Primer reporte de Escherichia coli diarreogénica en población pediátrica ambulatoria con diarrea atendida en la ciudad de La Plata, Argentina / First report of diarrheagenic Escherichia coli in pediatric outpatient population with diarrhea in La Plata, Argentina

Resumen Escherichia coli diarreogénica comprende un grupo heterogéneo de cepas que presentan diversos factores de virulencia y causan diferentes síndromes diarreicos. Los patotipos más estudiados son Escherichia coli enteropatogénica (EPEC), Escherichia coli enterotoxigé-nica (ETEC), Escherichia coli enteroagregativa (EAEC), Escherichia coli enteroinvasiva (EIEC) y Escherichia coli productora de toxina Shiga (STEC). El objetivo de este estudio fue estimar la frecuencia de infección de los diversos patotipos de E. coli diarreogénica en una población pediátrica ambulatoria con diarrea, atendida en el Hospital Sor María Ludovica de La Plata, Argentina, durante el período mayo-octubre de 2017. Los patotipos fueron detectados mediante la amplificación molecular de ocho genes de virulencia característicos. Se estudiaron las heces de 211 ninos (76% menores de 5 años). Se detectó infección con E. coli diarreogénica en el 12,3% (n = 26/211) de los niños con diarrea. Los patotipos identificados fueron EAEC, ETEC (todos lt positivos), EPEC y STEC (stx2 y eae positivos). El patotipo EAEC fue prevalente en todos los grupos etarios, mientras que los patotipos ETEC, EPEC y STEC solamente se observaron en niñnos menores de 5 anños. Este estudio constituye el primer reporte de detección por técnicas de amplificación molecular de Escherichia coli diarreogénica en una población pediátrica ambulatoria con diarrea, de la zona de La Plata. Se necesitan estudios más amplios que incluyan la caracterización de los aislamientos abarcando un mayor número de genes, controles asintomáticos, distintas épocas del ano y población de diversas áreas geográficas para esclarecer la relevancia de la infección por E. coli diarreogénica en niños de Argentina.

Abstract Diarrheagenic Escherichia coli is a heterogeneous group of strains that presents various virulence factors and causes different diarrheal syndromes. The most studied pat-hotypes are enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enteroaggregative Escherichia coli (EAEC), enteroinvasive Escherichia coli (EIEC) and Shiga toxin-producing Escherichia coli (STEC). The objective was to estimate the frequency of infec-tion of diarrheagenic E. coli pathotypes in children with diarrhea, attended at the Sor María Ludovica Hospital in La Plata, Argentina, during the period May-October 2017. E. coli pathotypes were detected by molecular amplification of eight characteristic virulence genes. The feces of 211 children (76% under 5 years) were studied. Infection with diarrheagenic E. coli was detected in 12.3% of the samples. The pathotypes were EAEC (10.43%), ETEC (1.42%, all of them positive for thermolabile toxin), EPEC (0.95%) and STEC (0.47%, positive for Shiga toxin 2). The EAEC pathotype was prevalent in children of all age groups, while ETEC, EPEC and STEC were only observed in children under 5 years of age. This study constitutes the first report of diarrheagenic Escherichia coli detection in an outpatient pediatric population with diarrhea from La Plata, using molecular amplification techniques. Broader future studies, including the charac-terization of the isolates with the largest number of genes, asymptomatic controls, different times of the year and population from different geographic areas will be necessary to clarify the relevance of diarrheagenic E. coli infection in children from Argentina.

Escherichia coli diarrogénicos, identificación de patotipos y fenotipos de resistencia antimicrobiana en aislados cubanos / Diarrheagenic Escherichia coli: identification of pathotypes and antimicrobial resistance phenotypes in Cuban isolates

Introducción: En los países en vías de desarrollo las enfermedades diarreicas agudas son causa frecuente de morbilidad y mortalidad. Entre las primeras causas se encuentra Escherichia coli diarrogénicos, que afecta a pacientes en edades extremas de la vida y con inmunodeficiencias. Objetivo: Identificar los patotipos de Escherichia coli diarrogénicos que más inciden y los fenotipos de resistencia antimicrobiana expresados por el patotipo predominante. Métodos: Se estudiaron 184 aislamientos procedentes de 15 centros provinciales de Higiene, Epidemiología y Microbiología de Cuba e Isla de la Juventud. La investigación se realizó desde julio de 2012 hasta febrero de 2014. La identificación de género, especie y patotipos fue realizada por métodos de diagnóstico convencional y molecular. La determinación de la susceptibilidad antimicrobiana se realizó por el método de Bauer y Kirby, y las normas del Clinical and Laboratory Institute Standards de 2013. Resultados: Se identificaron 108 (58 por ciento) Escherichia coli diarrogénicos. Los patotipos confirmados fueron: en la PCR múltiple 1, 5 (6 por ciento) de Escherichia coli enteropatogénico, 4 (4 por ciento) de enterotoxigénico y 0 (0 por ciento) de enterohemorrágico. La PCR múltiple 2 reveló 72 (82 por ciento) Escherichia coli enteroagregativo, que resultó el predominante en el estudio. La PCR 3 (simple) detectó 7 (8 por ciento) de enteroinvasivo. El 100 por ciento del patotipo predominante mostró resistencia, al menos a un antimicrobiano de los probados, un solo patron de resistencia a dos antimicrobianos, y nueve patrones de multirresistencia. Conclusiones: El estudio demuestra la importancia del uso de pruebas moleculares rápidas para la confirmación de los patotipos de E. coli diarrogénicos, los que provocan deshidratación ligera, complicaciones graves y la muerte. Se logra identificar los cuatro patotipos más frecuentes y E. coli enteroagregativo, el de mayor incidencia en la población estudiada. El patotipo predominante mostró altos porcentaje de resistencia antimicrobiana a betalactámicos y buena sensibilidad antimicrobiana a los aminoglucósidos y cefalosporinas de tercera generación. La investigación aporta conocimientos, no revelados en estudios anteriores con aislados cubanos, lo que es considerado de alto valor para los clínicos, pediatras y epidemiólogos del país(AU)

Introduction: Acute diarrheal disease is a frequent cause of morbidity and mortality in developing countries. One of the leading causes is diarrheagenic Escherichia coli, which affects patients at extreme ages and with immunodeficiencies. Objective: Identify the most active pathotypes of diarrheagenic Escherichia coli and the antimicrobial resistance phenotypes expressed by the prevailing pathotype. Methods: A study was conducted from July 2012 to February 2014 of 184 isolates obtained from 15 provincial Hygiene, Epidemiology and Microbiology Centers in Cuba and the Isle of Youth. Identification of the genus, species and pathotypes was based on conventional and molecular diagnostic methods. Determination of antimicrobial susceptibility was performed by the Bauer-Kirby method in compliance with guidelines from the Clinical and Laboratory Standards Institute of 2013. Results: A total 108 (58 percent) diarrheagenic Escherichia coli were identified. The following pathotypes were confirmed: Multiplex PCR 1 revealed 5 (6 percent) enteropathogenic, 4 (4 percent) enterotoxigenic and 0 (0 percent ) enterohemorrhagic Escherichia coli. Multiplex PCR 2 found 72 (82 percent) enteroaggregative Escherichia coli, which was the prevailing type in the study. PCR 3 (simple) detected 7 (8 percent) enteroinvasive Escherichia coli. 100 percent of the prevailing pathotype displayed resistance to at least one of the antimicrobials tested, a single resistance pattern to two antimicrobials, and nine multiresistance patterns. Conclusions: The study showed the importance of the use of rapid molecular tests to confirm diarrheagenic E. coli pathotypes, which cause mild dehydration, serious complications and death. Identification could be done of the four most common pathotypes and enteroaggregative E. coli, the one with the highest incidence in the study population. The prevailing pathotype displayed high percentages of antimicrobial resistance to beta-lactams and good antimicrobial sensitivity to third-generation cephalosporins and aminoglycosides. The study contributed knowledge not revealed by previous research about Cuban isolates. Such information is considered to be highly valuable for clinicians, pediatricians and epidemiologists in the country(AU)

Detection of Diarrheagenic Escherichia coli in Bovine Meat in the Northern Region of Paraná State, Brazil

Abstract Ground bovine meat is commonly consumed by the population of Brazil. However, it constitutes an excellent medium for the multiplication of microorganisms due to available nutrients and handling practices prior to consumption. Here, we examined 100 samples of ground beef for the presence of diarrheagenic Escherichia coli (DEC) pathotypes by PCR, and characterized isolates by analyzing their adherence to HEp-2 cells, serotype, antimicrobial susceptibility, and phylogeny. Enteroaggregative E. coli was detected in five (5%) meat samples, Shiga toxin-producing E. coli in three (3%), and atypical enteropathogenic E. coli in two (2%). According to the phylogeny, six isolates (60%) were classified in group A, two (20%) in group B1, and two (20%) in group E. The detected serotypes were O3:H2, O93:H9, O93:H46, O105ab:H7, O152:H8, O156:H10, and O175:H7. The antimicrobial susceptibility testing showed that one sample (10%) was resistant to ampicillin, two (20%) to sulfamethoxazole-trimethoprim, and two (20%) to cephalothin. Based on these results, bovine ground meat for human consumption can serve as a reservoir of DEC, which emphasizes the importance of appropriate hygienic-sanitary conditions during handling at every stage from slaughter to table.

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.

Identificação molecular de Escherichia coli diarreiogênica na Bacia Hidrográfica do Rio Xopotó na região do Alto Rio Doce / Molecular identification of diarrheagenic Escherichia coli in the watershed of Xopotó River, in Alto do Rio Doce, Brazil

RESUMO Esta pesquisa científica teve como principal objetivo identificar patótipos de Escherichia coli diarreiogênica nas águas superficiais da Bacia Hidrográfica do Rio Xopotó, na região do Alto Rio Doce, Minas Gerais. Os estudos referentes às estirpes diarreiogênicas de E. coli no meio ambiente no Brasil são escassos. A bacia hidrográfica escolhida para o estudo sofre intensa degradação ambiental devido ao lançamento de esgoto in natura em seus corpos d'água e às atividades antrópicas, como a agropecuária. As coletas de água nos 13 pontos amostrais foram realizadas em duas épocas do ano de 2015 (abril e julho). Para identificação dos genes de E. coli diarreiogênica, utilizou-se o método de reação em cadeia da polimerase (PCR). A bacia hidrográfica apresentou contaminação diarreiogênica de patótipos E. coli produtora de toxina Shiga (STEC), E. coli enteropatogênica (EPEC) e E. coli enterotoxigênica (ETEC), tal ocorrência foi constante em ambas as campanhas. Os genes de virulência observados foram: na STEC, toxina Shiga (Stx1), responsável por causar doenças renais graves, como a síndrome hemolítico-urêmica (SHU); já a EPEC apresentou somente o gene virulento eae, característico do subgrupo atípico (aEPEC); a ETEC apresentou toxinas termolábeis (LT). A presença desses patótipos representa potencial risco de doenças diarreiogênicas na população que utiliza os recursos hídricos, particularmente idosos e crianças, e evidencia o comprometimento da qualidade microbiológica dos cursos d'água constituintes da Bacia Hidrográfica do Rio Xopotó, decorrente principalmente da ausência de estações de tratamento de esgoto (ETEs).

ABSTRACT The main purpose of this scientific research was to identify pathotypes of diarrheogenic Escherichia coli in the watershed of Xopotó River, in Alto do Rio Doce, Minas Gerais region, Brazil. The studies referring to the stirps diarrheagenics of E. coli in the environment in Brazil are scarce. The watershed chosen for the study suffers intense environmental degradation due the release of raw sewage into their water bodies and human activities, such as agriculture. The water collecting at 13 sampling points were held in two periods of the year 2015 (April and July). For the identification of the genes of diarrheagenic E. coli, the method of reaction polymerase chain (PCR) was used. The watershed presented pathogenic contamination of pathotypes Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC) such a constant occurrence in both campaigns. The virulence genes observed were: STEC, Shiga toxin (Stx1), responsible for causing severe kidney disease as hemolytic-uremic syndrome (HUS). Besides that, EPEC presented only the virulent gene eae, characteristic of the atypical subgroup (aEPEC), as well as thermolabile toxins (LT). The presence of these pathotypes represents potential risk of diarrheagenic diseases in the population that uses the water resources, particularly the elderly and children, and demonstrates the commitment of the microbiological quality of constituents watercourses of the watershed of Xopotó River, mainly because the lack of sewage treatment facilities.

Monitoring of diarrheagenic Escherichia coli infections in patients in Guangdong Province during 2013 to 2016 / 中华微生物学和免疫学杂志

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 bio-chemical 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 en-teroinvasive 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 cefo-taxime. 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.

Placental and colostral transfer of antibodies reactive with enteropathogenic Escherichia coli intimins α, ß, or γ / Transferência placentária e colostral de anticorpos reativos a Escherichia coli enteropatogênica com expressão das intiminas α, ß, or γ

Abstract Objective: Intimins are protein adhesins of enteropathogenic Escherichia coli and enterohemorrhagic E. coli capable of inducing attachment and effacement lesions in enterocytes. Anti-intimin antibodies are important for the protection from enteropathogenic E. coli and enterohemorrhagic E. coli infections because these antibodies inhibit bacterial adhesion and impair the initial step of the pathogenesis. We studied the transfer of maternal anti-intimin antibodies from healthy Brazilian mothers to their newborns through the placenta and colostrum. Methods: Serum immunoglobulin G and secretory immunoglobulin A antibodies against conserved and variable regions of intimins α, β, and γ were analyzed using an enzyme linked-immunosorbent assay in the blood and colostrum from 45 healthy women as well as cord blood serum samples from their newborns. Results: The concentrations of antibodies reactive with α intimin were significantly lower than those of anti-γ and anti-conserved intimin antibodies in the colostrum samples. IgG serum antibodies reactive with all the subtypes of intimins were transferred to the newborns, but the concentrations of anti-conserved intimin serum antibodies were significantly higher in mothers and newborns than concentrations of antibodies against variable regions. The patterns of IgG transfer from mothers to newborns were similar for all anti-intimin antibodies. These values are similar to the percentage transference of total IgG. Conclusions: Anti-intimin antibodies are transferred from mothers to newborns through the placenta, and reinforce the protection provided by breastfeeding against diarrheagenic E. coli infections.

Resumo Objetivo: As intiminas são adesinas proteicas de Escherichia coli enteropatogênicas (EPEC) e enterro-hemorrágicas (EHEC) capazes de induzir as lesões attaching and effacing nos enterócitos. Anticorpos anti-intiminas são importantes para a proteção contra infecções por EPEC e EHEC porque esses anticorpos inibem a adesão bacteriana e impedem o passo inicial do mecanismo patogênico dessas bactérias. Nós estudamos a transferência de anticorpos maternos anti-intiminas de mães brasileiras saudáveis para os seus recém-nascidos através da placenta e do colostro. Métodos: Anticorpos séricos da classe IgG e secretórios da classe IgA (SIgA) reativos com as porções conservada (cons) e variáveis das intiminas α (vα), β (vβ) e γ (vγ) foram analisados pelo teste de ELISA no sangue e no colostro de 45 parturientes saudáveis e no sangue de cordão umbilical dos seus respectivos recém-nascidos. Resultados: As concentrações de anticorpos reativos com intimina vα foram significativamente mais baixas que as dos anticorpos anti-vγ e anti-cons nas amostras de colostro. Anticorpos IgG séricos reativos com todas as intiminas foram transferidos para os recém-nascidos, mas as concentrações de anti-cons foram significativamente mais altas tanto nas mães como nos recém-nascidos do que os anticorpos reativos com as regiões variáveis das intiminas. O padrão de transferência de IgG das mães para os recém-nascidos foi muito semelhante para todos os anticorpos anti-intiminas. Os valores de porcentagem de transferência foram semelhantes à transferência de IgG total. Conclusões: Anticorpos anti-intimina são transferidos das mães para os recém-nascidos pela placenta e corroboram a proteção contra infecções por Escherichia coli diarreiogênicas (DEC) conferida pelo aleitamento materno.

Detección molecular de Escherichia coli diarreogénica en pacientes pediátricos con síndrome diarreico agudo en Paraguay / Molecular detection of diarrheogenic Escherichia coli in pediatric patients with acute diarrheal syndrome in Paraguay

La Escherichia coli diarreogénica (ECD) se ha clasificado con base en criterios clínicos, epidemiológicos y moleculares en cinco grupos, cada uno con factores de virulencia específicos. El objetivo fue determinar la prevalencia de ECD en pacientes pediátricos con enfermedad diarreica aguda del Laboratorio Central de Salud Publica en el periodo 2012- 2015. Se procesaron muestras de heces con síndrome diarreico agudo, provenientes de pacientes pediátricos, en los cuales se buscó algún gen de virulencia ECD utilizando métodos convencionales de siembra y screening molecular, mediante PCR múltiple con cebadores diseñados específicamente para amplificar los genes de virulencia elt, est, eae, stx, ipaH y aggR. Del total de muestras analizadas, 13% (180/1379) de las muestras presentó algún factor de virulencia compatible con algún patotipo ECD con mayor predominio en niños de 1 a 3 años. La frecuencia de los distintos patotipos fue la siguiente: 61 (34%) ETEC, 40 (22%) EAEC, 41 (23%) EPEC, 27 (15%) EIEC, 7 (4%) STEC y 3 (2%) ETEC/EAEC, 1 (0.5%) ETEC/EAEC/EIEC. El porcentaje de E. coli diarreogénicas detectado tiene similitud con lo reportado en otros países de la región, lo que nos indica que estos patógenos son parte importante de la etiología de la enfermedad diarreica aguda infecciosa en la población infantil en nuestro país. Se debe destacar que para el diagnóstico de las diferentes categorías ECD, es necesario disponer de un procedimiento diagnóstico específico dirigido a la detección de los factores de virulencia utilizando métodos moleculares o métodos inmunológicos.

Diarrheagenic Escherichia coli (DEC) has been classified based on clinical, epidemiological and molecular criteria in five groups, each with specific virulence factors. The objective was to determine the prevalence of DEC in pediatric patients with acute diarrheal disease of the Central Laboratory of Public Health in the 2012-2015 period. A total of 1447 fecal samples of acute diarrheal syndrome from pediatric patients were processed in which a DEC virulence gene was searched using conventional screening and molecular screening methods with multiple PCR primers specifically designed to amplify virulence genes, st, lt, eae, stx, ipaH and aggR. From the total of analyzed samples, 13% (180/1379) of the samples presented some virulence factor compatible with a DEC pathogen type with greater predominance in children from 1 to 3 years. The frequency of the different pathogen types was as follows: 61 (34%) ETEC, 40 (22%) EAEC, 41 (23%) EPEC, 27 (15%) EIEC, 7 (4%) STEC and 3 (2% ETEC/EAEC, 1 (0.5%) ETEC/EAEC/EIEC. The percentage of DEC detected is similar to that reported in other countries of the region, which indicates that these pathogens are an important part of the etiology of acute infectious diarrheal disease in children in our country. It should be noted that for the diagnosis of different DEC categories, it is necessary to have a specific diagnostic procedure aimed at the detection of virulence factors using molecular methods or immunodiagnostic methods.

Perfil de susceptibilidade antimicrobiana e identificação de patotipos diarreiogênicos entre amostras de Escherichia coli isoladas de alimentos / Antimicrobial susceptibility profile and identification of pathotypes diarrheagenic between Escherichia coli strains isolated from food

O presente estudo objetivou avaliar o perfil de susceptibilidade antimicrobiana e a ocorrência de patotipos diarreiogênicos (E. coli entoropatogênica/EPEC, E. coli enterotoxigênica/ETEC, E. coli produtora de shiga toxina/STEC, E. coli enteroinvasiva/EIEC e E. coli enteroagregativa/EAEC) em amostras de Escherichia coli, isoladas de diversos alimentos destinados ao consumo humano e naqueles envolvidos em surtos de Doença de Transmissão Alimentar (DTA), em Minas Gerais, Brasil. O perfil de susceptibilidade antimicrobiana das amostras de E. coli foi avaliado por meio da técnica de Concentração Mínima Inibitória e os patotipos de E. coli diarreiogênica pela Reação em Cadeia de Polimerase, utilizando-se os marcadores de virulência eae, bfpA, eltB, estA, st1, stx1, stx2, ipaH e aatA. Dentre as 220 amostras de E. coli analisadas, 60 (27,3%) apresentaram-se resistentes ou com resistência intermediária. Entre estas, oito (13,3%) foram consideradas multirresistentes. Os fármacos amicacina e gentamicina foram os mais eficazes, diferentemente da ampicilina, que apresentou o maior percentual de amostras resistentes (10,9%). Não foram identificados os patotipos de EPEC, STEC, EIEC e EAEC. Duas amostras de E. coli (0,9%), isoladas de um salgado (pão de queijo) e de uma refeição mista (galinhada), foram classificadas como ETEC. Ambas albergavam os marcadores de virulência eltB, estA e st1. Estas foram caracterizadas como pertencentes sorotipos O9:H10 e O9:H33. Conclui-se que entre as amostras de E. coli, isoladas de alimentos destinados ao consumo humano em Minas Gerais e naqueles envolvidos em surtos de DTA, existem de amostras consideradas multirresistentes e diarreiogênicas.

This study aimed to evaluate the profile of antimicrobial susceptibility and the occurrence of diarrheagenic pathotypes (E. colientoropatogênic/EPEC, E. coli enterotoxigenic/ETEC, E. coli producing shiga toxin/STEC, E. coli enteroinvasive/EIEC and E. colienteroaggregative/EAEC) in Escherichia coli isolated from different foods intended for human consumption and those involved in foodborne disease outbreaks, in Minas Gerais, Brazil. The antimicrobial susceptibility of strains of E. coli was evaluated by the Minimum Inhibitory Concentration technique and pathotypes of E. coli diarrheagenic by polymerase chain reaction, using the virulence markers eae, bfpA, eltB, st1, stx1, stx2, ipaH and aatA. Among the 220 strains of E. coli analyzed, 60 (27.3%) were resistant or intermediate resistance. Among these, eight (13.3%) were considered multiresistant. The amikacin and gentamicin drugs were the most effective, unlike ampicillin, which had the highest percentage of resistant strains (10.9%). Pathotypes of EPEC, STEC, EIEC and EAEC were not identified. Two strains of E. coli (0.9%) isolated from a salty (cheese bread) and a mixed meal (chicken meal) were classified as ETEC. Both harbored the virulence markers eltB, estA and st1. These were characterized as serotypes O9:H10 and O9:H33. We conclude that among samples of E. coli, isolated from foods for human consumption in Minas Gerais and those involved in outbreaks of DTA, there are samples considered multiresistant and diarrheogenic.

Surveillance of diarrheagenic Escherichia coli infection in Yanping and Yongan, Fujian Province, China / 中国人兽共患病学报

We investigated the epidemiological characteristics of diarrheagenic Escherichia coli (E.coli) infection in Fujian Province,providing reference for the prevention and control of diarrhea epidemic and outbreak.Yanping and Yongan areas were selected as surveillance sites from year 2010 to 2015,where 1 950 samples were collected from the sentinel hospitals,and then samples were isolated and cultured on MacConkey agar plates.Suspected strains were identified by routine and molecular diag nosis technique methods.Results were analyzed by SPSS 17.0 software package.A total of 129 strains of diarrheagenic E.coli were isolated with a total detection rate of 6.62％.The detection rates of enteropathogenic E.coli(EPEC),enterotoxigenic E.coli(ETEC) and enteroadhesive E.coli(EAEC) were 3.33％,1.64％ and 1.64％ respectively (including 61 strains of atypical EPEC,4 strains of typical EPEC,and 32 strains of ETEC and EAEC for each),while EHEC and EIEC were not found.There was no significant difference between the detection rates of male and female.All the patients were divided into five groups according to the age,and there were no significant differences between the detection rates of EPEC,EAEC and ETEC in the 5 groups.The detection rates were the highest in August and September.There was no significant difference between the detection rates of rural area and uban area.There was also no significant difference between the composition ratio of diarrheagenic E.coli (DEC) in the two surveillance sites.In conclusion,there were three kinds of DEC in Fujian Province,and EPEC was the dominant.August and September were the months with the highest detection rates.Children age less than five and adult aged 20 59 years were the high risk groups with DEC infection.More attention should be attached on the rational treatment by antibiotic for DEC.

Flooding adds pathogenic Escherichia coli strains to the water sources in southern Khyber Pakhtunkhwa, Pakistan.

Purpose: Seasonal rains in Pakistan result in heavy floods across the country, whereby faecal contaminants will be added to the water bodies and cause numerous food‑borne outbreaks. The present study was aimed to determine the prevalence of diarrheagenic Escherichia coli (DEC) strains in the water sources. Materials and Methods: Two hundred water samples collected during (2011–2012) were processed for the isolation of E. coli (EC) strains. EC strains were further analysed for antibiotic susceptibility patterns, and pathogroups‑specific virulence factors stx1, stx2, stx2c, eae, tir, hlyA, bfpA, estA and eltA were detected using multiplex polymerase chain reaction. Results: Thirty‑three percent of the water samples were contaminated with EC pathotypes. Fifty percent (33/66) of the DEC pathotypes were identified as enterotoxigenic EC (ETEC). Seventy‑two percent (13/18) of the enteropathogenic EC (EPEC) strains were identified as typical EPEC and 28% (5/18) as atypical EPEC. Eleven percent (7/66) of the Shiga toxin EC (STEC) isolates carried a combination of stx1 and stx2 genes. Summer was found as a peak season with 47% (31/66) for EC pathogroups’ activities. Eighty‑nine percent of the strains showed resistance against tetracycline. Conclusion: ETEC and EPEC are the primary causes of water contamination in southern regions of Khyber Pakhtunkhwa province, Pakistan. Firm adherence to the prescribed drugs can decrease trends in antibiotic resistance.

Pathogenic detection of infectious diarrhea in patients in a district of Beijing from 2011 to 2013 / 中国感染控制杂志

Objective To analyze the distribution and pulsed-field gel electrophoresis (PFGE)of pathogenic bacteria causing infectious diarrhea in a district of Beijing from 2011 to 2013,and provide basis for tracing infection sources.Methods A total of 1 179 stool specimens of infectious diarrhea from patients in a diarrhea outpatient department from January 2011 to December 2013 were collected,all isolated pathogens were identified by serotyping and PFGE analysis.Results 330 enteric pathogens were isolated from 1 179 specimens,the top 4 bacteria were Shi-gella spp .(28.18%,n=93),Salmonella spp .(20.91 %,n=69),Vibrio parahaemolyticus (13.33%,n =44),and diarrheagenic Escherichia coli (3.33%,n = 11 ).18 Shigella sonnei isolates were identified as 8 PFGE patterns, clustering similarity was close to 88%;69 Salmonella spp .strains belonged to 18 serotypes and 41 PFGE patterns, Salmonella senftenberg and Salmonella enteritidis had dominant patterns;no dominant PFGE patterns were obviously identified among 23 strains ofVibrio parahaemolyticus .Conclusion The serotypes and PFGE patterns of pathogenic bacteria in infectious diarrhea in past three years showed a wide distribution characteristics,the dominant PFGE patterns of Salmonella spp .and Shigella spp .need to be paid more attention,and outbreak of infectious diarrhea caused by Salmonella spp .and Shigella spp .should be alerted.

Clinical and etiologic features of diarrheagenic Escherichia coli-associated diarrhea in children / 中华传染病杂志

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,amoxicillinclavulanate,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.

Study of a multiplex PCR method for the detection of three Diarrheagenic Escherichia coli / 国际检验医学杂志

Objective To establish a multiplex polymerase chain reaction (PCR) method for rapid detection of three kinds of di‐arrheagenic Escherichia coli(EPEC ,EIEC ,EHEC)simultaneously .Methods The eae gene of EPEC ,ipaH gene of EIEC and stx1 gene of EHEC were selected to design primers ;the reaction system and condition were adjusted to optimize the multiplex PCR sys‐tem .Results The target gene fragments were amplified correctly with these primers .The three target bacteria could be detected at the same time by multiplex PCR .Conclusion A rapid multiplex PCR system were successfully established for detection of three di‐arrheagenic Escherichia coli ,and this system could be suitable for rapid screening in food safety .

Study on the genotype and epidemic characteristics of diarrheagenic Escherichia coli isolated from diarrheal patients in Beijing / 中华流行病学杂志

Objective To understand the distribution of virulence gene and the epidemiological characteristics of diarrheagenic Escherichia(E.) coli (DEC) from diarrheal patients in Beijing. Methods Stool specimens from diarrheal patients were cultured which were collected from the hospitals under sentinel surveillance program,during 2012-2013. DNA was examined by real-time PCR. Results 253 out of 6 370 specimens were positive for DEC detection with the rate as 4.0%. A total number of 262 DEC strains were isolated. Two different pathotypes of DEC strains with mixed infection,were isolated from 9 specimens. Different pathotypes would show the following profiles:42.8% for enteropathogenic E. coli (EPEC) including 42.0% atypical and 0.8% typical;38.9% for enterotoxigenic E. coli(ETEC)including 24.8%st positive,9.9%lt positive and 4.2%st and lt both positive;15.3% for enteroaggregative E. coli(EAEC);2.7% for enteroinvasive E. coli(EIEC);one strain STEC with serotype O26 ∶ K60. ETEC had obvious characteristics on age. All kinds of DEC were isolated throughout the year with seasonal fluctuation. Conclusion DEC isolates from diarrheal patients in Beijing were dominated by EPEC and ETEC,with atypical ones accounted for the majority of EPEC. One specimen was found under mixed infection. Pathotypes DEC were found to have different age and seasonal distributions.

Study on the genotype and epidemic characteristics of diarrheagenic Escherichia coli isolated from diarrheal patients in Beijing / 中华流行病学杂志

Objective To understand the distribution of virulence gene and the epidemiological characteristics of diarrheagenic Escherichia(E.) coli (DEC) from diarrheal patients in Beijing. Methods Stool specimens from diarrheal patients were cultured which were collected from the hospitals under sentinel surveillance program,during 2012-2013. DNA was examined by real-time PCR. Results 253 out of 6 370 specimens were positive for DEC detection with the rate as 4.0%. A total number of 262 DEC strains were isolated. Two different pathotypes of DEC strains with mixed infection,were isolated from 9 specimens. Different pathotypes would show the following profiles:42.8% for enteropathogenic E. coli (EPEC) including 42.0% atypical and 0.8% typical;38.9% for enterotoxigenic E. coli(ETEC)including 24.8%st positive,9.9%lt positive and 4.2%st and lt both positive;15.3% for enteroaggregative E. coli(EAEC);2.7% for enteroinvasive E. coli(EIEC);one strain STEC with serotype O26 ∶ K60. ETEC had obvious characteristics on age. All kinds of DEC were isolated throughout the year with seasonal fluctuation. Conclusion DEC isolates from diarrheal patients in Beijing were dominated by EPEC and ETEC,with atypical ones accounted for the majority of EPEC. One specimen was found under mixed infection. Pathotypes DEC were found to have different age and seasonal distributions.

Detection of 13 Enteric Bacteria and 5 Viruses Causing Acute Infectious Diarrhea Using Multiplex PCR from Direct Stool Specimens / 대한임상미생물학회지

BACKGROUND: We investigated the prevalence of various pathogens (13 enteric bacteria and 5 viruses) which cause diarrhea using multiplex PCR of stool specimens and compared two multiplex PCR methods for detecting diarrheagenic Escherichia coli. METHODS: A total of 405 stool specimens submitted between November 2010 to February 2011 for routine culture of enteric pathogens were included and screened for five viruses (astrovirus, Group A rotavirus, enteric adenovirus, norovirus G1/G2) and eight bacteria (Salmonella spp., Shigella spp., Campylobacter spp., Vibrio spp., C. difficile Toxin B, C. perfringens, Y. enterolytica, Aeromonas spp.) using the Seeplex(R) Diarrhea ACE detection kit (Seegene). In addition, virulence-associated genes of enteropathogenic E. coli, (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli, (EIEC), enterotoxigenic E. coli (ETEC), and enteroaggressive E. coli (EAEC) were detected using 16-plex PCR and a commercial diarrheagenic E. coli detection (DEC) PCR kit (SSI Diagnostica). RESULTS: Overall, 138 (34.1%) of 405 samples was positive for pathogen. The positive rate for virus was 18.5%. norovirus G2, Group A rotavirus, enteric adenovirus, astrovirus and norovirus G1 were detected in 40, 23, 8, 3 and 1 samples, respectively. The positive rate for bacteria was 24.4% (99/405). C. difficile toxin B was the most frequently detected, followed by C. perfringens, EPEC, and EAEC. The agreements of the two multiplex PCR methods for detecting EPEC and EHEC were 99.3% and 100%, respectively. CONCLUSION: The detection rate was high (34.1%) including various diarrheagenic E. coli (6.2%) and C. perfringens (5.2%). Multiplex PCR is thus useful for detecting various pathogens.

Escherichia coli diarreogénica asociada a casos de diarrea aguda en niños de Cumaná, Venezuela / Diarrheagenic Escherichia coli associated with acute diarrhea in children of Cumana, Venezuela

Para establecer la prevalencia de cepas de E. coli diarreogénicas (ECD) asociadas a casos de diarrea aguda infantil en Cumaná, Venezuela; se tomaron muestras de heces de 200 niños con enfermedad diarreica aguda, menores de 5 años, y de 30 niños sanos incluidos como control. El aislamiento e identificación bacteriana se realizó por coprocultivos y pruebas bioquímicas convencionales. Para determinar la presencia de los genes de patogenicidad de cada tipo de ECD se usó la técnica de reacción en cadena de la polimerasa (PCR), determinando los genes eae y bfp (ECEP), st y lt (ECET), ipaH y virF (ECEI), Stx1/Stx2 (ECST), aafII (ECEA) y daaE (ECAD). Se realizaron 169 aislamientos de E. coli, de las cuales se determinó por PCR que el 10,65% fueron positivas para ECEP (1,18% “típicas”; 9,47% “atípicas”); ECET (5,91%); ECEA (1,78%); ECEI (0,59%). No se observaron diferencias estadísticas significativas en cuanto a la frecuencia de cada “patotipo” en relación a la edad, pero si en relación con el sexo (p< 0,05). Las características clínicas más relevantes fueron: fiebre, vómito y dolor abdominal y el mayor porcentaje de los niños afectados estuvo en las clases obrera y marginal. Estos resultados muestran que las cepas de ECD, son agentes etiológicos importantes en la enfermedad diarreica aguda infecciosa en la población infantil de Cumaná.

To establish the prevalence of strains of diarrheagenic Escherichia coli (DEC) associated to acute diarrhea in childrem of Cumaná, Venezuela, stool samples were taken from 200 children aged < 5 years with acute diarrheal disease, and from 30 healthy children used as control. Isolation and bacterial identification was performed by conventional biochemical tests and stool cultures. The presence of pathogenic genes of each type of DEC was investigated by the technique of polymerase chain reaction (PCR), determining the genes eae and bfp (EPEC), st and lt (ETEC), ipaH and virF (EIEC) Stx1/Stx2 (STEC), aafII (EAEC) and daaE (ADEC). From 169 E. coli, isolates we determined by PCR 10.65% positive for EPEC (1.18% “typical”, 9.47% “atypical”); ETEC (5.91%); EAEC (1.78 %), EIEC (0.59%). There were no statistically significant differences regarding the frequency of each “pathotype” in relation to age, but it did occur when related to the sex (p<0.05). The most relevant clinical features were: fever, vomiting and abdominal pain and the greatest percentage of children affected were of the working and marginal classes. These results shown that the strains of DEC are important etiological agents in acute infectious diarrhea in children of Cumaná.

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4 percent (259 patients) and 18.7 percent (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7 percent), followed by atypical EPEC (9.4 percent), ETEC (3.7 percent), and STEC (0.6 percent). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.