Characterization of Diarrheagenic Enteroaggregative Escherichia coli in Danish Adults—Antibiotic Treatment Does Not Reduce Duration of Diarrhea

Enteroaggregative Escherichia coli (EAEC) is frequently isolated from sporadic cases of diarrhea and in outbreaks of gastroenteritis in several regions of the world. The pathophysiology of EAEC continues to be enigmatic, and the efficacy of antibiotic treatment in EAEC-associated diarrhea has been discussed. Since the level of antibiotic resistance is increasing, it is essential to restrict the use of antibiotics to prevent further resistance development. We aimed to investigate EAEC strains in adult Danish patients suffering from diarrhea and from healthy controls. We examined the antibiotic resistance in EAEC strains, the clinical response to antibiotic treatment in EAEC diarrheal cases, and the distribution of virulence genes in diarrheal cases. The EAEC strains were collected from patients suffering from diarrhea in a Danish multicenter study. A medical doctor interviewed the patients by using a questionnaire regarding gastrointestinal symptoms, exposures, and use of antibiotic and over-the-counter antidiarrheal drugs. Follow-up was performed after 3–5 months to inquire about differential diagnosis to gastrointestinal disease. A multiplex polymerase chain reaction characterized virulence genes in diarrheal cases. Finally, the level of antibiotic resistance was examined by using the disc diffusion method. Asymptomatic carriage of EAEC in the adult Danish population was rare, in contrast to findings in healthy Danish children. The duration of diarrhea was not shortened by antibiotic treatment, specifically ciprofloxacin treatment, or by over-the-counter antidiarrheal drugs. Follow-up revealed no pathology in diarrheal patients apart from irritable bowel syndrome in two patients. A high number of patients suffered from long-term diarrhea, which was associated with the enterotoxin EAST-1 and a high virulence factor score. A high level of antibiotic resistance was observed and 58% of the EAEC strains were multidrug resistant. Multidrug resistance was most pronounced in cases of travelers' diarrhea, and it was seen that antibiotic treatment did not reduce the duration of diarrhea

Children Attending Day Care Centers are a Year-round Reservoir of Gastrointestinal Viruses

Abstract Viral gastroenteritis causes high morbidity worldwide. In this study, stool samples from 179 children aged 0–6 years attending Danish day care centers were investigated for gastrointestinal viruses. Each child was observed for one year with submission of samples and questionnaires every two months. Adenovirus, norovirus, rotavirus, and sapovirus were detected in samples using real-time PCR. A total of 229 (33%) of the 688 samples collected tested positive for at least one virus. At the first sampling point, adenovirus was shed by 6%, norovirus genotype I by 3% and genotype II by 12%, rotavirus A by 9%, and sapovirus by 21% of the 142 children included in the risk factor analyses. Increasing age was identified as a protective factor against testing positive for gastrointestinal virus, whereas nausea during the previous two months was positively associated with testing positive. Odds of shedding adenovirus were 9.6 times higher among children treated with antibiotics within the previous two months than among children who were not. Gastrointestinal viruses were shed year-round and high viral loads were observed in samples from both symptomatic and asymptomatic children, suggesting children in day care as a reservoir and a possible source of spreading of viruses into the community

Epidemiology and clinical manifestations of enteroaggregative Escherichia coli

SUMMARY: Enteroaggregative Escherichia coli (EAEC) represents a heterogeneous group of E. coli strains. The pathogenicity and clinical relevance of these bacteria are still controversial. In this review, we describe the clinical significance of EAEC regarding patterns of infection in humans, transmission, reservoirs, and symptoms. Manifestations associated with EAEC infection include watery diarrhea, mucoid diarrhea, low-grade fever, nausea, tenesmus, and borborygmi. In early studies, EAEC was considered to be an opportunistic pathogen associated with diarrhea in HIV patients and in malnourished children in developing countries. In recent studies, associations with traveler's diarrhea, the occurrence of diarrhea cases in industrialized countries, and outbreaks of diarrhea in Europe and Asia have been reported. In the spring of 2011, a large outbreak of hemolytic-uremic syndrome (HUS) and hemorrhagic colitis occurred in Germany due to an EAEC O104:H4 strain, causing 54 deaths and 855 cases of HUS. This strain produces the potent Shiga toxin along with the aggregative fimbriae. An outbreak of urinary tract infection associated with EAEC in Copenhagen, Denmark, occurred in 1991; this involved extensive production of biofilm, an important characteristic of the pathogenicity of EAEC. However, the heterogeneity of EAEC continues to complicate diagnostics and also our understanding of pathogenicity

The Lectin Complement Pathway Is Involved in Protection Against Enteroaggregative Escherichia coli Infection

Enteroaggregative Escherichia coli (EAEC) causes acute and persistent diarrhea worldwide. Still, the involvement of host factors in EAEC infections is unresolved. Binding of recognition molecules from the lectin pathway of complement to EAEC strains have been observed, but the importance is not known. Our aim was to uncover the involvement of these molecules in innate complement dependent immune protection toward EAEC. Binding of mannose-binding lectin, ficolin-1, -2, and -3 to four prototypic EAEC strains, and ficolin-2 binding to 56 clinical EAEC isolates were screened by a consumption-based ELISA method. Flow cytometry was used to determine deposition of C4b, C3b, and the bactericidal C5b-9 membrane attack complex (MAC) on the bacteria in combination with different complement inhibitors. In addition, the direct serum bactericidal effect was assessed. Screening of the prototypic EAEC strains revealed that ficolin-2 was the major binder among the lectin pathway recognition molecules. However, among the clinical EAEC isolates only a restricted number (n = 5) of the isolates bound ficolin-2. Using the ficolin-2 binding isolate C322-17 as a model, we found that incubation with normal human serum led to deposition of C4b, C3b, and to MAC formation. No inhibition of complement deposition was observed when a C1q inhibitor was added, while partial inhibition was observed when ficolin-2 or factor D inhibitors were used separately. Combining the inhibitors against ficolin-2 and factor D led to virtually complete inhibition of complement deposition and protection against direct bacterial killing. These results demonstrate that ficolin-2 may play an important role in innate immune protection against EAEC when an appropriate ligand is exposed, but many EAEC strains evade lectin pathway recognition and may, therefore, circumvent this strategy of innate host immune protection

Genetic Virulence Profile of Enteroaggregative Escherichia coli Strains Isolated from Danish Children with Either Acute or Persistent Diarrhea

Enteroaggregative Escherichia coli (EAEC) is frequently found in diarrheal stools worldwide. It has been associated with persistent diarrhea, weight loss, and failure to thrive in children living in developing countries. A number of important EAEC virulence genes are identified; however, their roles in acute and persistent diarrhea have not been previously investigated. The aim of this study was to identify specific EAEC virulence genes associated with duration and type of diarrhea in Danish children. We aimed to improve the current diagnostics of EAEC and enable targeting of strains with an expected severe disease course. Questionnaires answered by parents provided information regarding duration of diarrhea and presence of blood or mucus. A total of 295 EAEC strains were collected from children with acute (≤7 days) and persistent diarrhea (≥14 days) and were compared by using multiplex PCR targeting the genes sat, sepA, pic, sigA, pet, astA, aatA, aggR, aaiC, aap, agg3/4C, ORF3, aafA, aggA, agg3A, agg4A, and agg5A. Furthermore, the distribution of EAEC genes in strains collected from cases of bloody, mucoid, and watery diarrhea was investigated. The classification and regression tree analysis (CART) was applied to investigate the relationship between EAEC virulence genes and diarrheal duration and type. Persistent diarrhea was associated with strains lacking the pic gene (p = 0.002) and with the combination of the genes pic, sat, and absence of the aggA gene (p = 0.05). Prolonged diarrhea was associated with the combination of the genes aatA and astA (p = 0.03). Non-mucoid diarrhea was associated with strains lacking the aatA gene (p = 0.004). Acute diarrhea was associated with the genes aggR, aap, and aggA by individual odds ratios. Resistance toward gentamicin and ciprofloxacin was observed in 7.5 and 3% of strains, respectively. Multi-drug resistance was observed in 38% of strains. Genetic host factors have been associated with an increased risk of EAEC-associated disease. Therefore, we investigated a panel of risk factors in two groups of children—EAEC-positive and EAEC-negative—to identify additional factors predisposing to disease. The duration of breastfeeding was positively correlated with the likelihood of belonging to the EAEC-negative group of children

Stability and resilience of the intestinal microbiota in children in daycare – a 12 month cohort study

Abstract Background We performed a 12-month cohort study of the stability and resilience of the intestinal microbiota of healthy children in daycare in Denmark in relation to diarrheal events and exposure to known risk factors for gastrointestinal health such as travelling and antibiotic use. In addition, we analyzed how gut microbiota recover from such exposures. Results We monitored 32 children in daycare aged 1–6 years. Fecal samples were submitted every second month during a one-year observational period. Information regarding exposures and diarrheal episodes was obtained through questionnaires. Bacterial communities were identified using 16S rRNA gene sequencing. The core microbiota (mean abundance > 95%) dominated the intestinal microbiota, and none of the tested exposures (diarrheal events, travel, antibiotic use) were associated with decreases in the relative abundance of the core microbiota. Samples exhibited lower intra-individual variation than inter-individual variation. Half of all the variation between samples was explained by which child a sample originated from. Age explained 7.6–9.6% of the variation, while traveling, diarrheal events, and antibiotic use explained minor parts of the beta diversity. We found an age-dependent increase of alpha diversity in children aged 1–3 years, and while diarrheal events caused a decrease in alpha diversity, a recovery time of 40–45 days was observed. Among children having had a diarrheal event, we observed a 10x higher relative abundance of Prevotella. After travelling, a higher abundance of two Bacteroides species and 40% less Lachnospiraceae were seen. Antibiotic use did not correlate with changes in the abundance of any bacteria. Conclusion We present data showing that Danish children in daycare have stable intestinal microbiota, resilient to the exposures investigated. An early age-dependent increase in the diversity was demonstrated. Diarrheal episodes decreased alpha diversity with an estimated recovery time of 40–45 days

Enteroaggregative <i>Escherichia coli</i> in Daycare:A 1-Year Dynamic Cohort Study

Enteroaggregative Escherichia coli (EAEC) has been associated with persistent diarrhea, reduced growth acceleration, and failure to thrive in children living in developing countries and with childhood diarrhea in general in industrialized countries. The clinical implications of an EAEC carrier-status in children in industrialized countries warrants clarification. To investigate the pathological significance of an EAEC carrier-state in the industrialized countries, we designed a one-year dynamic cohort study and performed follow-up every second month, where the study participants submitted a stool sample and answered a questionnaire regarding gastrointestinal symptoms and exposures. Exposures included foreign travel, consumption of antibiotics and contact with a diseased animal.In the capital area of Denmark, a total of 179 children aged 0–6 years were followed in a cohort study, in the period between 2009 and 2013. This is the first investigation of the incidence and pathological significance of EAEC in Danish children attending daycare facilities. Conventional microbiological detection of enteric pathogens was performed at Statens Serum Institut, Copenhagen, Denmark and at Hvidovre Hospital, Copenhagen, Denmark. Parents completed questionnaires regarding gastrointestinal symptoms. The EAEC strains were further characterized by serotyping, phylogenetic analysis, and susceptibility testing. EAEC was detected in 25 (14%) of the children during the observational period of one year. One or more gastrointestinal symptoms were reported from 56% of the EAEC-positive children. Diarrhea was reported in six (24%) of the EAEC positive children, but no cases of weight loss and general failure to thrive were observed. The EAEC strains detected comprised a large number of different serotypes, confirming the genetic heterogeneity of this pathotype. EAEC was highly prevalent (n = 25, 14%) in Danish children in daycare centers and was accompanied by gastrointestinal symptoms in 56% of the infected children. No serotype or phylogenetic group was specifically linked to children with disease