Enteroaggregative Escherichia Coli Research Articles

Travelers\u2019 diarrhea: update on the incidence, etiology and risk in military and similar populations \u2013 1990-2005 versus 2005\u20132015, does a decade make a difference?

BackgroundTravelers’ 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.MethodsWe 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.ResultsMilitary 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.ConclusionsIn 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.

Etiological agents of diarrhea in hospitalized pediatric patients with special emphasis on diarrheagenic Escherichia coli in North India.

INTRODUCTION:Infectious diarrhea is leading infectious cause of childhood morbidity, hospitalizations, and mortality particularly in children living in developing countries like India. The etiological agents differ depending on geographical area, and recent data suggest increase in drug resistance to various enteropathogens.AIMS AND OBJECTIVES:The aim of the study was to investigate emerging diarrheal agents and antimicrobial resistance profile of bacterial pathogens from children (<12 years of age) hospitalized with acute diarrhea.MATERIALS AND METHODS:A cross-sectional, hospital-based observational study was conducted over 1 year in which 100 children <12 years who were hospitalized due to diarrhea were recruited. Diarrhea was defined as the passage of three or more liquid stools in a 24-h period using the World Health Organization guidelines. Samples were processed for detection of various bacterial, viral, and parasitic agents by standard microbiological, serological, and molecular tests. Antimicrobial resistance testing was performed with the Kirby–Bauer disk diffusion method. ELISA was performed for Rotavirus and Escherichia coli O157. Multiplex polymerase chain reaction test was performed to detect diarrheagenic E. coli (DEC).RESULTS:Pathogenic diarrheal agents were found in 63% patients. Rotavirus was identified in 52.5%, DEC in 29%, Vibrio cholerae in 4%, Shigella flexneri in 3%, Aeromonas sp. in 1%, Giardia lamblia in 4%, and Entamoeba histolytica in 1% cases. Enteropathogenic E. coli (EPEC) in 19 (65.5%) cases was the most common agent followed by Enteroaggregative E. coli (EAEC) in 5 (17.2%), Enterotoxigenic E. coli (ETEC) in 2 (6%), and Enteroinvasive E. coli (EIEC) in 3 (10.3%) cases. Resistance rates of DEC to first-line therapeutic drugs were high, 97.3% to ampicillin and 95.95% to co-trimoxazole. DEC was susceptible to chloramphenicol in 58.11%, gentamicin in 48.19%, and amikacin in 58.11% cases. Shigella sp. and V. cholerae isolates were 100% sensitive to gentamicin and ofloxacin.CONCLUSION:EPEC is the most common DEC pathotype and EAEC, ETEC, and EIEC are also emerging as dominant diarrheal agents. Rotavirus was the most common causative agents of diarrhea especially in children <5 years. Most of the bacterial isolates showed high level of drug resistance to first-line empirical drugs and were multidrug resistant making them unsuitable for empiric treatment. Laboratory monitoring of drug susceptibility of stool isolates appears necessary to formulate antibiotic policy for treating diarrheal illness at the local level. There is an urgent need to strengthen diarrheal surveillance to monitor susceptibility to commonly prescribed antibiotics.

Molecular Pathotyping of Escherichia Coli Isolates and Detection of Residual Antibiotics in Raw Cow Milk in Iran

Escherichia coli is an important species within the Enterobacteriaceae including at least six pathogenic classes of enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC). Therefore, the aims of the current study were molecular characterization of E. coli pathotypes in raw cow milk samples in Borujerd, Iran, and detection of antibiotic residues in those samples due to the importance for the public health. Totally, 150 raw cow milk samples were collected from Borujerd, Iran, 2015. Eleven genes of bfpA, eaeA, east1, flicH7, hlyA, ial, lt, st, stx1, stx2 and O157 were detected in isolates using PCR to molecularly typing the E. coli isolates. Of 150 samples, 31 E. coli strains (totally 20.6%) were isolated, including 16 ETEC (51.6%), nine EPEC (29%) and six EHEC (19.4%). No EIEC (0%) or EAEC (0%) was isolated. In PCR, 13 eaeA (29.5%), eight lt (18.18%), eight st (18.18%), four stx1 (9.09%), three stx2 (6.81%), three hlyA (6.81%), two bfpA (4.54%), two O157 (4.54%) and one flicH7 (2.27%) encoding genes were found in E. coli isolates. Furthermore, 30 samples (20%) included antibiotic residues. In General, pathotyping of the bacterial isolates can better reveal possible correlations between these pathotypes and foodborne outbreaks.

DIARRHEAGENIC Escherichia coli IN RAW MILK, WATER, AND CATTLE FECES IN NON-TECHNIFIED DAIRY FARMS

Abstract This study focused on detecting diarrheagenic Escherichia coli, enteropathogenic E. coli (EPEC), Shiga-toxin-producing E. coli (STEC), enterohemorrhagic E. coli (EHEC or STEC:EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC) in raw milk, water, and cattle feces sampled from non-technified dairy farms located in the northeastern São Paulo State, Brazil. Thirty-six water samples were collected at different points, namely, water wells (8 samples), water intended for human consumption (8 samples), water from milking parlor (8 samples), and water intended for animal consumption (7 samples), headwaters (1 sample), rivers (3 samples), and reservoirs (1 sample). Three raw milk samples were taken directly from bulk tanks in each farm, totalizing 24 samples. Feces samples were collected using rectal swabs from 160 bovines (20 animals per farm). E. coli was detected in 128 feces samples (80%), 16 raw milk samples (66.67%), and 20 water samples (55.56%). STEC (26 samples, 16.25%), EPEC (10 samples, 6.25%), STEC: EPEC (5 samples, 3.13%), and STEC: ETEC (1 sample, 0.63%) were the most prevalent strains detected in samples from cattle feces. EPEC, STEC, and STEC: EPEC strains were detected in 4.17% (1 sample), 16.67% (4 samples), and 4.17% (1 sample) of raw milk samples, respectively. STEC strains were detected in water used in the milking parlor, while no EAEC strain was detected. As a conclusion, cattle feces are important contamination sources of pathogenic E. coli in non-technified dairy farms and, consequently, cross-contamination among feces, water, and/or raw milk can occur. The use of quality water and hygienic practices during milking are recommended to avoid contamination since pathogens can be transmitted to humans via raw milk or raw milk cheese ingestion.

Occurrence of putative virulence-related genes, aatA, aggR and aaiC, of Enteroaggregative Escherichia coli (EAEC) among adults with travelers’ diarrhea acquired in Guatemala and Mexico

Enteroaggregative E. coli strains are important causes of diarrhea worldwide and are the second most important bacterial cause of travelers' diarrhea (TD). Pathogenicity of EAEC is not completely understood. We investigated the occurrence of putative virulence related genes (VRG), aatA, aggR and aaiC, in a nested case-control study of a cohort of US travelers >18 years of age, visited either Guatemala or Mexico. Fecal samples were collected between 2008 and 2012 from patients with TD from whom a HEp-2 cell adherent EAEC strain was identified (Cases) and from healthy subjects in the same locale without diarrhea from whom enteric pathogens were not isolated (Controls). Thirty-one subjects with acquired TD at destination was compared with 32 healthy controls. aaiC was the most expressed virulence related gene in 21 (67.7%) cases vs. 2 (6.3%) controls, (P < 0.000). aggR was found in 18 (58.1%) cases vs. 1 (3.1%) control, (P < 0.000). aatA in 9 (29.0%) cases vs. 1 (3.1%) control (P < 0.006). With genes combined, aaiC+aggR were seen together in 18 (58.1%) cases vs. 1 (3.1%) control (P < 0.000); aaiC+aatA were identified in 9 (29.0%) cases vs. 1 (3.1%) control (P < 0.006); aggR+aatA were present in 9 (29.0%) cases vs. 1 (3.1%) control, (P < 0.006). All three putative genes, aaiC+aggR+aatA were found in 9 (29.0%) cases vs. 1 (3.1%) control, (P < 0.006). The PCR products showed that aaiC, aggR, and aatA occurred in higher frequency and were more commonly associated with EAEC in cases of TD acquired in the two countries of study, as compared to controls. aaiC was found in all cases from Guatemala. Further research is needed to study geographic and host factors in EAEC-causing travelers’ diarrhea.

Evaluation of virulence factors profiles and antimicrobials resistance of Escherichia coli isolated from bulk tank milk and raw milk filters

Data on the presence of pathogenic Escherichia coli in bulk tank milk (BTM) and raw milk filters (RMF) are not available in Italy and there are few studies worldwide. Therefore, a study under field condition was conducted to assess the presence of E.coli pathogenic and commensal (CoEC) strains in BTM and RMF samples and their associated AMR pattern.One hundred forty-nine E.coli isolates were characterized. Among all the isolates, 53 (35.6%) were classified as pathogenic while the other ones were classified as CoEC. Among the pathogenic ones, 23 (54.7%) were classified as enterotoxigenic E.coli (ETEC), 6 (11.3%) as enteroinvasive E.coli (EIEC), 2 (3.8%) as enteroaggregative E.coli (EAEC), 12 (22.6%) harboured virulence factors (VF) common to ETEC+EIEC, and 2 (3.8%) common to ETEC+EAEC. To our knowledge, it is the first time that ETEC isolates harboring VF associated with EAEC or EIEC are observed in raw milk. These data support the presence of transmission of VFs genes among isolates.None of the isolates showed resistance to three or more antimicrobials. The CoEC role as a vector of AMR was confirmed by the presence of 18% ampicillin- and cephalexin-resistant isolates. The presence of AMR in CoEC supports the role of these bacteria as source of resistance genes.Monitoring raw milk by either BTM or RMF analysis, and the relatively cheap procedure applied to identify E.coli pathotypes can be useful to identify hazards related to the spread of enteric diseases and antimicrobial resistance.

Enteric pathogen co-infections in the paediatric population from rural communities in the Vhembe District, South Africa

Background. Enteric pathogens co-infections are a serious health risk in children under the age of 5 years. Objective. The study aimed to determine the prevalence of diarrhoea-causing pathogens in children suffering from diarrhoea in rural communities of the Vhembe District. Methods. A cross-sectional study was conducted from July 2014 to June 2015. Diarrhoeal stool specimens ( N =237) were collected from children attending primary healthcare facilities in rural communities of the Vhembe District. Stools were screened for enteric viral adenovirus 40/41, rotavirus and norovirus pathogens by means of enzyme immuno-assay (EIA) and enteric bacterial Escherichia coli spp. (diarrhoeagenic pathotypes), Shigella spp., Salmonella spp. and Vibrio spp. pathogens by means of multiplex polymerase chain reaction. Results. A total of 59.1% (140/237) were positive for at least one or more enteric pathogens. Enterotoxigenic E. coli (ETEC) (27.9%), enteroaggregative E. coli (EAEC) (26.8%) and atypical enteropathogenic E. coli (EPEC) (17.9%) were frequently detected in children less than 2 years of age. Bacterial-bacteria co-infections were detected in 24.5% ( n =58) and bacterial-viral co-infections in 14.3% ( n =34) of the stool specimens. Conclusion. The findings indicated that enteric pathogen co-infections are major causes of diarrhoea in children less than 2 years of age in the Vhembe District.

Distribution of the pilS gene in Escherichia coli pathovars, its transfer ability and influence in the typical enteropathogenic E. coli adherence phenotype

Typical enteropathogenic Escherichia coli strains (tEPEC) cause attaching/effacing lesions in eukaryotic cells and produce the bundle-forming pilus (BFP), which interweaves and aggregates bacteria, resulting in the localized adherence (LA) pattern on eukaryotic cells. Previously, we identified tEPEC strains (serotype O119:H6) that exhibited LA simultaneously with an aggregative adherence (AA)-like pattern (LA/AA-like+). Remarkably, AA is characteristically produced by strains of enteroaggregative E. coli (EAEC), another diarrheagenic E. coli pathovar. In one LA/AA-like + strain (Ec404/03), we identified a conjugative plasmid containing the pil operon, which encodes the Pil fimbriae. Moreover, a pil operon associated with an AA pattern and plasmid transfer had been previously described in the EAEC C1096 strain. In this study, we investigated the occurrence of the two pilS alleles (pilSEc404 and pilSC1096) in tEPEC strains of different serotypes, origins and years of isolation. We also examined the potential relationship of pilS with the AA-like phenotype, its ability to be transferred by conjugation, and occurrence among strains of the other E. coli pathovars. The pilS alleles were found in 90 (55.2%) of 163 tEPEC strains, with pilSEc404 occurring more often (30.7%) than pilSC1096 (25.1%). About 21 tEPEC serotypes carried pilS. The pilS alleles were found in tEPEC strains from Chile, Peru and different Brazilian cities, with the oldest strain being isolated in 1966. No absolute correlation was found between the presence of pilS and the AA-like pattern. Conjugative pilS transfer was detected in 26.2% of pilSEc404+ strains and in 65.1% of pilSC1096+ strains, but only pilSEc404+ transconjugants were AA-like+, thus suggesting that the latter allele might need a different genetic background to express this phenotype. pilS was found in all other E. coli pathovars, where it was most prevalent in enterotoxigenic E. coli. More studies are needed to understand the mechanisms involved in the regulation of Pil expression and production.

CHARACTERIZATION OF ENTEROAGGREGATIVE ESCHERICHIA COLI AMONG DIARRHEAL CHILDRENIN WESTERN BRAZILIAN AMAZON.

Enteroaggregative Escherichia coli (EAEC) is one of the main acute and chronic diarrhea causes both in children and adults, mainly in developing countries. The aim of the present study is to characterize EAEC strains isolated from faecal samples and to identify genes potentially contributing to virulence, biofilm production and antimicrobial resistance in children admitted to a pediatric hospital in Porto Velho, Rondônia State. The total of 1,625 E. coli specimens were isolated from 591 children in the age group 6 years or younger who were hospitalized in Cosme and Damião Children Hospital in Porto Velho, between February 2010 and February 2012, with acute gastroenteritis. Colonies suggestive of E. coli were subjected to polymerase chain reaction testing in order to identify the virulence factors. The in vitro adhesion assays using HEp-2 adherence were tests. Biofilm detection through spectrophotometry and antimicrobial susceptibility tests were conducted in the disk diffusion method. The mentioned study examined 591 stool samples from children with diarrhea. Diarrheogenic E. coli was found in 27.4% (162/591) of the children. EAEC was the diarreagenic E. coli most frequently associated with diarrhea 52.4% (85/162), which was followed by enteropathogenic E. coli 43.8% (71/162), enterotoxigenic E. coli 2.4% (4/162), and enterohemorrhagic E. coli 1.2% (2/162). The aggR gene was detected in 63.5% (54/85) of EAEC isolates; moreover, statistically significant correlation was observed among typical EAEC (aggR) and aatA (P<0.0001), irp2 (P=0.0357) and shf (P=0.0328). It was recorded that 69% (59/85) of the 85 analyzed EAEC strains were biofilm producers; 73% (43/59) of the biofilm producers carried the aggR gene versus 42.3% (11/26) of non-producers (P=0.0135). In addition, there was association between the aatA gene and biofilm production; 61% (36/59) of the samples presented producer strains, versus 19.2% (5/26) of non-producers (P<0.0004). Antibiotic sensitivity test evidenced that most EAEC were ampicillin 70.6% (60/85), sulfamethoxazole 60% (51/85), tetracycline 44.7% (38/85) and cefotaxime 22.4% (19/85) resistant. As far as it is known, the present study is pioneer in Northern Brazil to investigate EAEC virulence factors and to show the antimicrobial susceptibility of EAEC strains isolated from children with diarrhea.

1108. Diagnostic Yield of the BioFire FilmArray Gastrointestinal Panel in Hospitalized Children at an Academic Children’s Center

BackgroundThe BioFire FilmArray Gastrointestinal Panel (BioFire Diagnostics) (GIP) is a multiplex stool PCR test that detects 22 organisms. Studies in adults suggest that the diagnostic yield of the GIP in hospitalized patients is low. The utility of the GIP among hospitalized pediatric patients and indications for diagnostic stewardship of this test are not well described.MethodsWe conducted a retrospective chart review of hospitalized pediatric patients who had a GIP ordered between October 2015 and October 2017. Demographic, clinical, and laboratory information was extracted from the medical record. Statistical analysis was completed using JMP Pro 13.0.0 (SAS Institute Inc.).ResultsOver the 2-year study period, 193 GIPs were obtained on 155 individual pediatric patients. The mean patient age was 8 years and 59% were male. Forty-four percent of patients were immunocompromised and 21% had inflammatory bowel disease. The pediatric infectious disease (PID) team was consulted in 15% of patients at the time the test was ordered. The overall positivity rate of the GIP for one or more pathogens was 42% (Figure 1), with 76% of GIPs positive for one, 23% for two, and 1% for three pathogens. No parasitic infections were diagnosed. The GIP was more likely to be positive if GI symptom onset was prior to admission (48% vs. 24%, P = 0.004), if GI symptoms had been present for < 2 weeks vs. ≥2 weeks (52% vs. 20%, P = 0.0001), and if GI symptoms were the primary reason for the hospital admission (50% vs. 32%, P = 0.012). Only Clostridioides difficile or viral pathogens were detected in patients whose symptoms began in the hospital (Figure 2). Among patients with a positive test, 40% received treatment targeted at one or more of the detected pathogens (Figure 1). Enteropathogenic E.coli (EPEC) and Enteroaggregative E.coli (EAEC) were never treated (Figure 3).ConclusionThe GIP was positive for one or more pathogens in 42% of hospitalized children for whom the test was ordered, and led to specific therapy in 40% of those with a positive test. EPEC and EAEC were not treated. The diagnostic yield of the GIP was higher if GI symptoms were present for <2 weeks, began before hospitalization, and were the primary reason for admission. The GIP was frequently obtained without guidance from the PID team. Disclosures R. Patel, CD Diagnostics, BioFire, Curetis, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, and The Medicines Company: Grant Investigator, Research grant – monies paid to Mayo Clinic. Curetis, Specific Technologies, Selux Dx, GenMark Diagnostics, PathoQuest and Genentech: Consultant and Scientific Advisor, Consulting fee – monies paid to Mayo Clinic. ASM and IDSA: Travel reimbursement and editor’s stipends, Travel reimbursement and editor’s stipends. NBME, Up-to-Date and the Infectious Diseases Board Review Course: Varies, Honoraria. Mayo Clinic: Employee, Salary.

Evaluation of PCR-based methods for the identification of enteroaggregative hemorrhagic Escherichia coli in sprouts

In this study real-time PCR assays were evaluated for the detection of enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104:H4 in artificially contaminated mung bean and/alfalfa sprouts inoculated with 1, 10, and 100 CFU of EAHEC O104:H4 per 25 g sample (20, 10, and 2 replicates respectively). After selective culture enrichment the samples were tested using commercial real-time PCR kits detecting aggR/aaiC, stx/eae, and wzxO104. Using the commercial real-time PCR kits, the artificially contaminated samples were detected in the range of 75–80% positive results when contaminated with approximately 1 CFU, and 100% at 10 and 100 CFU. Microbiological detection employing O104-specific immunomagnetic capture and plating onto chromogenic media (modified Rainbow Agar and CHROMagar STEC) and confirmation by latex agglutination and PCR gave similar results (Cohen's kappa value between 0.61 and 1). In addition, the real-time PCR assay targeting the aggR and aaiC genes, indicative of enteroaggregative Escherichia coli (EAggEC), was tested against a panel of 60 bacterial strains and demonstrated 100% exclusivity (54 strains) and 100% inclusivity (6 strains). This study demonstrates the efficacy of the real-time PCR assays for the specific and sensitive detection of EAHEC from spouts.

Biogenesis and structure of a type VI secretion baseplate.

To support their growth in a competitive environment and cause pathogenesis, bacteria have evolved a broad repertoire of macromolecular machineries to deliver specific effectors and toxins. Among these multiprotein complexes, the type VI secretion system (T6SS) is a contractile nanomachine that targets both prokaryotic and eukaryotic cells. The T6SS comprises two functional subcomplexes: a bacteriophage-related tail structure anchored to the cell envelope by a membrane complex. As in other contractile injection systems, the tail is composed of an inner tube wrapped by a sheath and built on the baseplate. In the T6SS, the baseplate is not only the tail assembly platform, but also docks the tail to the membrane complex and hence serves as an evolutionary adaptor. Here we define the biogenesis pathway and report the cryo-electron microscopy (cryo-EM) structure of the wedge protein complex of the T6SS from enteroaggregative Escherichia coli (EAEC). Using an integrative approach, we unveil the molecular architecture of the whole T6SS baseplate and its interaction with the tail sheath, offering detailed insights into its biogenesis and function. We discuss architectural and mechanistic similarities but also reveal key differences with the T4 phage and Mu phage baseplates.

Enteroaggregative Escherichia coli subclinical and clinical infections.

The current review is to update the results on epidemiology, pathobiology, and genes related to virulence, clinical presentation, molecular diagnosis, antimicrobial resistance, and extraintestinal infection of enteroaggregative Escherichia coli (EAEC). EAEC subclinical infection was significantly associated with reduced length at 2 years of age and EAEC and coinfections were associated with reduced delta weight-for-length and weight-for-age z-scores in the first 6 months of age in the MAL-ED birth cohort study. EAEC was associated with malnutrition in children 6-24 months of age in prospective case-control studies in Bangladesh and Brazil. Virulence gene-based studies have suggested aggregative fimbriae II may be a major contributor to disease, whereas AggR-activated regulator a marker of less severe disease. The high ability of EAEC colonization likely exacerbates effects of other microbial virulence strategies. Molecular diagnosis has been useful for understanding EAEC burden, although different criteria may relate to different pathogenic outcomes. EAEC gained special interest in the past few years, especially due to association with growth decrements in children with subclinical infections and its important role as a copathogen. Understanding of EAEC pathogenesis advanced but further research is needed for elucidating both microbial and host factors influencing infection outcomes.

Real-Time Multiplex Polymerase Chain Reaction with High-Resolution Melting-Curve Analysis for the Diagnosis of Enteric Infections Associated with Diarrheagenic Escherichia coli

Introduction: Although diarrheagenic Escherichia coli (DEC) strains are important bacterial causative agents of diarrhoea, they are not routinely sought as stool pathogens in clinical laboratories as conventional microbiological testing are unable to distinguish between normal flora and pathogenic strains of E. coli. This study was undertaken to determine the prevalence of DEC pathotypes amongst children with and without diarrhoea and to detect specific virulent genes present in different DEC pathotypes, using real-time multiplex polymerase chain reaction (PCR) with high-resolution melting (HRM) technology. Materials and Methods: Stool samples were obtained from cases and controls. Using a set of conventional biochemical tests, E. coli strains were identified. Further, these isolates were subjected to multiplex PCR system for the detection of virulence genes of different pathotypes of DEC. Real-time multiplex PCR was performed for the detection of specific virulent genes of DEC pathotypes, using Rotor-Gene Q instrument (Qiagen) having High-resolution Melt analyser using Type-it HRM PCR kit (Qiagen) containing EvaGreen fluorescent intercalating dye. Results: In this study, we had successfully standardised two multiplex PCR assays which were found to be effective for direct detection of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC) and enteroinvasive E. coli (EIEC). A total of 42 DEC strains were detected at an overall rate of 19.3% (n = 42), from the total 217 E. coli isolates recovered from the cases (n = 39, 17.9%) and control (n = 3, 3.8%) groups. Amongst the 42 DEC pathotypes (39 from cases and 3 from controls), EPEC (10%), EAEC (8.82%), ETEC (2.94%) and EIEC (1.18%) were found in children with diarrhoea (cases) and in children without diarrhoea (control) only EAEC (2.13%) and EPEC (4.26%) were detected. Age distribution, gender variation, seasonal variation and clinical features were also analysed Conclusion: This study helped evaluate the prevalence of DEC amongst children (<18 years of age) with and without diarrhoea using multiplex real-time PCR with HRM analysis.

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.

Diarrhoeagenic Escherichia coli and Escherichia albertii in Brazil: pathotypes and serotypes over a 6-year period of surveillance.

Diarrhoeagenic Escherichia coli (DEC) is a leading cause of infectious diarrhoea worldwide. In recent years, Escherichia albertii has also been implicated as a cause of human enteric diseases. This study describes the occurrence of E. coli pathotypes and serotypes associated with enteric illness and haemolytic uremic syndrome (HUS) isolated in Brazil from 2011 to 2016. Pathotypes isolated included enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC). PCR of stool enrichments for DEC pathotypes was employed, and E. albertii was also sought. O:H serotyping was performed on all DEC isolates. A total of 683 DEC and 10 E. albertii strains were isolated from 5047 clinical samples. The frequencies of DEC pathotypes were 52.6% (359/683) for EPEC, 32.5% for EAEC, 6.3% for ETEC, 4.4% for EIEC and 4.2% for STEC. DEC strains occurred in patients from 3 months to 96 years old, but EPEC, EAEC and STEC were most prevalent among children. Both typical and atypical isolates of EPEC and EAEC were recovered and presented great serotype heterogeneity. HUS cases were only associated with STEC serotype O157:H7. Two E. albertii isolates belonged to serogroup O113 and one had the stx2f gene. The higher prevalence of atypical EPEC in relation to EAEC in community-acquired diarrhoea in Brazil suggests a shift in the trend of DEC pathotypes circulation as previously EAEC predominated. This is the first report of E. albertii isolation from active surveillance. These results highlight the need of continuing DEC and E. albertii surveillance, as a mean to detect changes in the pattern of pathotypes and serotypes circulation and provide useful information for intervention and control strategies.

Association of intestinal pathogens with faecal markers of environmental enteric dysfunction among slum-dwelling children in the first 2 years of life in Bangladesh.

ObjectiveEnvironmental Enteric Dysfunction (EED) can be assessed by faecal biomarkers such as Myeloperoxidase (MPO), Neopterin (NEO) and Alpha‐1 anti‐trypsin (AAT). We aimed to test the association of intestinal pathogens with faecal markers of EED among slum‐dwelling children in first 2 years of life.MethodsThe MAL‐ED birth cohort data of Bangladesh site were used to conduct this analysis. Multivariable analyses using Generalized Estimating Equations (GEE) were performed to test the association between intestinal pathogens and faecal markers of EED.ResultsGiardiasis, ascariasis and trichuriasis were the most frequent parasitic infections and Campylobacter spp., Enteroaggregative Escherichia coli (EAEC) and Enterotoxigenic Escherichia coli (ETEC) were the common bacterial pathogens observed in stool samples of the children. Overall, 71%, 97% and 58% of stool samples were above values considered normal in non‐tropical settings for MPO, NEO and AAT respectively. Giardiasis was found to be significantly associated with MPO (Coefficient = 0.55; 95% CI = 0.15, 0.95; P‐value = 0.008) and AAT concentrations (Coefficient = 0.34; 95% CI = 0.04, 0.63; P‐value = 0.03). A significant association was found between trichuriasis and NEO (Coefficient = 0.90; 95% CI = 0.19, 1.61; P‐value = 0.01). Trichuriasis (Coefficient = 1.71; 95% CI = 0.32, 3.11; P‐value = 0.02) and giardiasis (Coefficient = 1.51; 95% CI = 0.79, 2.23; P‐value <0.001) were significantly associated with EED score. Children with EAEC had significantly higher MPO concentrations (Coefficient = 0.33; 95% CI = 0.06, 0.61; P‐value = 0.02).ConclusionThe study results imply the importance of intestinal pathogens in contributing to intestinal inflammation and increased intestinal permeability in young children.

Characterization of diarrheagenic Escherichia coli strains associated with diarrhea in children, Khouzestan, Iran.

Diarrheagenic Escherichia coli (DEC) is a major etiologic agent among the pathogens that cause diarrhea in children. To investigate the presence and pathotypes of DEC in children under five years of age, living in the province of Khouzestan, Iran. 208 diarrhea stool samples were screened by multiplex-PCR. The isolated DEC isolates were investigated for resistance to various antimicrobials including the production of extended-spectrum beta-lactamases (ESBLs) and phylogenetic groups were determined. DEC isolates were identified in 54 (26%) diarrhea samples, and 4 (7%) cases contained two DEC pathotypes. DEC isolated included 35 (16.8%) enteroaggregative E. coli (EAEC), ten (4.8%) enteropathogenic E. coli (EPEC), six (2.9%) enteroinvasive E. coli (EIEC), six (2.9%) enterotoxigenic E. coli (ETEC) and one (0.48%) LEE-positive EAEC. Shiga-toxin producing E. coli (STEC) was not identified in any diarrheal samples. The most prevalent resistance was observed with ceftazidime (88%), followed by ceftizoxime (83%) and ceftriaxone (71%). The majority of isolates (> 75%) were sensitive to Imipenem, ciprofloxacin, and amikacin. More than 65% of the pathogenic isolates showed a multidrug-resistant phenotype. ESBL-producing strains was observed in 79.3% of all DEC isolates. Phylogenetic group B2 was the most predominant group with a frequency of 44.8%. A significant association was observed between the B2 phylogenetic group and the DEC isolates (P < 0.05). Overall, our findings highlight the importance of the role of DEC isolates in the etiology of diarrhea in children in Iran. The progressive increase in antimicrobial resistance among DEC isolates makes it imperative to implement policies to control the spread of resistant bacteria.

Virulence gene profiles and molecular genetic characteristics of diarrheagenic Escherichia coli from a hospital in western China

BackgroundDiarrheagenic Escherichia coli (DEC) is one of the most important etiological agents of diarrheal diseases. In this study we investigated the prevalence, virulence gene profiles, antimicrobial resistance, and molecular genetic characteristics of DEC at a hospital in western China.MethodsA total of 110 Escherichia coli clinical isolates were collected from the First Affiliated Hospital of Chengdu Medical College from 2015 to 2016. Microbiological methods, PCR, antimicrobial susceptibility test, pulsed-field gel electrophoresis and multilocus sequence typing were used in this study.ResultsMolecular analysis of six DEC pathotype marker genes showed that 13 of the 110 E. coli isolates (11.82%) were DEC including nine (8.18%) diffusely adherent Escherichia coli (DAEC) and four (3.64%) enteroaggregative Escherichia coli (EAEC). The adherence genes fimC and fimH were present in all DAEC and EAEC isolates. All nine DAEC isolates harbored the virulence genes fyuA and irp2 and four (44.44%) also carried the hlyA and sat genes. The virulence genes fyuA, irp2, cnf1, hlyA, and sat were found in 100%, 100%, 75%, 50%, and 50% of EAEC isolates, respectively. In addition, all DEC isolates were multidrug resistant and had high frequencies of antimicrobial resistance. Molecular genetic characterization showed that the 13 DEC isolates were divided into 11 pulsed-field gel electrophoresis patterns and 10 sequence types.ConclusionsTo the best of our knowledge, this study provides the first report of DEC, including DAEC and EAEC, in western China. Our analyses identified the virulence genes present in E. coli from a hospital indicating their role in the isolated DEC strains’ pathogenesis. At the same time, the analyses revealed, the antimicrobial resistance pattern of the DEC isolates. Thus, DAEC and EAEC among the DEC strains should be considered a significant risk to humans in western China due to their evolved pathogenicity and antimicrobial resistance pattern.

Prevalence and potential virulence of Escherichia coli in ready-to-eat raw mixed vegetable salads in collective catering in Abidjan, Côte d’Ivoire

Purpose Vegetable salads, despite their recognized health benefits, are an increasingly common cause of foodborne illness worldwide. The purpose of this paper is to determine the prevalence of E. coli with virulence genes in ready-to-eat raw mixed vegetable salads sold in collective catering in Abidjan. Design/methodology/approach A total of 436 strains of E. coli were isolated from 306 ready-to-eat raw mixed vegetables salads and then identified biochemically and molecularly based on the uidA gene responsible for beta-glucuronidase activity. The virulence genes were determined by polymerase chain reaction. Findings The prevalence in vegetable salads of E. coli with virulence genes was 35.3 percent. The distribution of pathovars was 21.2 percent enterotoxigenic (ETEC), 4.9 percent enteropathogenic (EPEC), 0.7 percent Shigatoxigenic (STEC), and 7.5 percent Enteroaggregative E. coli (EAEC). It appears from the study that vegetable salads sold in collective catering in Abidjan are at risk for contamination by E. coli pathovars. Originality/value Processing conditions for these salads during preparation appear to be hygienically insufficient, so measures to control the risk of contamination are necessary.