Diarrheagenic Escherichia Coli Infections Research Articles

Sequence-subtype association of multi-drug-resistant diarrheagenic Escherichia coli

Background and Objectives: Multi-drug-resistant pathogens pose a significant threat as they can rapidly spread, leading to severe healthcare-associated invasive infections. In developing countries, diarrheagenic Escherichia coli (DEC) is a major bacterial pathogen responsible for causing diarrhea. However, the outbreak of resistant strains has made the treatment of DEC infections much more challenging. This study aimed to investigate the relationship between antibiotic resistance genes and other virulence categories in E. coli strains that cause diarrhea, particularly DEC.
 Materials and Methods: The phylogenetic grouping was defined using PCR and multi-locus sequence type (MLST) meth- ods.
 Results: Among the isolates analyzed, 14 were identified as resistant and were classified into eight distinct sequence types: ST3, ST53, ST77, ST483, ST512, ST636, ST833, and ST774, indicating genetic diversity among the resistant strains. Certain sequence types, notably ST512 and ST636, were found to be associated with multiple antibiotic resistance in DEC. Regard- ing antibiotic susceptibility, strains showed the highest resistance to amoxicillin, suggesting that this antibiotic may not be effective in treating DEC infections. On the other hand, the isolates demonstrated susceptibility to amikacin and chloram- phenicol, implying that these antibiotics could be more suitable treatment options for DEC infections.
 Conclusion: The findings underscore the importance of promptly identifying antibiotic resistance patterns and their correla- tion with specific pathogenic virulence categories, as this knowledge can aid in selecting the most appropriate antibiotics for treating DEC infections. Considering the antibiotic resistance profiles and associated resistance genes is crucial in managing and containing diarrheal outbreaks and in selecting effective antibiotic therapies for DEC infections.

Why are so many enteric pathogen infections asymptomatic? Pathogen and gut microbiome characteristics associated with diarrhea symptoms and carriage of diarrheagenic E. coli in northern Ecuador

ABSTRACT A high proportion of enteric infections, including those caused by diarrheagenic Escherichia coli (DEC), are asymptomatic for diarrhea. The factors responsible for the development of diarrhea symptoms, or lack thereof, remain unclear. Here, we used DEC isolate genome and whole stool microbiome data from a case–control study of diarrhea in Ecuador to examine factors associated with diarrhea symptoms accompanying DEC carriage. We investigated i) pathogen abundance, ii) gut microbiome characteristics, and iii) strain-level pathogen characteristics from DEC infections with diarrhea symptoms (symptomatic infections) and without diarrhea symptoms (asymptomatic infections). We also included data from individuals with and without diarrhea who were not infected with DEC (uninfected cases and controls). i) E. coli relative abundance in the gut microbiome was highly variable, but higher on-average in individuals with symptomatic compared to asymptomatic DEC infections. Similarly, the number and relative abundances of virulence genes in the gut were higher in symptomatic than asymptomatic DEC infections. ii) Measures of microbiome diversity were similar regardless of diarrhea symptoms or DEC carriage. Proteobacterial families that have been described as pathobionts were enriched in symptomatic infections and uninfected cases, whereas potentially beneficial taxa, including the Bacteroidaceae and Bifidobacteriaceae, were more abundant in individuals without diarrhea. An analysis of high-level gene functions recovered in metagenomes revealed that genes that were differentially abundant by diarrhea and DEC infection status were more abundant in symptomatic than asymptomatic DEC infections. iii) DEC isolates from symptomatic versus asymptomatic individuals showed no significant differences in virulence or accessory gene content, and there was no phylogenetic signal associated with diarrhea symptoms. Together, these data suggest signals that distinguish symptomatic from asymptomatic DEC infections. In particular, the abundance of E. coli, the virulence gene content of the gut microbiome, and the taxa present in the gut microbiome have an apparent role.

Molecular Characterization of Hetero-Pathogenic and Diarrheagenic Escherichia coli Pathotypes in Diarrheic Children under Five Years and Exposure Environment in Ogun State, South-West Nigeria.

Diarrheagenic Escherichia coli (DEC) is one of the most common etiological agents of moderate-to-severe diarrhea in Low- and Middle-Income Countries (LMICs). Therefore, determining the source(s) of DEC in index cases and exposure environment is important for developing a prevention strategy. The current study aims to investigate the prevalence of DEC among children under 5 years and their exposure environment in Ogun State, Nigeria. Samples from 228 diarrheic children and their exposure environment were collected and screened for E. coli. Bio-chemically compatible distinct colonies were molecularly characterized using a 7-virulence-gene multiplex PCR with virulence factors (VFs) indicative of four pathotypes of E. coli: enterotoxigenic (ETEC), verotoxigenic (VTEC), enteropathogenic (EPEC), and enteroinvasive (EIEC). Representative pathotypes were subjected to antimicrobial susceptibility and over-expressed efflux pump assays. One or more VFs typical of specific pathotypes were detected in 25.9% (59/228) diarrhea cases consisting of ETEC (21.5%) and EPEC (0.4%), while hetero-pathogenic pathotypes were found in 4.0% of cases. Of the food sources, 27.9% (101/362) were positive for DEC, of which ETEC accounted for 21.0%, VTEC 1.9%, EPEC 0.6%, EIEC 0.6%, and hetero-pathogenic pathotypes were 3.9%. Furthermore, ETEC was the only pathotype detected in the wastewater (4/183). Interestingly, the consumption of street-vended foods was the most significant (p = 0.04) risk factor for DEC infection in the study area. A total of 73.3% of selected DEC pathotypes showed resistance to antimicrobials, while 27.5% demonstrated over-expression of efflux pump activity. The high prevalence of ETEC across all sources and the occurrence of hetero-pathogenic DEC in diarrheic children and food sources emphasizes the importance of establishing a better strategy for the control and prevention of diarrhea among children in low- and medium-income households.

Subjects’ Sociodemographics Influence the Transmission Patterns of Diarrheagenic Escherichia coli Pathotypes among Children under 5 Years in Nakuru County

Background: Diarrheagenic Escherichia coli (DEC) infections constitute the leading causes of morbidity and mortality among children in Sub-Saharan Africa. However, little has so far been done to properly reveal the pathogenic endowments of DEC in these populations. Aims and Objectives: We evaluated 4 DEC strains among children under 5 years. Materials and Methods: A cross-sectional study design was employed among 384 positive cases. Results: There was a significant decline in infections associated with DEC as the children grew older (χ2[12] = 87.366: P = [0.000]. A total of 56 (14.6%) cases were 0–12 months, 168 (43.8%) were 13–24 months, 88 (22.9%) were 25–36 months, 40 (10.4%) were 37–48 months, and 32 (8.3%) were 49–60 months. A total of 248 (64.6%) male subjects exhibited more susceptibility to DEC infections than their female counterparts (n = 136 [35.4%]) (χ2[3] =13.313: P = [0.004]. Subjects from urban areas (n = 248 [64.6%]), significantly bored the brunt of infections than those from rural areas (n = 136 [35.4%]) (χ2[3] = 35.147: P = [0.000]. The prevalence of DEC appeared significantly higher during rainy seasons (n = 269 [70.1%]). Conclusion: Young age, male gender, crowding, and rainy season play a central role in the transmission of DEC pathotypes.

Prevalence and Antimicrobial Resistance Profile of Diarrheagenic Escherichia coli from Fomites in Rural Households in South Africa.

Diarrheagenic Escherichia coli (DEC) pathotypes are the leading cause of mortality and morbidity in South Asia and sub-Saharan Africa. Daily interaction between people contributes to the spreading of Escherichia coli (E. coli), and fomites are a common source of community-acquired bacterial infections. The spread of bacterial infectious diseases from inanimate objects to the surrounding environment and humans is a serious problem for public health, safety, and development. This study aimed to determine the prevalence and antibiotic resistance of diarrheagenic E. coli found in toilets and kitchen cloths in the Vhembe district, South Africa. One hundred and five samples were cultured to isolate E. coli: thirty-five samples were kitchen cloths and seventy-five samples were toilet swabs. Biochemical tests, API20E, and the VITEK®-2 automated system were used to identify E. coli. Pathotypes of E. coli were characterised using Multiplex Polymerase Chain Reaction (mPCR). Nine amplified gene fragments were sequenced using partial sequencing. A total of eight antibiotics were used for the antibiotic susceptibility testing of E. coli isolates using the Kirby-Bauer disc diffusion method. Among the collected samples, 47% were positive for E. coli. DEC prevalence was high (81%), with ETEC (51%) harboring lt and st genes being the most dominant pathotype found on both kitchen cloths and toilet surfaces. Diarrheagenic E. coli pathotypes were more prevalent in the kitchen cloths (79.6%) compared with the toilet surfaces. Notably, hybrid pathotypes were detected in 44.2% of the isolates, showcasing the co-existence of multiple pathotypes within a single E. coli strain. The antibiotic resistance testing of E. coli isolates from kitchen cloths and toilets showed high resistance to ampicillin (100%) and amoxicillin (100%). Only E. coli isolates with hybrid pathotypes were found to be resistant to more than three antibiotics. This study emphasizes the significance of fomites as potential sources of bacterial contamination in rural settings. The results highlight the importance of implementing proactive measures to improve hygiene practices and antibiotic stewardship in these communities. These measures are essential for reducing the impact of DEC infections and antibiotic resistance, ultimately safeguarding public health.

Factors Associated with Diarrheagenic Escherichia Coli Infection among Children with Diarrhea in Kwali, Federal Capital Territory, Nigeria

Factors Associated with Diarrheagenic Escherichia Coli Infection among Children with Diarrhea in Kwali, Federal Capital Territory, Nigeria

Development and Effective Utilization of a Rapid Multiplex Real-Time PCR of Diarrheagenic Escherichia coli for Food Poisoning Cases.

Diarrheagenic Escherichia coli (DEC) causes diarrheal symptoms in humans. The comprehensive detection of DEC from feces using SYBR Green real-time PCR assay requires multiple runs. Moreover, PCR screening can have discrepancies related to the conformance between the results from PCR screening and culturing. We aimed to develop a real-time PCR for the comprehensive testing of DEC for diagnostic support that can be used in any general laboratory and proposed its effective utilization. We tested specificity for the designed primer sets using 100 strains. Moreover, screening and isolation of DEC were performed using the proposed multiplex real-time PCR system for 308 fecal samples collected from 37 food poisoning incidences that occurred in Gifu Prefecture, Japan from 2017 to 2019. Furthermore, the factor of discrepant results between PCR screening and culturing was analyzed by quantifying the number of DEC cell and whole E. coli cell using real-time PCR for 47 PCR screening-positive fecal samples. The results obtained from the developed multiplex real-time PCR system were in 99% concordance with those from the conventional techniques. A total of 49 fecal samples were detected with virulence genes for the screening. Of the samples which were positive with virulence genes by PCR screening, 38.3% could not be detected from the strain for bacterial culture. We found that the culturing positive samples were significantly high in numbers for the DEC cells, but no significant difference was noted in the whole E. coli cells with culturing negative samples. The multiplex real-time PCR developed in this study was found to be rapid and practical for DEC testing. The PCR screening for DEC using this method can provide rapid information toward the diagnostic support of DEC infection.

Rapid Detection of Diarrheagenic Escherichia coli by a New Multiplex Real-Time Quantitative PCR Assay

Diarrheagenic Escherichia coli (DEC) is a group of important foodborne pathogens that can spread and cause infection in humans. As a significant public health issue, DEC infection may lead to a series of diseases such as abdominal pain, diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Therefore, its rapid and accurate detection has become an important concern for food safety issues and healthcare-associated infections. We have developed a multiplex real-time quantitative PCR assay (qPCR), DEC Sensor Test, to simultaneously recognize 5 major DEC pathotypes in a single tube, enterotoxigenic, enterohemorrhagic, enteropathogenic, enteroaggregative and enteroinvasive E. coli. In this assay, 13 DEC virulence genes, stp, sth, lt, stx1, stx2, eae, escV, bfpB, aggR, astA, pic, invE, and ipaH, can be simultaneously detected along with a spiked-in internal amplification control to avoid false negative results. All the constituent PCR for the 13 DEC virulence genes had high analytical sensitivity and specificity. Detection of all virulence genes by DEC Sensor Test can be much easier, cheaper and faster to perform than conventional tests. It also has added advantages of being able to distinguish live or dead bacteria as well as eliminating the possibility of switching partial results between different samples associated with multi-tube tests. Therefore, the application of this assay may significantly enhance our ability to prevent and fight DEC-associated diseases and to improve public health.

The frequency of virulent genes and antimicrobial resistance patterns of diarrheagenic Escherichia coli isolated from stools of children presenting with diarrhea in a tertiary hospital in Abakaliki, Nigeria

Aim: This study was aimed to determine the virulent genes and antibiotic resistance patterns among circulating diarrheagenic Escherichia coli (DEC) pathotypes in a tertiary care health center in east of Nigeria. Materials and Methods: Diarrheal stool samples were obtained from 80 children under 5 years and E. coli was isolated and identified using standard biochemical and molecular methods. Multiplex polymerase chain reaction (PCR) was used to detect eight virulent genes of DEC. Disk diffusion method was used to determine the antibiotic susceptibility of DEC. Results: DEC infection was observed in 54 (68%) children among which ial gene for enteroinvasive E. coli (EIEC) (40% [n=22]) was commonly detected followed by eltA/eltB for enterotoxigenic E. coli (ETEC) (30% [n=16]), pCVD for enteroaggregative E. coli (EAEC) (20% [n=11]), and eaeA/bfpA for typical enteropathogenic E. coli (EPEC) (10% [n=5]). The DEC isolates phenotypically exhibited resistance for ampicillin (AMP) (44 [81%]), followed by ciprofloxacin (CIP)/ levofloxacin (LEV) (28 [52%]), cefoxitin (FOX) (11 [20%]), and amoxicillin-clavulanic acid (AMC) (6 [11%]). About 60% isolates of stable toxins-ETEC were resistant to AMC, CIP, and LEV while all the labile toxin-ETEC exhibited resistance to AMP. About 60% (n=6) resistance were seen in EAEC against ampicillin, AMC, FOX, CIP, and LEV. In EIEC, all the isolates (n=22) were resistant to AMP while 50% (n=11) were resistant to both CIP and LEV. All EPEC (n=5) were resistant to AMP, FOX, CIP, and LEV. Conclusion: High frequency of virulent ial and eltA/eltB genes for EIEC and ETEC, respectively, suggests that they are the primary etiological agents of diarrhea in children among DEC pathotypes. Resistance of DEC to more than two classes of antibiotics indicate possible emergence of multidrug resistance.

Diarrheagenic Escherichia coli Pathotypes From Children Younger Than 5 Years in Kano State, Nigeria.

Diarrheagenic Escherichia coli (DEC) is one of the leading causes of gastrointestinal disorders worldwide and an important public health challenge. DEC infection is often underdiagnosed during routine microbiological analysis, especially in resource constrained settings; the use of molecular tests could however help to determine the distribution of DEC and its clinical significance. Here, a study to assess the prevalence of DEC in clinical samples from patients <5 years attending three hospitals in Kano state, Nigeria, was carried out. Samples from 400 patients and 50 controls were collected and screened for E. coli. Compatible colonies from 248 individuals (215 patients and 33 controls) were characterized using biochemical test, a set of real-time PCRs for detection of nine virulence factors (VF: eae, bfpA, elt, est, stx1, stx2, ehxA, aggR, and invA) associated with five DEC pathotypes (EPEC, ETEC, EHEC, EAEC, and EIEC) and antimicrobial susceptibility tests. One or more VFs typical of specific pathotypes were detected in 73.8% (183/248) of the isolates, with those associated with EAEC (36.3%), ETEC (17.3%), and EPEC (6.0%) being the most common, although proportion of specific pathotypes differed between hospitals. est was the only VF detected in a significantly higher proportion in cases compared to controls (P = 0.034). Up to 86.9% of DEC were resistant to at least one class of antibiotics, with trimethoprim-sulfamethoxazole being the least effective drug (77.6% resistance). Our results demonstrate the widespread circulation of different DEC pathotypes that were highly resistant to trimethoprim-sulfamethoxazole among children in Kano state, and highlight the need of characterizing the causative agents in cases of gastrointestinal disorders.

Identification of diarrheagenic Escherichia coli by a new multiplex PCR assay and capillary electrophoresis

Diarrheagenic Escherichia coli (DEC) is a set of the most common pathogens causing diarrhea. DEC strains are classified into five pathotypes based on the possession of different virulence genes: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) or Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC). The development of an easy-to-use method to detect the specific virulence genes and distinguish the pathotypes is essential for the diagnosis and surveillance of DEC infections. In this study, a multiplex PCR assay (mPCR) specific to nine virulence genes and an internal control was designed for the identification of five DEC pathotypes. A temperature switch PCR (TSP) strategy was used in the PCR amplification. The PCR products were detected by capillary electrophoresis. The limit of detection (LOD) of the 10-plex reaction was 5 × 103 copies/reaction for stx2 and 5 × 102 copies/reaction for the other targets. The mPCR showed very high specificity, and inclusivity and exclusivity were both 100%. When the mPCR assay was used for the detection of 221 cryopreserved diarrhea specimens, DEC colonies were detected from 49 specimens, and the positive rate was 22.2%. The mPCR assay was sensitive and specific, and the amplified product could be analyzed easily. Thus, this method could be used effectively to identify the suspected colonies of DEC in the primary culture of the specimen.

Molecular epidemiology and antimicrobial resistance of diarrheagic Escherichia coli among children with acute diarrhea

Objective To learn the distribution, epidemiology and antimicrobial susceptibility of diarrheagenic Escherichia coli (DEC) isolated from patients with acute diarrhea among children less than 5 years old. Methods Totally 684 stool samples collected from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between August 1, 2015 and September 30, 2016 were tested by culture and identified the common pathogens. PCR was applied to detect the virulence genes of DEC. Meanwhile, serotyping of enteropathogenic E. coli (EPEC) was performed by slide agglutination tests for all the isolates of EPEC. An antimicrobial sensitivity test was performed using the agar dilution method. Results A total of 149 (21.7%) enteric bacteria pathogens were isolated from 684 specimens. DEC was found in 54 cases, ranked 2nd among the pathogenic bacteria. DEC tended to occur in summer/autumn periods. EPEC was the most frequent DEC genotype, accounted for 50% (27/54). Among EPEC, atypical EPEC was dominant, accounted for 77.8% (21/27) and typical EPEC only accounted for 22.2% (6/27). Followed by enteroaggregative E.coli 20.4% (11/54), enterotoxigenic E.coli 14.8% (8/54), enteroinvasive E. coli and Shiga toxin-producing E.coli 3.7% (2/54), 7.4% (4/54) cases were co-infected with more than one DEC genotypes. About 17/18 of suspicious DEC isolates can get the same genotypes by commercial multiplex PCR kit and single PCR test. Among the 27 EPEC strains, only 11(40.7%) strains can be detected by the slide agglutination serotyping method. More than 50% (27/54) of DEC isolates were resistant to conventional first-line antibiotics (ampicillin, trimethoprim-sulfamethoxazole) and cefazolin, cefuroxime, cefotaxime, but relatively low resistance to cefoxitin, amikacin, piperacillin/tazobactam, imipenem and meropenem. However, there was still a 9.2% (5/54) resistance rate to carbapenems. Conclusions DEC strains exhibited a high frequency of resistance to many antibiotics. Empirical antimicrobial therapy of severe DEC infection faces the challenge from the high resistance to ampicillin, trimethoprim-sulfamethoxazole. Even worse, some strains were resistant to relatively efficient drugs imipenem and meropenem. It is necessary to strengthen the epidemiological survey and antimicrobial resistance of DEC. Key words: Diarrhea; Escherichia coli; Virulence; Escherichia coli infections; Drug resistance, bacterial; Epidemiologic studies

An overview of diarrheagenic Escherichia coli in Iran: A systematic review and meta-analysis.

Background:Diarrheagenic Escherichia coli (DEC) is a common enteric pathogen that causes a wide spectrum of gastrointestinal infections, particularly in developing countries. This is a systematic review and meta-analysis to determine the prevalence of DEC in various geographical regions in Iran.Materials and Methods:English (PubMed, Web of Science, Scopus, Embase, Cochrane Library, and Google Scholar) and Persian (IranMedex, SID, Magiran, and Iran Doc) databases were comprehensively searched from January 1990 to April 2017. Study selection and data extraction were performed by two independent reviewers. After assessing heterogeneity among studies, a random effects model was applied to estimate pooled prevalence. Data analyses were done with the Stata software (version 12.0). This meta-analysis was registered with PROSPERO, number CRD42017070411.Results:A total of 73 studies with 18068 isolates were eligible for inclusion within the meta-analysis. The results of random effects model showed that the most prevalent DEC pathotypes were enterotoxigenic E. coli (ETEC) (16%; 95% confidence interval [CI]: 11%–23%), enteroaggregative E. coli (11%; 95% CI: 8%–15%), atypical enteropathogenic E. coli (EPEC) (11%; 95% CI: 8%–14%), Shiga toxin-producing E. coli (9%; 95% CI: 6%–13%), diffuse adherent E. coli (6%; 95% CI: 6%–12%), enteroinvasive E. coli (4%; 95% CI: 2%–6%), and typical EPEC (3%; 95% CI: 1%–5%).Conclusion:This study showed that DEC infections in the Iranian population have low frequency. Our data suggest that the ETEC pathotype can be regarded as one of the most important etiological agents of diarrhea in this country. However, the prevalence of DEC pathotypes is diverse in different regions of Iran.

Monitoring of diarrheagenic Escherichia coli infections in patients in Guangdong Province during 2013 to 2016

Objective To investigate the infection status, serotype distribution, drug sensitivity and molecular characteristics of diarrheagenic Escherichia coli (DEC) in patients with diarrhea in Guangdong Province. Methods Fecal samples were collected, cultured and isolated by traditional methods. Suspected Escherichia coli isolates were confirmed by multiplex PCR used for detecting specific virulence genes and biochemical methods. Positive strains were serotyped, characterized for drug sensitivity and analyzed by pulsed-field gel electrophoresis (PFGE). Results The total positive rate of DEC in patients with diarrhea was 6.26%. The positive rates of enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), enteroadherent Escherichia coli (EAEC) and enteroinvasive Escherichia coli (EIEC) were 2.47%, 1.54%, 1.32%, 0.62% and 0.09%, respectively, with infections primarily in children aged 0-<7 years. The total seropositive rate was 52.54%, with EHEC accounting for 15.00%. DEC showed high sensitivity to imipenem, ciprofloxacin, ceftazidime and cefotaxime. The multidrug resistance rate of DEC was 58.45%, with EPEC being the most serious for multidrug resistance. PFGE results showed that ETEC, EHEC, EPEC and EAEC had a high degree of polymorphism. Conclusion EPEC is the predominant type of DEC circulating in Guangdong Province. Third-generation cephalosporins are the first drugs of choice for treating infections in children. Ciprofloxacin can be used to treat adults. The problem of multiple drug resistance of DEC is severe and efforts to monitor DEC infections and drug resistance should be strengthened. Key words: Diarrheagenic Escherichia coli; Virulence gene; Pathogen surveillance; Antibiotic resistance surveillance; Pulsed-field gel electrophoresis

Characteristics of diarrheagenic Escherichia coli among children under 5 years of age with acute diarrhea: a hospital based study

BackgroundDiarrhea is the leading infectious cause of childhood morbidity and mortality. Among bacterial agents, diarrheagenic Escherichia coli (DEC) is the major causal agent of childhood diarrhea in developing countries, particularly in children under the age of 5 years. Here, we performed a hospital-based prospective study to explore the pathotype distribution, epidemiological characteristics and antibiotic resistance patterns of DEC from < 5-year-old diarrheal children.MethodsBetween August 2015 and September 2016, 684 stool samples were collected from children (< 5 years old) with acute diarrhea. All samples were cultured and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and biochemical tests. PCR was used for subtyping, and enteropathogenic E. coli (EPEC) isolates were identified simultaneously with serology. Furthermore, antimicrobial sensitivity tests and sequencing of antibiotic resistance-related genes were conducted.ResultsDEC strains were identified in 7.9% of the 684 stool samples. Among them, the most commonly detected pathotype was EPEC (50.0% of DEC), of which 77.8% were classified as atypical EPEC (aEPEC). Age and seasonal distribution revealed that DEC tended to infect younger children and to occur in summer/autumn periods. Multidrug-resistant DEC isolates were 66.7%; resistance rates to ampicillin, co-trimoxazole, cefazolin, cefuroxime, cefotaxime, and ciprofloxacin were ≥ 50%. Among 5 carbapenem-resistant DEC, 60.0% were positive for carbapenemase genes (2 blaNDM-1 and 1 blaKPC-2). Among 30 cephalosporin-resistant DEC, 93.3% were positive for extended-spectrum β-lactamase (ESBL) genes, with blaTEM-1 and blaCTX-M-55 being the most common types. However, no gyrA or gyrB genes were detected in 16 quinolone-resistant isolates. Notably, aEPEC, which has not received much attention before, also exhibited high rates of drug resistance (81.0%, 66.7%, and 14.3% for ampicillin, co-trimoxazole, and carbapenem resistance, respectively).ConclusionsEPEC was the most frequent DEC pathotype in acute diarrheal children, with aEPEC emerging as a dominant diarrheal agent in central China. Most DEC strains were multidrug-resistant, making even ciprofloxacin unsuitable for empiric treatment against DEC infection. Among carbapenem-resistant DEC strains, those harboring blaNDM-1 and blaKPC-2 were the main causal agents. blaTEM-1 and blaCTX-M-55 were the major genetic determinants associated with high levels of cephalosporin resistance.

Molecular characterization of diarrheagenic Escherichia coli pathotypes: Association of virulent genes, serogroups, and antibiotic resistance among moderate-to-severe diarrhea patients.

Diarrheagenic Escherichia coli (DEC) signifies as an important etiological agent of moderate-to-severe diarrhea. This study was primarily focused on molecular identification of DEC pathotypes; their association with serogroups and estimates of resistance profiles against different antibiotics regime. Five hundred seventy-two stool specimens from diarrhea patients were investigated for DEC pathotypes. Molecular pathotypes were identified by amplification of virulence genes associated with distinct pathotypes followed by sequencing. Diarrhea is a self-limiting disease, however, severity and persistence of infection suggest antibiotic use. Therefore, AST and MIC were determined against common antibiotic regimen. Correlations between molecular pathotypes and serogroups were analyzed by somatic "O" antigen serotyping. The present findings reveal incidence of DEC as an etiological agent up to a level of 21% among all diarrheal age groups. DEC infection rate was higher in children. Enteropathogenic E.coliEPEC, a molecular pathotype of DEC, was found as a predominant pathotype with highest frequency of 13.7%. Two other molecular pathotypes enterotoxigenic E.coli (ETEC) and enteroaggregative E.coli (EAEC) accounted for 5.7% and 1.3%, respectively for all diarrhea incidences. Serological analysis deciphered somatic antigens O26, O2, and O3 as major serogroups identified among EPEC, ETEC, and EAEC pathotypes, respectively. All DEC pathotypes exhibited high levels of antibiotic resistance except for cotrimoxazole and norfloxacin. Comprehensive molecular characterization of DEC pathotypes, their incidence estimates, and antibiogram patterns will help in ascertaining better diagnostic and therapeutic measures in management of diarrheal diseases.

Distinctive Gut Microbiota Is Associated with Diarrheagenic Escherichia coli Infections in Chilean Children.

Background: Diarrheagenic Escherichia coli (DEC) strains are a major cause of diarrhea in children under 5 years of age worldwide. DEC pathogenicity relies on the interaction of bacteria with environmental factors, including the host's resident gut microbiota. Previous reports have shown changes in the gut microbiota's composition during episodes of diarrhea, which may increase the pathogenicity of DEC strains. More intense and detailed identification of microbiota strains specifically associated with DEC infections and disease is needed to pinpoint their role in DEC pathogenicity.Aim: To identify resident indicative bacterial taxa in DEC-positive diarrhea stool samples of Chilean children.Methods: We analyzed 63 diarrheal stool samples from children 1–5 years of age by FilmArray® GI in order to identify a potential pathogen and to group diarrhea episodes into those caused by DEC as sole pathogen (DEC group, 32 samples) and those caused by an enteric virus as sole pathogen (viral group, 31 samples). In addition, 30 stool samples from healthy children, negative for enteric pathogens, were evaluated (healthy group). The 16S rRNA gene was amplified and sequenced using 454 pyrosequencing. Sequences were clustered into operational taxonomic units (OTUs) at 99% identity and their representatives were used to assign them to operational phylogenetic units (OPUs) using a phylogenetic inference approach.Results: Taxa assignment using the OPU approach resulted in a lower number of units but with higher accuracy compared to the OTU approach. Data analysis indicated an increase in sequences belonging to the phylum Proteobacteria in the DEC group compared to the viral and healthy groups. Samples displayed a statistically different community structure by sample grouping by redundancy analysis and ANOVA. Escherichia albertii (p = 0.001), Citrobacter werkmanii (p = 0.001), Yersinia enterocolitica, subsp. paleartica (p = 0.048), and Haemophilus sputorum (p = 0.028) were indicative species for the DEC group as compared to the viral and healthy groups.Conclusion: Gut microbiota in Chilean children with DEC-positive diarrhea differed from microbiota associated with enteric virus and healthy children. Indicative species found in this study may prove relevant in advancing our understanding of the relationship between resident gut microbiota and DEC leading to the occurrence of disease.

High Prevalence of Diarrheagenic Escherichia coli among Children with Diarrhea in Kenya.

Diarrheagenic Escherichia coli (DEC) is an important agent of endemic and epidemic diarrhea worldwide, particularly in developing countries. DEC cannot be differentiated from commensal E. coli on selective media, although there are a few exceptions. Most studies use the colony isolation method, which cannot detect low numbers of DEC, and therefore, these studies might underestimate the incidence of DEC. In the present study, we employed a colony sweep method with real-time PCR targeting virulence genes of 5 categories of DEC; this technique can detect very low numbers of DEC among hundreds of commensal E. coli. DEC was detected in 171 (55.9%) of 306 children with diarrhea in Kenya. The prevalence of DEC in Kenya was notably higher than that (30 in 143, 21.0%) in Indonesia. Occurrences of multiple DEC infection in Kenya were frequent (69 in 306, 23.2%), suggesting that the source of DEC infection may be related to grossly contaminated food and water. In contrast, only 9 (6.0%) of 150 healthy adults in Kenya carried DEC. Considering that healthy adults naturally harbor non-DEC, it is interesting how children exclude DEC but not non-DEC as they grow up. Several mechanisms, such as mucosal immunity and intestinal microbiota, might be involved in the exclusion of DEC.

Detection of Shiga toxin-producing and other diarrheagenic Escherichia coli by the BioFire FilmArray® Gastrointestinal Panel in human fecal samples.

The purpose of this investigation was the evaluation of the performance of the BioFire FilmArray® Gastrointestinal (FA-GI) Panel, a multiplexed molecular stool screening assay, for the detection of diarrheagenic Escherichia coli (DEC), with emphasis on Shiga toxin-producing E. coli (STEC). A dilution series of 12 STEC reference strains was tested with the FA-GI Panel to assess the analytical sensitivity. A total of 389 patient samples were analyzed with the FA-GI Panel and confirmation of the detected DEC was attempted with an in-house culture-based polymerase chain reaction (PCR) method. All Shiga toxin genes, except the one encoding Stx2f, were detected in bacterial dilutions ranging from 10(4) to 10(2)colony-forming units (CFU)/ml. eae + stx2f + STEC was misclassified as enteropathogenic E. coli (EPEC). Different sensitivities for various gene targets present in one isolate led to differing identifications depending on the concentration. Using the in-house method as a reference, the FA-GI Panel had a sensitivity of 90.6% [confidence interval (CI) 75.0%-98.0%] and a specificity of 97.2% (CI 94.9%-98.6%) for STEC detection in feces. At least one DEC was reported in 35.5% (171/389) of the patient specimens, with EPEC being the most prevalent (n = 71). Only 59.7% of the detected DEC could be confirmed, presumably because the comparator method was not applied directly on feces. The FA-GI Panel could not detect the stx2f subtype, misclassified certain pathogens, and the high detection rate of EPEC needs further investigation. Nevertheless, we believe that this sensitive and convenient system will prove to be an invaluable tool for the rapid diagnosis of most DEC infections, but culturing of the detected microorganisms should always be attempted.

Clinical and etiologic features of diarrheagenic Escherichia coli-associated diarrhea in children

Objective To investigate the pathotypes, epidemiological characteristics and antimicrobial resistance of diarrheagenic Escherichia coli (DEC) in children with acute bacterial diarrhea in Shanghai. Methods A total of 2 071 outpatient children with probable acute bacterial diarrhea referred to the enteric clinic of Children′s Hospital of Fudan University during June 2012 to June 2014 were enrolled in our study. The stool samples were processed for routine microbiologic and biochemistry tests to identify enteric bacteria, including enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli(EIEC) and enterohemorrhagic Escherichia coli (EHEC). Kirby-Bauer method was used to identify the antibiotic sensitivity. Difference of means between groups was compared by chi-square test. Results Of 2 071 enrolled children, DEC were identified in 145 (7.0%) cases. 148 strains were isolated with three of mix infection strains. All DEC isolates in this study included 106 (71.6%) EPEC, 24 (16.2%) ETEC, 16(10.8%) EIEC and 2(1.4%) EHEC. The median ages of diarrheal children with DEC infections were 14 months (range: 3 months to 13 years) and 62.8% of them were <2 years. Among 125 DEC isolates tested for antimicrobial susceptibility, the rates of resistance to ampicillin, trimethoprim-sulfamethoxazole, cefotaxime, cefepime, gentamicin, ceftazidime, amoxicillin-clavulanate, ciprofloxacin, and ofloxacin in a descending order were 55.2%, 35.2%, 28.0%, 27.2%, 23.2%, 8.8%, 5.6%, 4.0% and 4.0%, respectively. Resistance rates of EIEC to cefotaxime, cefepime and ceftazidime were 50.0%, 43.8% and 25.0%, respectively, which were higher than those of EPEC, ETEC and EHEC. Conclusion DEC is the important enteric bacteria that causes bacterial diarrhea in children in this study. Key words: Diarrhea; Diarrheagenic Escherichia coli; Epidemiology; Child; Drug resistance, microbial