Clinical severity of enteric viruses detected using a quantitative molecular assay compared to conventional assays in the Global Enteric Multicenter Study.

BACKGROUND: Quantitative molecular assays are increasingly used for detection of enteric viruses. METHODS: We compared the clinical severity using modified Vesikari score (mVS) of enteric viruses detected by conventional assays (enzyme immunoassays [EIA] for rotavirus and adenovirus 40/41 and conventional polymerase chain reaction for astrovirus, sapovirus, and norovirus) and a quantitative molecular assay (TaqMan Array Card [TAC]) among children aged 0-59 months in the Global Enteric Multicenter Study. For rotavirus and adenovirus 40/41, we compared severity between EIA-positive and TAC-positive cases assigned etiologies using different cycle threshold (CT) cutoffs. RESULTS: Using conventional assays, the median (interquartile range) mVS was 10 (8, 11) for rotavirus, 9 (7, 11) for adenovirus 40/41, 8 (6, 10) for astrovirus, sapovirus, and norovirus GII, and 7 (6, 9) for norovirus GI. Compared to rotavirus EIA-positive cases, the median mVS was 2 and 3 points lower for EIA-negative/TAC-positive cases with CT<32.6 and 32.6≤CT<35, respectively (p-value<.0001). Adenovirus 40/41 EIA-positive and EIA-negative/TAC-positive cases were similar, regardless of CT cutoff. CONCLUSIONS: Quantitative molecular assays compared to conventional assays, such as EIA, may influence severity of identified cases, especially for rotavirus. Cutoffs to assign etiology for quantitative assays should be considered in the design and interpretation of enteric virus studies.

Highly-conserved regulatory activity of the ANR family in the virulence of diarrheagenic bacteria through interaction with master and global regulators.

ANR (AraC negative regulators) are a novel class of small regulatory proteins commonly found in enteric pathogens. Aar (AggR-activated regulator), the best-characterized member of the ANR family, regulates the master transcriptional regulator of virulence AggR and the global regulator HNS in enteroaggregative Escherichia coli (EAEC) by protein-protein interactions. On the other hand, Rnr (RegA-negative regulator) is an ANR homolog identified in attaching and effacing (AE) pathogens, including Citrobacter rodentium and enteropathogenic Escherichia coli (EPEC), sharing only 25% identity with Aar. We previously found that C. rodentium lacking Rnr exhibits prolonged shedding and increased gut colonization in mice compared to the parental strain. To gain mechanistic insights into this phenomenon, we characterized the regulatory role of Rnr in the virulence of prototype EPEC strain E2348/69 by genetic, biochemical, and human organoid-based approaches. Accordingly, RNA-seq analysis revealed more than 500 genes differentially regulated by Rnr, including the type-3 secretion system (T3SS). The abundance of EspA and EspB in whole cells and bacterial supernatants confirmed the negative regulatory activity of Rnr on T3SS effectors. We found that besides HNS and Ler, twenty-six other transcriptional regulators were also under Rnr control. Most importantly, the deletion of aar in EAEC or rnr in EPEC increases the adherence of these pathogens to human intestinal organoids. In contrast, the overexpression of ANR drastically reduces bacterial adherence and the formation of AE lesions in the intestine. Our study suggests a conserved regulatory mechanism and a central role of ANR in modulating intestinal colonization by these enteropathogens despite the fact that EAEC and EPEC evolved with utterly different virulence programs.

Mucosal Immune Profiles Associated with Diarrheal Disease Severity in Shigella- and Enteropathogenic Escherichia coli-Infected Children Enrolled in the Global Enteric Multicenter Study.

Enteropathogenic Escherichia coli (EPEC) and Shigella are etiologic agents of diarrhea in children <5 years old living in resource-poor countries. Repeated bouts of infection lead to lifelong morbidity and even death. The goal of this study was to characterize local mucosal immune responses in Shigella- and EPEC-infected children <5 years of age with moderate to severe diarrhea (MSD) enrolled in the Global Enteric Multicenter Study (GEMS). We hypothesized that infection with each of these pathogens would induce distinct gut mucosal immune profiles indicative of disease etiology and severity. To test this hypothesis, innate and adaptive immune markers were measured in stools from children with diarrhea due to EPEC, Shigella, or other organisms and in children who had no diarrhea. Shigella-positive diarrhea evoked robust proinflammatory and TH1/TH2 cytokine responses compared to diarrhea caused by EPEC or other organisms, with the exception of interleukin 5 (IL-5), which was associated with EPEC infection. The presence of IL-1ß, IL-4, IL-16, and tumor necrosis factor beta (TNF-ß) was associated with the absence of dysentery. EPEC-positive diarrhea evoked high levels of IL-1ß, vascular endothelial growth factor (VEGF), and IL-10. Granulocyte-macrophage colony-stimulating factor (GM-CSF) had opposing roles in disease severity, being associated with absence of diarrhea in EPEC-infected children and with dysenteric Shigella infection. High levels of antigen-specific antibodies were detected in the controls and children with Shigella without dysentery, which suggests a protective role against severe disease. In summary, this study identified distinct local immune responses associated with two clinically relevant diarrheagenic pathogens, Shigella and EPEC, in children and identified protective immune phenotypes that can inform the development of preventive measures. IMPORTANCE Shigella and enteropathogenic Escherichia coli are primary agents of moderate to severe diarrhea in children <5 years of age living in resource-poor countries. Repeated bouts of illness lead to lifelong health impairment and even death. Aiming to understand the local host immunity to these pathogens in relation to disease prognosis and to identify prophylaxis and therapeutic targets, we investigated innate and adaptive immune profiles in stools from children infected with EPEC with and without diarrhea, Shigella with and without dysentery, and controls in well characterized clinical samples obtained during the Global Enteric Multicenter Study. For the first time, we report pathogen-specific mucosal immune profiles associated with severity or absence of disease in children <5 years of age that can inform prevention and treatment efforts.

A Novel Adult Murine Model of Typical Enteroaggregative Escherichia coli Infection Reveals Microbiota Dysbiosis, Mucus Secretion, and AAF/II-Mediated Expression and Localization of ß-Catenin and Expression of MUC1 in Ileum.

Typical enteroaggregative Escherichia coli (tEAEC) is a diarrheagenic E. coli pathotype associated with pediatric and traveler's diarrhea. Even without diarrhea, EAEC infections in children also lead to increased gut inflammation and growth shortfalls. EAEC strain's defining phenotype is the aggregative adherence pattern on epithelial cells attributable to the aggregative adherence fimbriae (AAF). EAEC only causes diarrhea in humans; therefore, not much is known of the exact intestinal region of infection and damage or its interactions with intestinal enterocytes in vivo and in situ. This study aimed to develop a new tEAEC mouse model of infection, characterize the microbiota of infected mice, and evaluate in situ the expression of host adherence and surface molecules triggering EAEC infection and the role of the EAEC AAF-II in adherence. Six-week-old C57BL/6 mice, without previous antibiotic treatment, were orally challenged with EAEC 042 strain or EAEC 042 AAF-II mutant (ΔAAF/II) strain, or DAEC-MXR strain (diffusely adherent E. coli clinical isolate), and with saline solution (control group). Paraffin sections of the colon and ileum were stained with H&E and periodic acid-Schiff. ZO-1, ß-catenin, MUC1, and bacteria were analyzed by immunofluorescence. EAEC-infected mice, in comparison with DAEC-MXR-infected and control mice, significantly lost weight during the first 3 days. After 7 days post-infection, mucus production was increased in the colon and ileum, ZO-1 localization remained unaltered, and morphological alterations were more pronounced in the ileum since increased expression and apical localization of ß-catenin in ileal enterocytes were observed. EAEC-infected mice developed dysbiosis 21 days post-infection. At 4 days post-infection, EAEC strain 042 formed a biofilm on ileal villi and increased the expression and apical localization of ß-catenin in ileal enterocytes; these effects were not seen in animals infected with the 042 ΔAAF/II strain. At 3 days post-infection, MUC1 expression on ileal enterocytes was mainly detectable among infected mice and colocalized with 042 strains on the enterocyte surface. We developed a novel mouse model of EAEC infection, which mimics human infection, not an illness, revealing that EAEC 042 exerts its pathogenic effects in the mouse ileum and causes dysbiosis. This model is a unique tool to unveil early molecular mechanisms of EAEC infection in vivo and in situ.

Molecular Epidemiology of Rotavirus Strains in Symptomatic and Asymptomatic Children in Manhiça District, Southern Mozambique 2008-2019.

Group A rotaviruses remain the leading cause of diarrhoea in children aged <5 years. Mozambique introduced rotavirus vaccine (Rotarix®) in September 2015. We report rotavirus genotypes circulating among symptomatic and asymptomatic children in Manhiça District, Mozambique, pre- and post-vaccine introduction. Stool was collected from enrolled children and screened for rotavirus by enzyme-immuno-sorbent assay. Positive specimens were genotyped for VP7 (G genotypes) and VP4 (P genotypes) by the conventional reverse transcriptase polymerase chain reaction. The combination G12P[8] was more frequently observed in pre-vaccine than in post-vaccine introduction, in moderate to severe diarrhoea (34%, 61/177 vs. 0, p < 0.0001) and controls (23%, 26/113 vs. 0, p = 0.0013) and mixed genotypes (36%, 24/67 vs. 7% 4/58, p = 0.0003) in less severe diarrhoea. We observed changes in post-vaccine compared to pre-vaccine introduction, where G3P[4] and G3P[8] were prevalent in moderate to severe diarrhoea (10%, 5/49 vs. 0, p = 0.0002; and 14%, 7/49 vs. 1%, 1/177, p < 0.0001; respectively), and in less severe diarrhoea (21%, 12/58 vs. 0, p = 0.003; and 24%, 14/58 vs. 0, p < 0.0001; respectively). Our surveillance demonstrated the circulation of similar genotypes contemporaneously among cases and controls, as well as switching from pre- to post-vaccine introduction. Continuous surveillance is needed to evaluate the dynamics of the changes in genotypes following vaccine introduction.

Role of the YehD fimbriae in the virulence-associated properties of enteroaggregative Escherichia coli.

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new YehD fimbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.

Pathogens Associated With Linear Growth Faltering in Children With Diarrhea and Impact of Antibiotic Treatment: The Global Enteric Multicenter Study.

BACKGROUND: The association between childhood diarrheal disease and linear growth faltering in developing countries is well described. However, the impact attributed to specific pathogens has not been elucidated, nor has the impact of recommended antibiotic treatment. METHODS: The Global Enteric Multicenter Study enrolled children with moderate to severe diarrhea (MSD) seeking healthcare at 7 sites in sub-Saharan Africa and South Asia. At enrollment, we collected stool samples to identify enteropathogens. Length/height was measured at enrollment and follow-up, approximately 60 days later, to calculate change in height-for-age z scores (ΔHAZ). The association of pathogens with ΔHAZ was tested using linear mixed effects regression models. RESULTS: Among 8077 MSD cases analyzed, the proportion with stunting (HAZ below -1) increased from 59% at enrollment to 65% at follow-up (P < .0001). Pathogens significantly associated with linear growth decline included Cryptosporidium (P < .001), typical enteropathogenic Escherichia coli (P = .01), and untreated Shigella (P = .009) among infants (aged 0-11 months) and enterotoxigenic E. coli encoding heat-stable toxin (P < .001) and Cryptosporidium (P = .03) among toddlers (aged 12-23 months). Shigella-infected toddlers given antibiotics had improved linear growth (P = .02). CONCLUSIONS: Linear growth faltering among children aged 0-23 months with MSD is associated with specific pathogens and can be mitigated with targeted treatment strategies, as demonstrated for Shigella.

Rotavirus disease burden pre-vaccine introduction in young children in Rural Southern Mozambique, an area of high HIV prevalence.

BACKGROUND: Rotavirus vaccines have been adopted in African countries since 2009, including Mozambique (2015). Disease burden data are needed to evaluate the impact of rotavirus vaccine. We report the burden of rotavirus-associated diarrhea in Mozambique from the Global Enteric Multicenter Study (GEMS) before vaccine introduction. METHODS: A case-control study (GEMS), was conducted in Manhiça district, recruiting children aged 0-59 months with moderate-to-severe diarrhea (MSD) and less-severe-diarrhea (LSD) between December 2007 and November 2012; including 1-3 matched (age, sex and neighborhood) healthy community controls. Clinical and epidemiological data and stool samples (for laboratory investigation) were collected. Association of rotavirus with MSD or LSD was determined by conditional logistic regression and adjusted attributable fractions (AF) calculated, and risk factors for rotavirus diarrhea assessed. RESULTS: Overall 915 cases and 1,977 controls for MSD, and 431 cases and 430 controls for LSD were enrolled. Rotavirus positivity was 44% (217/495) for cases and 15% (160/1046) of controls, with AF = 34.9% (95% CI: 32.85-37.06) and adjusted Odds Ratio (aOR) of 6.4 p< 0.0001 in infants with MSD compared to 30% (46/155) in cases and 14% (22/154) in controls yielding AF = 18.7%, (95% CI: 12.02-25.39) and aOR = 2.8, p = 0.0011 in infants with LSD. The proportion of children with rotavirus was 32% (21/66) among HIV-positive children and 23% (128/566) among HIV-negative ones for MSD. Presence of animals in the compound (OR = 1.9; p = 0.0151) and giving stored water to the child (OR = 2.0, p = 0.0483) were risk factors for MSD; while animals in the compound (OR = 2.37, p = 0.007); not having routine access to water on a daily basis (OR = 1.53, p = 0.015) and washing hands before cooking (OR = 1.76, p = 0.0197) were risk factors for LSD. CONCLUSION: The implementation of vaccination against rotavirus may likely result in a significant reduction of rotavirus-associated diarrhea, suggesting the need for monitoring of vaccine impact.

Characteristics of Salmonella Recovered From Stools of Children Enrolled in the Global Enteric Multicenter Study.

BACKGROUND: The Global Enteric Multicenter Study (GEMS) determined the etiologic agents of moderate-to-severe diarrhea (MSD) in children under 5 years old in Africa and Asia. Here, we describe the prevalence and antimicrobial susceptibility of nontyphoidal Salmonella (NTS) serovars in GEMS and examine the phylogenetics of Salmonella Typhimurium ST313 isolates. METHODS: Salmonella isolated from children with MSD or diarrhea-free controls were identified by classical clinical microbiology and serotyped using antisera and/or whole-genome sequence data. We evaluated antimicrobial susceptibility using the Kirby-Bauer disk-diffusion method. Salmonella Typhimurium sequence types were determined using multi-locus sequence typing, and whole-genome sequencing was performed to assess the phylogeny of ST313. RESULTS: Of 370 Salmonella-positive individuals, 190 (51.4%) were MSD cases and 180 (48.6%) were diarrhea-free controls. The most frequent Salmonella serovars identified were Salmonella Typhimurium, serogroup O:8 (C2-C3), serogroup O:6,7 (C1), Salmonella Paratyphi B Java, and serogroup O:4 (B). The prevalence of NTS was low but similar across sites, regardless of age, and was similar among both cases and controls except in Kenya, where Salmonella Typhimurium was more commonly associated with cases than controls. Phylogenetic analysis showed that these Salmonella Typhimurium isolates, all ST313, were highly genetically related to isolates from controls. Generally, Salmonella isolates from Asia were resistant to ciprofloxacin and ceftriaxone, but African isolates were susceptible to these antibiotics. CONCLUSIONS: Our data confirm that NTS is prevalent, albeit at low levels, in Africa and South Asia. Our findings provide further evidence that multidrug-resistant Salmonella Typhimurium ST313 can be carried asymptomatically by humans in sub-Saharan Africa.

Dose escalation study of bovine lactoferrin in preterm infants: getting the dose right.

Lactoferrin as a nutritional enteral supplement has emerged as a novel preventative therapy against serious infections in preterm infants, although neonatal studies have demonstrated variable results, in part due to the lack of pharmacokinetic data and differences in the products tested. We conducted a prospective, dose escalation (100, 200, and 300 mg·kg-1·day-1) safety study of bovine lactoferrin (Glanbia Nutritionals, USA) dissolved in sterile water (100 mg·mL-1) for 30 days in preterm infants with birth weight <1500 g. Safety related to adverse events (AEs), tolerability, and exposure-response of lactoferrin was assessed. We enrolled 31 patients [10, 10, and 11 patients, for the lactoferrin treatment groups (100, 200, and 300 mg·kg-1·day-1, respectively)] over a 10-month period. No AEs related to the study solution occurred, and lactoferrin was tolerated by each group. During lactoferrin supplementation, one bloodstream infection occurred in each group, but there were no incidences of urinary tract infections and no cases of necrotizing enterocolitis. Postnatal cytomegalovirus acquisition was detected in the group treated with 200 mg·kg-1·day-1 (n = 2). There were no adverse effects on hepatic, renal, or hematologic function. All of the patients survived to discharge. Bovine lactoferrin at doses up to 300 mg·kg-1·day-1 is safe in preterm infants. Future studies examining higher doses of lactoferrin, length of treatment, and potency of different products will aid in determining the optimal approach for the use of lactoferrin to prevent infections in preterm infants.

The Clinical Presentation of Culture-positive and Culture-negative, Quantitative Polymerase Chain Reaction (qPCR)-Attributable Shigellosis in the Global Enteric Multicenter Study and Derivation of a Shigella Severity Score: Implications for Pediatric Shigella Vaccine Trials.

BACKGROUND: Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials. METHODS: We compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, quantitative polymerase chain reaction (qPCR)-attributable Shigella (defined by an ipaH gene cycle threshold <27.9). Among Shigella MSD cases, we determined risk factors for death and derived a clinical severity score. RESULTS: Compared to culture-positive Shigella MSD cases (n = 745), culture-negative/qPCR-attributable Shigella cases (n = 852) were more likely to be under 12 months, stunted, have a longer duration of diarrhea, and less likely to have high stool frequency or a fever. There was no difference in dehydration, hospitalization, or severe classification from a modified Vesikari score. Twenty-two (1.8%) Shigella MSD cases died within the 14-days after presentation to health facilities, and 59.1% of these deaths were in culture-negative cases. Age <12 months, diarrhea duration prior to presentation, vomiting, stunting, wasting, and hospitalization were associated with mortality. A model-derived score assigned points for dehydration, hospital admission, and longer diarrhea duration but was not significantly better at predicting 14-day mortality than a modified Vesikari score. CONCLUSIONS: A composite severity score consistent with severe disease or dysentery may be a pragmatic clinical endpoint for severe shigellosis in vaccine trials. Reliance on culture for microbiologic confirmation may miss a substantial number of Shigella cases but is currently required to measure serotype specific immunity.

Role of the YehD fimbriae in the virulence-associated properties of enteroaggregative Escherichia coli

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new YehD fimbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.

New Insights Into DAEC and EAEC Pathogenesis and Phylogeny.

Diarrheagenic E. coli can be separated into six distinct pathotypes, with enteroaggregative (EAEC) and diffusely-adherent E. coli (DAEC) among the least characterized. To gain additional insights into these two pathotypes we performed whole genome sequencing of ten DAEC, nine EAEC strains, isolated from Mexican children with diarrhea, and one EAEC plus one commensal E. coli strains isolated from an adult with diarrhea and a healthy child, respectively. These genome sequences were compared to 85 E. coli genomes available in public databases. The EAEC and DAEC strains segregated into multiple different clades; however, six clades were heavily or exclusively comprised of EAEC and DAEC strains, suggesting a phylogenetic relationship between these two pathotypes. EAEC strains harbored the typical virulence factors under control of the activator AggR, but also several toxins, bacteriocins, and other virulence factors. DAEC strains harbored several iron-scavenging systems, toxins, adhesins, and complement resistance or Immune system evasion factors that suggest a pathogenic paradigm for this poorly understood pathotype. Several virulence factors for both EAEC and DAEC were associated with clinical presentations, not only suggesting the importance of these factors, but also potentially indicating opportunities for intervention. Our studies provide new insights into two distinct but related diarrheagenic organisms.

Redefining enteroaggregative Escherichia coli (EAEC): Genomic characterization of epidemiological EAEC strains.

Although enteroaggregative E. coli (EAEC) has been implicated as a common cause of diarrhea in multiple settings, neither its essential genomic nature nor its role as an enteric pathogen are fully understood. The current definition of this pathotype requires demonstration of cellular adherence; a working molecular definition encompasses E. coli which do not harbor the heat-stable or heat-labile toxins of enterotoxigenic E. coli (ETEC) and harbor the genes aaiC, aggR, and/or aatA. In an effort to improve the definition of this pathotype, we report the most definitive characterization of the pan-genome of EAEC to date, applying comparative genomics and functional characterization on a collection of 97 EAEC strains isolated in the course of a multicenter case-control diarrhea study (Global Enteric Multi-Center Study, GEMS). Genomic analysis revealed that the EAEC strains mapped to all phylogenomic groups of E. coli. Circa 70% of strains harbored one of the five described AAF variants; there were no additional AAF variants identified, and strains that lacked an identifiable AAF generally did not have an otherwise complete AggR regulon. An exception was strains that harbored an ETEC colonization factor (CF) CS22, like AAF a member of the chaperone-usher family of adhesins, but not phylogenetically related to the AAF family. Of all genes scored, sepA yielded the strongest association with diarrhea (P = 0.002) followed by the increased serum survival gene, iss (p = 0.026), and the outer membrane protease gene ompT (p = 0.046). Notably, the EAEC genomes harbored several genes characteristically associated with other E. coli pathotypes. Our data suggest that a molecular definition of EAEC could comprise E. coli strains harboring AggR and a complete AAF(I-V) or CS22 gene cluster. Further, it is possible that strains meeting this definition could be both enteric bacteria and urinary/systemic pathogens.

Mucus layer modeling of human colonoids during infection with enteroaggragative E. coli.

EAEC is a common cause of diarrheal illness worldwide. Pathogenesis is believed to occur in the ileum and colon, where the bacteria adhere and form a robust aggregating biofilm. Among the multiple virulence factors produced by EAEC, the Pic serine protease has been implicated in bacterial colonization by virtue of its mucinolytic activity. Hence, a potential role of Pic in mucus barrier disruption during EAEC infection has been long postulated. In this study, we used human colonoids comprising goblet cells and a thick mucin barrier as an intestinal model to investigate Pic's roles during infection with EAEC. We demonstrated the ability of purified Pic, but not a protease defective Pic mutant to degrade MUC2. Western blot and confocal microscopy analysis revealed degradation of the MUC2 layer in colonoids infected with EAEC, but not with its isogenic EAECpic mutant. Wild-type and MUC2-knockdown colonoids infected with EAEC strains exposed a differential biofilm distribution, greater penetration of the mucus layer and increased colonization of the colonic epithelium by Wild-type EAEC than its isogenic Pic mutant. Higher secretion of pro-inflammatory cytokines was seen in colonoids infected with EAEC than EAECpic. Although commensal E. coli expressing Pic degraded MUC2, it did not show improved mucus layer penetration or colonization of the colonic epithelium. Our study demonstrates a role of Pic in MUC2 barrier disruption in the human intestine and shows that colonoids are a reliable system to study the interaction of pathogens with the mucus layer.

Aggregative Adherence Fimbriae II of Enteroaggregative Escherichia coli Are Required for Adherence and Barrier Disruption during Infection of Human Colonoids.

Symptomatic and asymptomatic infection with the diarrheal pathogen enteroaggregative Escherichia coli (EAEC) is associated with growth faltering in children in developing settings. The mechanism of this association is unknown, emphasizing a need for better understanding of the interactions between EAEC and the human gastrointestinal mucosa. In this study, we investigated the role of the aggregative adherence fimbriae II (AAF/II) in EAEC adherence and pathogenesis using human colonoids and duodenal enteroids. We found that a null mutant in aafA, the major subunit of AAF/II, adhered significantly less than wild-type (WT) EAEC strain 042, and adherence was restored in a complemented strain. Immunofluorescence confocal microscopy of differentiated colonoids, which produce an intact mucus layer comprised of the secreted mucin MUC2, revealed bacteria at the epithelial surface and within the MUC2 layer. The WT strain adhered to the epithelial surface, whereas the aafA deletion strain remained within the MUC2 layer, suggesting that the presence or absence of AAF/II determines both the abundance and location of EAEC adherence. In order to determine the consequences of EAEC adherence on epithelial barrier integrity, colonoid monolayers were exposed to EAEC constructs expressing or lacking aafA Colonoids infected with WT EAEC had significantly decreased epithelial resistance, an effect that required AAF/II, suggesting that binding of EAEC to the epithelium is necessary to impair barrier function. In summary, we show that production of AAF/II is critical for adherence and barrier disruption in human colonoids, suggesting a role for this virulence factor in EAEC colonization of the gastrointestinal mucosa.

Escherichia coli ST131 clones harbouring AggR and AAF/V fimbriae causing bacteremia in Mozambican children: Emergence of new variant of fimH27 subclone.

Multidrug-resistant Escherichia coli ST131 fimH30 responsible for extra-intestinal pathogenic (ExPEC) infections is globally distributed. However, the occurrence of a subclone fimH27 of ST131 harboring both ExPEC and enteroaggregative E. coli (EAEC) related genes and belonging to commonly reported O25:H4 and other serotypes causing bacteremia in African children remain unknown. We characterized 325 E. coli isolates causing bacteremia in Mozambican children between 2001 and 2014 by conventional multiplex polymerase chain reaction and whole genome sequencing. Incidence rate of EAEC bacteremia was calculated among cases from the demographic surveillance study area. Approximately 17.5% (57/325) of isolates were EAEC, yielding an incidence rate of 45.3 episodes/105 children-years-at-risk among infants; and 44 of isolates were sequenced. 72.7% (32/44) of sequenced strains contained simultaneously genes associated with ExPEC (iutA, fyuA and traT); 88.6% (39/44) harbored the aggregative adherence fimbriae type V variant (AAF/V). Sequence type ST-131 accounted for 84.1% (37/44), predominantly belonging to serotype O25:H4 (59% of the 37); 95.6% (35/44) harbored fimH27. Approximately 15% (6/41) of the children died, and five of the six yielded ST131 strains (83.3%) mostly (60%; 3/5) due to serotypes other than O25:H4. We report the emergence of a new subclone of ST-131 E. coli strains belonging to O25:H4 and other serotypes harboring both ExPEC and EAEC virulence genes, including agg5A, associated with poor outcome in bacteremic Mozambican children, suggesting the need for prompt recognition for appropriate management.

Dual Function of Aar, a Member of the New AraC Negative Regulator Family, in Escherichia coli Gene Expression.

Enteroaggregative Escherichia coli (EAEC) is an E. coli pathotype associated with diarrhea and growth faltering. EAEC virulence gene expression is controlled by the autoactivated AraC family transcriptional regulator, AggR. AggR activates transcription of a large number of virulence genes, including Aar, which in turn acts as a negative regulator of AggR itself. Aar has also been shown to affect expression of E. coli housekeeping genes, including H-NS, a global regulator that acts at multiple promoters and silences AT-rich genes (such as those in the AggR regulon). Although Aar has been shown to bind both AggR and H-NS in vitro, functional significance of these interactions has not been shown in vivo In order to dissect this regulatory network, we removed the complex interdependence of aggR and aar by placing the genes under the control of titratable promoters. We measured phenotypic and genotypic changes on downstream genes in EAEC strain 042 and E. coli K-12 strain DH5α, which lacks the AggR regulon. In EAEC, we found that low expression of aar increases aafA fimbrial gene expression via H-NS; however, when aar is more highly expressed, it acts as a negative regulator via AggR. In DH5α, aar affected expression of E. coli genes in some cases via H-NS and in some cases independent of H-NS. Our data support the model that Aar interacts in concert with AggR, H-NS, and possibly other regulators and that these interactions are likely to be functionally significant in vivo.

Gram-negative Microbiota Blooms in Premature Twins Discordant for Parenteral Nutrition-associated Cholestasis.

Parenteral nutrition-associated cholestasis (PNAC) causes serious morbidity in the neonatal intensive care unit. Infection with gut-associated bacteria is associated with cholestasis, but the role of intestinal microbiota in PNAC is poorly understood. We examined the composition of stool microbiota from premature twins discordant for PNAC as a strategy to reduce confounding from variables associated with both microbiota and cholestasis. Eighty-four serial stool samples were included from 4 twin sets discordant for PNAC. Random Forests was utilized to determine genera most discriminatory in classifying samples from infants with and without PNAC. In infants with PNAC, we detected a significant increase in the relative abundance of Klebsiella, Veillonella, Enterobacter, and Enterococcus (P < 0.05). Bray-Curtis dissimilarities in infants with PNAC were significantly different (P < 0.05) from infants without PNAC. Our findings warrant further exploration in larger cohorts and experimental models of PNAC to determine if a microbiota signature predicts PNAC, as a basis for future interventions to mitigate liver injury.

Enteropathogenic Escherichia coli Infection Induces Diarrhea, Intestinal Damage, Metabolic Alterations, and Increased Intestinal Permeability in a Murine Model.

Enteropathogenic E. coli (EPEC) are recognized as one of the leading bacterial causes of infantile diarrhea worldwide. Weaned C57BL/6 mice pretreated with antibiotics were challenged orally with wild-type EPEC or escN mutant (lacking type 3 secretion system) to determine colonization, inflammatory responses and clinical outcomes during infection. Antibiotic disruption of intestinal microbiota enabled efficient colonization by wild-type EPEC resulting in growth impairment and diarrhea. Increase in inflammatory biomarkers, chemokines, cellular recruitment and pro-inflammatory cytokines were observed in intestinal tissues. Metabolomic changes were also observed in EPEC infected mice with changes in tricarboxylic acid (TCA) cycle intermediates, increased creatine excretion and shifts in gut microbial metabolite levels. In addition, by 7 days after infection, although weights were recovering, EPEC-infected mice had increased intestinal permeability and decreased colonic claudin-1 levels. The escN mutant colonized the mice with no weight loss or increased inflammatory biomarkers, showing the importance of the T3SS in EPEC virulence in this model. In conclusion, a murine infection model treated with antibiotics has been developed to mimic clinical outcomes seen in children with EPEC infection and to examine potential roles of selected virulence traits. This model can help in further understanding mechanisms involved in the pathogenesis of EPEC infections and potential outcomes and thus assist in the development of potential preventive or therapeutic interventions.