprisingly, we have found that AA probe-negative strains in Iran share virulence factors with AA probe-positive isolates and therefore may be more similar to probe-positive strains than previously believed.

Volunteer studies investigating the safety and efficacy of live oral El Tor Vibrio cholerae O1 vaccine strain CVD 111.

A live oral cholera vaccine should ideally protect against both classical and El Tor biotypes of Vibrio cholerae O1. An El Tor biotype vaccine strain, therefore, would complement classical cholera vaccine strain CVD 103-HgR, a strain already in use in some countries. In this study, 25 healthy adult volunteers received a single dose of 10s colony-forming units of El Tor vaccine strain CVD 111, a derivative of El Tor Ogawa strain N16117 deleted in the virulence cassette. Three (12%) volunteers developed mild diarrhea (mean stool volume = 813 ml) but no systemic symptoms; 23 (92%) of the 25 volunteers developed serum vibriocidal antibodies (geometric mean titer = 1:2,291). Five weeks after vaccination, 18 vaccines and eight uninimunized control volunteers underwent wild-type challenge with El Tor Ogawa strain 3008. Three (16.7%) of 18 vaccinees and seven (87.5%) of eight controls developed diarrhea (P = 0.001) (vaccine efficacy = 80.9%). Further studies are underway to determine a dosage of CVD 111 that will be more clinically acceptable but equally immunogenic and protective.

Identification of enteric pathogens in the small and large intestine of children with diarrhea.

Enteric pathogens were identified in children with diarrhea from duodenal specimens obtained with a string capsule and from fecal specimens. Rotavirus was identified in stools of 43 of 100 children, and was recovered from the small intestine from nine (21%) children who were excreting this virus. Shigella was isolated from stools from 22, Salmonella from 17, enterotoxigenic Escherichia coli from eight, and Aeromonas hydrophila from one of 100 children with diarrhea. In contrast to rotavirus, Salmonella, Shigella, enterotoxigenic Escherichia coli, and A. hydrophila were not isolated from the small intestine. Nonenterotoxigenic Aeromonas species were recovered from the small intestine, but not the stool of five children. These children were also infected with Shigella or with rotavirus; this suggests that Aeromonas was not the cause of their diarrhea. None of 51 Escherichia coli isolated with the string capsules, or 67 isolated from stool that agglutinated in commercial enteropathogenic Escherichia coli antisera were of classical enteropathogenic Escherichia coli serotypes. One hundred and five of these 118 Escherichia coli did not hybridize with a deoxyribonucleic acid probe for plasmid mediated factors conferring adherence to HeLa cells. Examination of specimens collected with a string capsule from children with diarrhea did not identify any more enteric pathogens than examining stools. Furthermore testing Escherichia coli for agglutination in commercial enteropathogenic Escherichia coli antisera did not identify Escherichia coli of enteropathogenic serotypes.

Extension of the volunteer challenge model to study South American cholera in a population of volunteers predominantly with blood group antigen O.

The volunteer challenge model was used to study the virulence of strains of Vibrio cholerae O1 El Tor recently isolated from cases of cholera in South America. Fifteen of the 24 volunteers (62%) were of blood group O, the group most prevalent in South America and the group at increased risk of more severe cholera. Two El T or Inaba strains and 2 El Tor Ogawa strains were given to volunteers at a dose of 1-2 x 10(6) colony-forming units. All 4 strains caused diarrhoea in 67-83% of volunteers. Volunteers with blood group antigen O had an increased attack rate for diarrhoea (P = 0.015) and a marginally increased mean diarrhoeal stool volume (P = 0.08) after challenge. One-third of the volunteers with blood group O, and none of the volunteers with other blood groups, developed severe diarrhoea (> 5 L) (P = 0.01). This study established a model of South American cholera that can be used to predict field efficacy of candidate vaccines among populations with a high prevalence of blood group antigen O.

Diagnosisand Investigation of Diarrheagenic Escherichia coli.

Although most Escherichia coli are harmless commensals of the human intestine, certain specific, highly-adapted E. coli strains are capable of causing urinary tract, systemic or enteric/diarrheagenic infection. Diarrheagenic E coli are divided into six distinct categories, or pathotypes, each with a distinct pathogenic scheme (Table 1). Combined, diarrheagenic E coli have emerged as perhaps the most important enteric pathogens of man. In the developing world, the E coli categories account for more cases of gastroenteiltis among infants than any other cause (1) In addition, E coli are also the most common cause of traveller's diarrhea, which afflicts more than one million travellers to the developing world annually (1). Enterohemorrhagic E coli (EHEC) are the cause of hemolytic uremic syndrome (HUS), which has become a major foodborne threat in many parts of the developed world (2). Table 1 Categories of Diarrheagenic E. coli Category Toxins Invasion Virulence plasmid Adhesin Clinical syndrome ETEC LT, ST - Many CFA/I, CFA/II, CFA/IV, others Watery diarrhea EPEC - + 60 MDa Bundle-forming pilus Watery diarrhea of infants EHEC SLT-1, SLT-2 - 60 MDa( a ) Intimin, Fimbriae( a ) Hemorrhagic colitis, HUS EAEC EAST1( a ) ? 65 MDa( a ) AAF/I, AAF/I Watery, persistent diarrhea EIEC EIET( a ) +++ 140 MDa Ipa's(?) Watery diarrhea, dysentery DAEC ? ? ? F1845( a ) Watery diarrhea ( a )Role in pathogenesis unproven.