First detection of diarrheagenic Escherichia coli pathotypes in the Mearim River Watershed, Maranhão State, Brazil

The Mearim River Watershed has multiple uses e.g. leisure, navigation, fishing and subsistence agriculture and constitutes the main source of supply for the populations of municipalities situated along its course. In addition to being a water supply source, the existence of the ‘pororoca’ (tidal bore) effect in a stretch of the lower course of the Mearim River attracts people from several Brazilian states and different countries, as it offers excellent conditions for surfing in fresh water. In this respect, given the importance of the watershed, this study was developed to report the detection of diarrheagenic Escherichia coli pathotypes in a stretch of the lower course of the Mearim River, located in the state of Maranhão, Brazil. Thirty water samples were collected from 10 sampling points. To quantify E. coli, the chromogenic enzymatic system was used and positive samples were isolated and biochemically identified. Pure cultures underwent DNA extraction by heating followed by polymerase chain reaction (PCR) characterization. At the time of the collections, an observation schedule was used to record information on the existence of rearing of livestock and domestic animals; businesses; residences; and fruit and vegetable farming on the riverbanks. The samples were analyzed for the mean populations of E. coli, which ranged from 444 to 2,585 MPN mL-1. Twenty bacterial isolates were identified and the diarrheal pathotypes ETEC, typical EPEC and atypical EPEC were detected. The detection of these pathotypes can represent an epidemiological risk and compromise several uses of this water resource, such as irrigation of fruits and vegetables consumed raw, fishing, animal watering and recreation. Structural investments in basic sanitation are essential to minimize environmental degradation resulting from anthropic activities and to act preventively in public health. In addition, the recovery of riparian forests along the watershed and the maintenance of vegetation in these areas are measures to reduce the transport of particles from the soil to the watercourses, improving the qualitative and quantitative characteristics of this water resource.

Enteropathogenic Escherichia coli—A Summary of the Literature

Diarrheal disease is still a major public health concern, as it is still considered an important cause of death in children under five years of age. A few decades ago, the detection of enteropathogenic E. coli was made by detecting the O, H, and K antigens, mostly by agglutination. The recent protocols recommend the molecular methods for diagnosing EPEC, as they can distinguish between typical and atypical EPEC by identifying the presence/absence of specific virulence factors. EPEC are defined as diarrheagenic strains of E. coli that can produce attaching and effacing lesions on the intestinal epithelium while being incapable of producing Shiga toxins and heat-labile or heat-stable enterotoxins. The ability of these strains to produce attaching and effacing lesions enable them to cause localized lesions by attaching tightly to the surface of the intestinal epithelial cells, disrupting the surfaces of the cells, thus leading to the effacement of the microvilli. EPEC are classified on typical and atypical isolates, based on the presence or absence of E. coli adherence factor plasmids. All the EPEC strains are eae positive; typical EPEC strains are eae+, bfpA+, while atypical strains are eae+, bfpA−. No vaccines are currently available to prevent EPEC infections.

Diarrheal Pathogens Associated with Growth and Neurodevelopment

Background Diarrheal pathogens have been associated with linear growth deficits. The effect of diarrheal pathogens on growth is likely due to inflammation which also adversely affects neurodevelopment. We hypothesized that diarrheagenic pathogens would be negatively associated with both growth and neurodevelopment. Methods We conducted a longitudinal birth cohort study of 250 children with diarrheal surveillance and measured pathogen burden in diarrheal samples using quantitative PCR. Pathogen attributable fraction estimates (AFe) of diarrhea over the first two years of life, corrected for socioeconomic variables, were used to predict both growth and scores on the Bayley III Scales of Infant and Toddler Development. Results 180 children were analyzed for growth and 162 for neurodevelopmental outcomes. Rotavirus, Campylobacter, and Shigella were the leading causes of diarrhea in year 1 while Shigella, Campylobacter, and ST-ETEC were the leading causes in year 2. Norovirus was the only pathogen associated with LAZ at 24 months and was positively associated (RC 0.42, CI 0.04, 0.80). Norovirus (RC 2.46, CI 0.05 – 4.87) was also positively associated with cognitive scores while sapovirus (RC -2.64, CI -4.80 – -0.48) and Typical EPEC (RC -4.14, CI -8.02 – -0.27) were inversely associated. No pathogens were associated with language or motor scores. Significant maternal, socioeconomic, and perinatal predictors were identified for both growth and neurodevelopment. Conclusion Maternal, prenatal, and socioeconomic factors were common predictors of growth and neurodevelopment. Only a limited number of diarrheal pathogens were associated with these outcomes.

Prevalence, phylogeny, and antimicrobial resistance of Escherichia coli pathotypes isolated from children less than 5 years old with community acquired- diarrhea in Upper Egypt

Background Diarrhoea, affecting children in developing countries, is mainly caused by diarrheagenic Escherichia coli (DEC). This study principally aimed to determine the prevalence of DEC pathotypes and Extended-spectrum β-lactamase (ESBL) genes isolated from children under 5 years old with diarrhea. Methods A total of 320 diarrhoea stool samples were investigated. E. coli isolates were investigated for genes specific for enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and enterohemorrhagic E. coli (EHEC) using polymerase chain reaction (PCR). Furthermore, antimicrobial susceptibility testing, detection of antibiotic resistance-genes and phylogenetic typing were performed. Results Over all, DEC were isolated from 66/320 (20.6%) of the children with diarrhoea. EAEC was the predominant (47%), followed by typical EPEC (28.8%) and atypical EPEC (16.6%). Co-infection by EPEC and EAEC was detected in (7.6%) of isolates. However, ETEC, EIEC and EHEC were not detected. Phylogroup A (47%) and B2 (43.9%) were the predominant types. Multidrug-resistance (MDR) was found in 55% of DEC isolates. Extended-spectrum β-lactamase (ESBL) genes were detected in 24 isolates (24 blaTEM and 15 blaCTX-M-15). Only one isolate harbored AmpC β-lactamase gene (DHA gene). Conclusion The study concluded that, EAEC and EPEC are important causative agents of diarrhoea in children under 5 years. MDR among DEC has the potential to be a big concern.

Bfp Adhesions and EspA Filament by Enteropathogenic E.Coli in Relation to Pathogenesis of Infantile Diarrhea in Children Under 5 Years in Mansoura University Children Hospital

Background: Our study was applied aiming to detect the biofilm formation by the Enteropathogenic E. coli causing diarrhea and to investigate the relation between the Bfp adhesions and EspA filament containing strains with the severity of diarrhea in children under 5 years in Mansoura University Children Hospital, Egypt. Methods: It was carried out by identification of EPEC, Serological agglutination test, Biofilm Formation under static condition at 3,6,9,24 hours. Results: Out of 103 children with diarrhea, 50 E. coli were isolated, 16 (32%) were EPEC and 34 (68%) were non-EPEC. The CFU mean± SD of biofilm of 16 EPEC isolates at 4 time points 3, 6, 9, 24h are 108- 1013, 69.5910±24.9310, 106-109, 20.2948±3.96778, 107-1011, 13.92×1010±3.376×1010, 109-1015, 6.402×1013± 2.496×1013. The relation of the biofilm formation and the severity of diarrhea in 16 EPEC isolates including 5 typical EPEC isolates and11 atypical EPEC reveals significant relation between the duration of the diarrhea and biofilm formation P=0.001**. Conclusion: EPEC is capable of forming biofilms on plastic under static and flow through-continuous conditions which correlate to the severity of the diarrhea in children under 5 years.

Fine Tuning Data Mining Algorithm for an Efficient Classification of E-Coli

E. coli is the first organisms to be sequenced as genome and the classification within the DEC pathotypes has epidemiologic and clinical implications for managing diarrheal diseases. In many developing countries E.coli leads to cause of diarrhea in children. where the mode of transmissions takes place via food and water. based on their pathogenic phenotype and diseases they cause it can be classified into 6 groups. consequently, our awareness of the spectrum of diseases and syndromes that they cause is quite limited. Also, because we cannot readily identify infected patients, there are many complexities in defining the modes of attainment, prevention and treatment strategies, and estimating the burden of infectious squealed. These infections create many challenges, and no progress will take place until the diagnostic potential for these agents got improved. Identifying E. coli isolate co-express LA reiterates the difficulty of assigning bacteria to groups on the basis of their adherence phenotype or genotype. Therefore the analysis of E -coli with molecular methods demonstrates that strains carry will represent more characteristics of typical EPEC and also the lack of AggR regulon, we propose a novel classification approach for classifying E-coli therefore to recognize pathogens. In addition, the ability to simultaneously induce attaching effacing lesions and biofilm production may enhance the potential of the strains to cause diarrhea and prolong bacterial residence in the intestines, thus worsening malnutrition in the patients.

Antibiotic resistance and biofilm formation in children with Enteropathogenic Escherichia coli (EPEC) in Brazilian Amazon

Introduction: Enteropathogenic Escherichia coli is an important causative agent of diarrhea in both developed and developing countries. Methodology: We assessed the antibiotic resistance profile and the ability of 71 Enteropathogenic Escherichia coli (EPEC) isolates from children in the age group 6 years, or younger, to form biofilm. These children were hospitalized in Cosme and Damião Children Hospital in Porto Velho, Western Brazilian Amazon, between 2010 and 2012, with clinical symptoms of acute gastroenteritis. Results: The highest frequency of atypical EPEC (aEPEC) isolates reached 83.1% (59/71). Most EPEC isolates presented Localized Adherence Like (LAL) pattern in HEp-2 cells (57.7% - 41/71). Biofilm production was observed in 33.8% (24/71) of EPEC isolates, and it means statistically significant association with shf gene (p = 0.0254). The highest antimicrobial resistance rates and a large number of multiresistant isolates 67.6% (48/71), regarded cefuroxime (CXM), ampicillin (AMP), trimethoprim-sulfamethoxazole (SXT) and tetracycline (TET), respectively, mainly in typical EPEC (tEPEC). Furthermore, 96% (68/71) of EPEC isolates in the present study were resistant to at least one antibiotic, whereas only 3 isolates were sensitive to all the tested drugs. Conclusion: Based on our findings, there was increased aEPEC identification. EPEC isolates showed high resistance rate; most strains showed multiresistance; thus, they work as warning about the continuous need of surveillance towards antimicrobial use. Besides, the ability of forming biofilm was evidenced by the EPEC isolates. This outcome is worrisome, since it is a natural resistance mechanism of bacteria.

The Complete Genome of the Atypical Enteropathogenic Escherichia coli Archetype Isolate E110019 Highlights a Role for Plasmids in Dissemination of the Type III Secreted Effector EspT

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT-containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT-containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness.

1100. Characterization of Enteropathogenic Escherichia coli (EPEC) in Cancer Patients With Diarrhea

Background Biofire FilmArray multiplexed nucleic acid amplification tests (NAAT) for bacterial diarrhea include probes specific for EPEC. However, the platform does not differentiate typical EPEC (tEPEC, defined as carrying eaeA and bfp) which have strong epidemiologic associations with diarrhea from atypical EPEC (aEPEC, carrying eaeA but not bfp) for which there is a weaker association. Nevertheless, emerging data suggest that aEPEC subsets carrying efa1/lifA which encodes for adherence factor 1/lymphocyte inhibitory factor A, are associated with diarrhea. The role of EPEC and its subtypes as agents of bacterial diarrhea have not been well defined in immunosuppressed and cancer patients. Methods We characterized EPEC subtypes in stools from healthy individuals with no diarrhea (HI, N = 21), cancer patients with diarrhea and negative NAAT (DN, N = 25) and patients with diarrhea positive NAAT for EPEC (DP, N = 54). EPEC isolated from stool cultures were tested for eaeA and bfp, stx and other E. coli pathotypes. We estimated the number of fecal EPEC using a qPCR for eaeA, efa1/lifA that detected 5.6 × 101 to 5 × 107 cfu/mg of stool. Results Demographic characteristics and underlying malignancy were similar between DN and DP groups. DP were more likely to have diarrhea on admission than DN [46/52 (88%) vs. 13/25 (52%), P < 0.01]. Stool cultures confirmed EPEC in 24/52 (60%) DP of which 23/24 (96%) were aEPEC. Fecal qPCR for eaeA confirmed EPEC in 43/52 (83%) of DP, 0/25 DN and in 3/21 (14%) of HI (P < 0.001). DP excreted a higher number of EPEC cfu/mg of stool than HI (median 168 vs. 1.18 cfu/mg, P < 0.001) and only DP excreted EPEC efa1/lifA (+) [14/52 DP (27%) vs. 0/25 DN and 0/21 HI; P < 0.001]. When compared with DP EPEC efa1/lifA (-), DP EPEC efa1/lifA (+) had a longer median duration of illness (3 days vs. 1 days, P < 0.05); more likely to be hematopoietic stem cell transplant recipients [7/14 (50%) vs. 7/38 (18%), P < 0.05] and had a higher EPEC eaeA fecal burden (median 3885 vs. 84 cfu/mg, P < 0.05). Co-infections with other pathogens were equally represented in efa1/lifA (−) and efa1/lifA (+) DP subgroups [8/14 (57%) vs. 21/38 (55%) P = NS]. Conclusion Most EPEC in cancer patients with diarrhea are aEPEC acquired in the community and when carrying efa1/lifA (+), are associated with more severe disease. Disclosures All authors: No reported disclosures.

Molecular diagnosis of diarrheagenic Escherichia coli isolated from Psittaciformes of illegal wildlife trade

ABSTRACT: Diarrheagenic Escherichia coli (DEC) are considered one of the major causes of human diarrhea in developing countries. Some studies have pointed wild birds as important reservoirs for these pathogens. However, scarce species from the Psittaciformes order have been investigated. This study aimed to evaluate the presence of DEC strains in Psittaciformes from illegal wildlife trade. A total of 78 E. coli strains isolated from cloacal swab samples of 167 Psittaciformes in the Ceará State, Brazil, were evaluated regarding the presence of the following DEC virulence genes by polymerase chain reaction (PCR): eaeA and bfpA genes (Enteropathogenic E. coli - EPEC); stx1 and stx2 (Shiga toxin-producing E. coli - STEC); estA and eltB (Enterotoxigenic E. coli - ETEC); ipaH (Enteroinvasive E. coli - EIEC); aatA and aaiC (Enteroaggregative E. coli - EAEC). Positive strains for eaeA and bfpA genes were considered typical EPEC, while strain positive exclusively for the eaeA gene were classified as atypical EPEC. The eaeA gene was identified in 20 E. coli strains and bfpA in 22 isolates. In addition, 11 and 9 belonged to tEPEC and aEPEC, respectively. No strain was positive for stx1 or stx2. A total of 47 (60.3%) strains and a total of 136 birds (81.4%) were negative for the remaining DEC pathotypes investigated. In conclusion, psittacine from illegal wildlife trade in Ceará State, Brazil, presented a relevant prevalence of typical and atypical EPEC, potentially playing a role as reservoirs of DEC strains in the environment. Thus, proper control measures must be adopted to block the spread of these pathogens.