Enteroaggregative E. coli (EAEC) is a major cause of diarrhea worldwide. EAEC are highly adherent to cultured epithelial cells and make biofilms. Both adherence and biofilm formation rely on the presence of aggregative adherence fimbriae (AAF). We compared biofilm formation from two EAEC strains of each of the five AAF types. We found that AAF type did not correlate with the level of biofilm produced. Because the composition of the EAEC biofilm has not been fully described, we stained EAEC biofilms to determine if they contained protein, carbohydrate glycoproteins, and/or eDNA and found that EAEC biofilms contained all three extracellular components. Next, we assessed the changes to the growing or mature EAEC biofilm mediated by treatment with proteinase K, DNase, or a carbohydrate cleavage agent to target the different components of the matrix. Growing biofilms treated with proteinase K had decreased biofilm staining for more than half of the strains tested. In contrast, although sodium metaperiodate only altered the biofilm in a quantitative way for two strains, images of biofilms treated with sodium metaperiodate showed that the EAEC were more spread out. Overall, we found variability in the response of the EAEC strains to the treatments, with no one treatment producing a biofilm change for all strains. Finally, once formed, mature EAEC biofilms were more resistant to treatment than biofilms grown in the presence of those same treatments.

Twenty-two atypical enteroaggregative Escherichia coli isolates from a previous epidemiological study harboring EAEC virulence genes were examined for their adhesion properties. Nine strains showed a typical aggregative adherence (AA) pattern, while 13 strains showed variant AA, such as AA with lined up cells characteristic of the chain-like adhesion (CLA) and AA mainly to HeLa cells characteristic of the diffuse adherence (DA). The aggregative forming pilus (AFP) genes afpA2 and afpR were detected only in strain Q015B, which exhibited an AA/DA pattern. Using Tn5-based transposon mutagenesis on Q015B strain, we identified a 5517-bp open reading frame (ORF) encoding a predicted 1838-amino-acid polypeptide that is genetically related to a putative filamentous hemagglutinin identified in E. coli strain 7-233-03_S3_C2. Therefore, the ORF was named orfHA. The regions flanking orfHA were sequenced and two ORFs were found; upstream, an ORF that encodes a 603-amino-acid polypeptide with 99% identity to hemolysin secretion/activation proteins of the ShlB/FhaC/HecB family, and downstream, another ORF, which encodes a 632-amino-acid polypeptide with 72% identity to the glycosyltransferase EtpC. An orfHA mutant (Q015BΔorfHA) was constructed from strain Q015B. Q015BΔorfHA strain did not adhere to HeLa cells, whereas Q015BΔ orfHA transformed with a pACYC184 plasmid carrying orfHA restored the AA/DA phenotype of strain Q015B. Furthermore, the Q015ΔorfHA mutant had a marked effect on the ability of strain Q015B to kill the larvae of Galleria mellonella. Our results suggest that the AA/DA pattern of strain Q015B is mediated by a hemagglutinin-associated protein which also contributes to its virulence in the G. mellonella model.

Calf diarrhea induced by enteroaggregative E. coli (EAEC) spreads fast among young ruminants, causing continuous hazard to dairy industry. Antimicrobial drug abuse aggravates the incidence rate of multi-drug resistant (MDR) extended-spectrum β-lactamase-producing E. coli (ESBL-EC). However, knowledge of detection and significance of disease-related biomarkers in neonatal female calves are still limited. Gallic acid (GA), a natural secondary metabolite mostly derived from plants, has attracted increasing attention for its excellent anti-inflammatory and anti-oxidative properties. However, it is vague how GA engenders amelioration effects on clinical symptoms and colitis induced by ESBL-EAEC infection in neonatal animals. Here, differentiated gut microbiome and fecal metabolome discerned from neonatal calves were analyzed to ascertain biomarkers in their early lives. Commensal Collinsella and Coriobacterium acted as key microbial markers mediating colonization resistance. In addition, there exists a strongly positive relation between GA, short-chain fatty acid (SCFA) or other prebiotics, and those commensals using random forest machine learning algorithm and Spearman correlation analyses. The protective effect of GA pretreatment on bacterial growth, cell adherence, and ESBL-EAEC-lipopolysaccharide (LPS)-treated Caco-2 cells were first assessed, and results revealed direct antibacterial effects and diminished colonic cell inflammation. Then, oral GA mediated colitis attenuation and recovery of colonic short-chain fatty acid (SCFA) productions on neonatal mice peritonitis sepsis or oral infection model. To corroborate this phenomenon, fecal microbiota transplantation (FMT) method was adopted to remedy the bacterial infection. Of note, FMT from GA-treated neonatal mice achieved profound remission of clinical symptoms and colitis over the other groups as demonstrated by antibacterial capability and prominent anti-inflammatory abilities, revealing improved hindgut microbiota structure with enriched Clostridia_UCG-014, Lachnospiraceae, Oscillospiraceae, and Enterococcaceae, and upregulation of SCFA productions. Collectively, our findings provided the direct evidence of hindgut microbiota and intestinal metabolites, discriminating the health status of neonatal calves post ESBL-EAEC infection. The data provided novel insights into GA-mediated remission of colitis via amelioration of hindgut commensal structure and upregulation of SCFA productions. In addition, its eminent role as potential antibiotic alternative or synergist for future clinic ESBL-EAEC control in livestock.

BACKGROUND: Travelers\' diarrhea remains a prevalent illness impacting individuals visiting developing countries, however most studies have focused on this disease in the context of short term travel. This study aims to determine the regional estimates of travelers\' diarrhea incidence, pathogen-specific prevalence, and describe the morbidity associated with diarrheal disease among deployed military personnel and similar long term travelers.
METHODS: We updated a prior systematic review to include publications between January 1990 and June 2015. Point estimates and confidence intervals of travelers\' diarrhea and pathogen prevalence were combined in a random effects model and assessed for heterogeneity. Eighty-two studies were included in the analysis, including 29 new studies since the prior systematic review.
RESULTS: Military personnel were evaluated in 69% of studies and non-military long term travelers in 34%, with a median duration of travel of 4.9 months, and travel predominantly to the Middle East, Southeast Asia, and Latin America and the Caribbean. Sixty-two percent of tested cases were due to bacterial pathogens, with enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and Campylobacter predominating, and significant regional variability. The incidence of TD from studies with longitudinal data was 36.3 cases per 100 person-months, with the highest rates in Southeast Asia, Latin America and the Caribbean, and the Middle East, with higher estimates from those studies using self-reporting of disease. Morbidity remained significant, with 21% being incapacitated or placed sick in quarters (SIQ) by their illness, 15% requiring intravenous fluids, and 3% requiring hospitalization.
CONCLUSIONS: In comparison to results from the prior systematic review, there were no significant differences in incidence, pathogen prevalence, or morbidity; however there was a trend toward improved care-seeking by sick individuals.

Escherichia coli O104:H4 (E. coli O104:H4), which caused in 2011 a massive foodborne outbreak in Germany, is characterized by an unusual combination of virulence traits. E. coli O104:H4 contains a prophage-encoded Shiga toxin (Stx) gene, which is the cardinal virulence factor of enterohemorrhagic E. coli (EHEC). However, the outbreak strain shares highest DNA sequence similarity with enteroaggregative E. coli (EAEC) and displays the EAEC-characteristic tight adherence to epithelial cells. The virulence potential of the underlying EAEC background has not been investigated and it is therefore not clear whether E. coli O104:H4 displays distinct virulence characteristics in comparison to prototypical EAEC. In this study, we performed a detailed comparative phenotypic characterization of the Stx phage-cured E. coli O104:H4 strain C227-11φcu, the closely related EAEC strain 55989 and two other well-characterized EAEC strains 042 and 17-2 with focus on virulence traits. C227-11φcu displayed superior aggregative adherence phenotype to cultured HCT-8 epithelial cells, adhering with 3-6 times more bacteria per epithelial cells than the tested EAEC strains. Otherwise, C227-11φcu showed similar virulence characteristics to its closest relative 55989, i.e. strong acid resistance, good biofilm formation and cytotoxic culture supernatants. Furthermore, C227-11φcu was characterized by significantly weaker motility and pro-inflammatory properties than 55989 and 042, nevertheless stronger than 17-2. Taken together, C227-11φcu displayed mostly robust, but not outstanding virulence characteristics in comparison to the tested EAEC. Therefore, it appears likely that the combination of Stx production and EAEC characteristics in general, rather than an exceptionally potent EAEC background resulted in the unusual virulence of the E. coli O104:H4. Thus, the emergence of such hypervirulent strains in the future might be more likely than previously anticipated.

Diarrheagenic E. coli (DEC) are one of the most common causes for diarrhea worldwide, especially in children. We evaluated the rapid RIDA ® GENE (RG) real-time multiplex PCR assays (R-Biopharm, Darmstadt, Germany) for the detection of the most important diarrheagenic E. coli. Three hundred fifteen liquid or non-formed stool specimens were examined. The results of the RG multiplex assays were compared to specific PCR methods. The sensitivity and specificity of the RG PCRs were as follows, 100%/100% for the detection of EHEC, 96.3% and 99% for EPEC, 100% and 100% for the detection of EAEC, ETEC and EIEC, respectively. Overall, the RG real-time PCR system for the detection of DEC tested in this study provided reliable and rapid results and shows the ability as a useful addendum for the detection of diarrheagenic E. coli in the medical laboratory.