Aislamiento de Escherichia coli productora de toxina Shiga (STEC) en heces de ganado y detección de factores de virulencia asociados con su patogénesis / Isolation of Shiga toxin-producing Escherichia coli (STEC) in cattle stool and detection of virulence factors associated with its pathogenesis

Resumen Objetivo: Aislar STEC en las heces del ganado bovino en el municipio de Ulloa, Valle del Cauca y detectar factores de virulencia asociados con la patogénesis. Materiales y métodos: Se tomaron 21 muestras provenientes de bovinos, las cuales fueron tomadas directamente del recto del animal mediante hisopos. Las muestras se procesaron hasta obtener colonias puras a las cuales se les evaluó la presencia de los genes stx1, stx2, eae, saa y hlyA mediante PCR y posteriormente, se evaluó el efecto citotóxico de las muestras positivas sobre células Vero (ATCC-CCL-81.4). Resultados: De las 21 muestras de heces de bovinos,12 presentaron bacterias con uno o ambos genes stx. Se obtuvieron 106 aislamientos totales de STEC y se observaron diferencias en cuanto a la presencia y ausencia de los genes de virulencia evaluados en los aislamientos de cada bovino, obteniendo cinco combinaciones de genes. 48 aislamientos presentaron únicamente el gen stx2 y 58 presentaron tanto el gen stx1 como el gen stx2; de los 106 aislamientos, se detectaron 44 con el gen hlyA y 57 con el gen saa. Conclusiones: Todos los sobrenadantes de STEC analizados mostraron actividad citotóxica sobre las células Vero, mientras que en ausencia de STEC las células formaron monocapa después de 48 h de incubación. Este trabajo es el primer reporte en Colombia que aporta información sobre la presencia de STEC en el ganado bovino, la presencia de factores de virulencia y el potencial efecto citotóxico que poseen estas cepas nativas.

Abstract Objective: To isolate STEC in stool samples from cattle in Ulloa, Valle del Cauca, and to detect virulence factors associated with its pathogenesis. Materials and methods: We took 21 samples from cattle, which were taken directly from the rectum of the animal using swabs. The samples were processed until obtaining pure colonies and evaluated for the presence of the stx1, stx2, eae, saa and hlyA genes by PCR. Afterward, the cytotoxic effect of positive samples were evaluated on Vero cells (ATCC-CCL- 81.4). Results: We observed that from the 21 stools samples, 12 presented bacteria with one or both stx genes. A total of 106 isolates of STEC were obtained and differences among each other were observed regarding the presence and absence of the virulence genes, obtaining five combinations of genes. We found that 48 isolates presented only stx2 gene and 58 presented both the stx1 and stx2 gene. Regarding the other virulence genes, the hlyA gene was detected in 44 isolates and the saa gene was detected in 57 isolates. Conclusions: All the STEC supernatants showed cytotoxic activity on Vero cells, while in its absence the cells formed monolayer after 48 h of incubation. This work is the first report in Colombia that provides information about the presence of STEC in stool cattle, virulence genes and its potential cytotoxic effect in native strains.

Síndrome hemolítico urémico en una mujer adulta mayor: reporte de caso y revisión de la literatura / Hemolytic uremic syndrome in an older adult woman: case report and literature review

El síndrome hemolítico urémico (SHU) típico en adultos es una patología infrecuente. En la literatura se encuentran pocos reportes, y se ha documentado principalmente en la población pediátrica. Esta entidad se caracteriza por ser una microangiopatía trombótica (MAT) que compromete de manera característica los riñones. Es causada usualmente por la infección por Escherichia coli productora de toxina Shiga (STEC), específicamente el serotipo O157:H7. En Colombia no existen casos reportados sobre esta condición en adultos, lo cual llama la atención, pero puede deberse en parte a las dificultades en su diagnóstico, al no tenerse fácil acceso a algunas de las pruebas que orientan hacia esta enfermedad y confirman el diagnóstico. Se reporta el caso de una mujer adulta mayor colombiana, quien consultó por deposiciones diarreicas y hematoquecia, con el posterior desarrollo de trombocitopenia severa, lesión renal aguda, y evidencia de equinocitos y esquistocitos en extendido de sangre periférica, lo que llevó a sospechar una MAT. Se le solicitó FilmArray® gastrointestinal, el cual fue positivo para STEC, confirmando así el diagnóstico de un SHU típico. Se presenta también una breve revisión del tema de una entidad que requiere un diagnóstico temprano y certero que permita brindar un tratamiento eficaz y oportuno

The classic or typical hemolytic uremic syndrome (HUS) in adults is a rare disease. Few reports are found in the literature, and it has mainly been documented in the pediatric population. This condition is a form of thrombotic microangiopathy (TMA), which characteristically compromises the kidneys. It is mainly caused by infection with Shiga toxin-producing Escherichia coli (STEC), specifically the O157:H7 serotype. In Colombia there are no reports on this condition in adults, and may be due in part to difficulties in its diagnosis, as there is not easy access to some of the tests that guide towards this condition and confirm the diagnosis. The case of an elderly Colombian woman is reported, who presented diarrhea and hematochezia, and subsequently developed severe thrombocytopenia and acute kidney injury, with evidence of echinocytes and schistocytes in peripheral blood smears, which led to suspect TMA. A gastrointestinal FilmArray™ was ordered, which was positive for STEC, thus confirming the diagnosis of a typical HUS. A brief literature review is also presented, which covers general concepts of a condition that requires an early and accurate diagnosis in order to provide an effective and timely treatment

Infecciones por Escherichia coli productora de toxina Shiga O121: H19 en pacientes atendidos en Mar del Plata / Infections caused by Escherichia coli producing Shiga O121: H19 toxin in patients treated in Mar del Plata

Escherichia coli productora de toxina Shiga (STEC) O157:H7 es el serotipo más frecuentemente identificado como agente causal de colitis hemorrágica y síndrome urémico hemolítico (SUH), aunque se han descripto más de 100 serotipos con potencial patogénico similar. El objetivo del trabajo fue describir casos de enfermedad humana asociados a la infección por STEC O121:H19, atendidos en la ciudad de Mar del Plata y establecer la relación genética de los aislamientos mediante técnicas de epidemiología molecular. Se observó un amplio espectro en la severidad clínica de los ocho casos estudiados: dos fueron asintomáticos (contactos de SUH), un paciente tuvo diarrea sanguinolenta, y cinco presentaron SUH. Uno de los pacientes con SUH falleció. Las cepas O121:H19 portadoras del genotipo stx2a/eae/ehxA fueron sensibles a los antibióticos ensayados y presentaron por electroforesis en gel de campo pulsado (Xbal-PFGE) distintos patrones de macrorrestricción, con similitud del 84,25%. El patrón AREXKX01.0072, detectado en un SUH y en su contacto, es nuevo en la Base de Datos Nacional de STEC no-O157 de la Argentina. La utilización de métodos estandarizados de detección y tipificación de STEC permite a los laboratorios de referencia monitorear la frecuencia temporal y la distribución geográfica de las cepas circulantes para la prevención y control de estos patógenos asociados a enfermedad humana.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is the most frequent serotype identified as causative agent of sporadic cases and outbreaks of diarrhea with or without blood, hemorrhagic colitis and hemolytic uremic syndrome (HUS), although more than 100 serotypes have been described of similar pathogenic potential. The aim of the study was to describe cases of human disease associated with STEC O121:H19 infections, assisted in Mar del Plata City, and to establish the genetic relationship of the isolates by molecular epidemiology techniques. A wide spectrum was observed in the clinical severity of the eight cases studied: two were asymptomatic (contacts of HUS), one patient had bloody diarrhea, and five cases presented HUS. One HUS case died. All STEC O121:H19 strains carried the stx2a/eae/ehxA genotype, were sensitive to all antibiotics tested and showed different macrorestriction patterns by pulsed-field gel electrophoresis (Xbal-PFGE), with 84.25% similarity. The pattern AREXKX01.0072, detected in a HUS case and in his contact, is new in the Argentine National Database of non-O157 STEC. The use of standardized methods for detection and typing of STEC allows reference laboratories to monitor the temporal frequency and geographical distribution of circulating strains for the prevention and control of these pathogens associated with human diseases.

Escherichia coli produtora de toxina Shiga (STEC) O157:H7 é o sorotipo mais frequentemente identificado como o agente causador de colite hemorrágica e síndrome hemolítica urêmica (SHU), embora tenham sido descritas mais de 100 sorotipos com potencial patogênico semelhantes. O objectivo foi o de descrever os casos de doença humana associadas com a infecção por STEC O121:H19, assistido, na cidade de Mar del Plata e estabelecer relação genética de isolados utilizando epidemiologia molecular. Um amplo espectro foi observado na severidade clínica dos oito casos estudados, dois eram assintomáticos (contacto SHU), uma paciente teve diarreia com sangue, e cinco tiveram SHU. Um caso de SHU faleceu. As cepas O121:H19 portaram o genótipo stx2a/eae/ehxA, foram sensíveis aos antibióticos testados e apresentaram, por eletroforese em gel de campo pulsado (Xbal-PFGE), diferentes padrões de macrorestrição, com similaridade de 84,25%. O padrão AREXKX01.0072 detectado em SHU e em seu contato, é novo para a Base de Dados Nacional de STEC não-O157 na Argentina. O uso de métodos padrão de detecção e tipagem de STEC permite os laboratórios de referência monitorar frequência temporal e distribuição geográfica de estirpes circulantes para a prevenção e controlo destes agentes patogénicos associados com a doença humana.

Molecular epidemiology of sequence type 33 of Shiga toxin-producing Escherichia coli O91:H14 isolates from human patients and retail meats in Korea

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.

Eschechiria coli produtora da toxina shiga em bovinos: revisão / Eschechiria coli producing shiga toxin in cattle: review

Esta revisão apresenta os aspectos epidemiológicos da bactéria Escherichia coli produtora da toxina shiga, (STEC) que é reconhecida no mundo todo como agente causador de toxinfecção. Os produtos de origem animal são frequentemente associados aos surtos da doença, mas os alimentos hortifrutigranjeiros também podem ser contaminados e causar toxinfecção em seus consumidores. A incidência da infecção em humanos no Brasil (STEC) é baixa, foi observada essa prevalência nos rebanhos bovinos, ovinos e aves. Por isso, a importância das boas práticas de manipulação do produtor e do consumidor para evitar possível contaminação dos alimentos. Este trabalho teve por objetivo revisar a literatura sobre a ocorrência de Escherichia coli produtora da toxina shiga. Frente aos limitados estudos realizados sobre os agentes etiológicos, novos estudos fazem-se necessários.

This review presents the aspects of shiga toxin-producing Escherichia coli, which are recognized worldwide as toxinfection agents in the world. Products of animal origin are often associated with outbreaks of disease, but horticultural crops can be contaminated and cause toxinfection as well. This study aims to review the literature on the occurrence of shiga toxin-producing Escherichia coli. The incidence of infection in humans in Brasil by STEC and low, prevalence was observed in cattle, sheep and poultry. Therefore, the importance of good practices of manipulation of the producer and the consumer to avoid possible food contamination. Faced with the limited studies of etiological agents, it is necessary to study the subject.

Isolated abducens nerve palsy treated by immunoadsorption in a patient with diarrhea-associated hemolytic uremic syndrome / Parálisis ocular aislada del sexto par craneal tratada con inmunoadsorción en un paciente con síndrome urémico hemolítico asociado a diarrea

Shiga toxin-producing Escherichia coli (STEC) that causes a prodromal hemorrhagic enteritis is the main cause of hemolytic uremic syndrome (HUS) particularly in pediatric patients. It is characterized by acute kidney injury with microangiopathic hemolytic anemia and thrombocytopenia. The kidney and brain are the two major target organs, and neurological involvement is the most frequent cause of mortality. The time delay between bloody diarrhea and neurological symptoms ranges from few days to a month. Neurological disorders include disturbances in cognitive functions, focal neurological signs, epileptic seizures, myoclonus and neuropsychiatric symptoms. Cerebral magnetic resonance imaging reveals various patterns of hyperintensities distributed through cerebral matter or may be totally normal even the patient has severe neurological involvement. Electroencephalography usually show generalized or focal slowing of the background activity, spikes or sharp waves despite being normal in around 20% of patients. We present here an adult male patient referred to our center with requirement of hemodialysis due to diarrhea-associated HUS complicated by acute kidney injury. Later during the course of plasma exchange therapy the patient developed an isolated abducens nerve palsy. Complete renal recovery was achieved by plasma exchange therapy but abducens palsy remedied rescue introduction of immunoglobulin G (IgG) depletion by immunoadsorption

Escherichia coli, productor de toxina Shiga (STEC), que causa una enteritis hemorrágica en fase prodrómica, es la principal causa del síndrome urémico hemolítico (SUH), particularmente, en pacientes pediátricos. Se caracteriza por una lesión renal aguda con anemia hemolítica microangiopática y trombocitopenia. El riñón y el cerebro son los dos órganos principales a los que ataca, y la afectación neurológica es la causa más frecuente de mortalidad. El tiempo que transcurre entre la aparición de diarrea sanguinolenta y los síntomas neurológicos varía entre pocos días y un mes. Los trastornos neurológicos incluyen alteraciones en las funciones cognitivas, signos neurológicos focales, ataques epilépticos, mioclonías y síntomas neuropsiquiátricos. La resonancia magnética de cerebro revela varios patrones de hiperintensidades distribuidas a través de la materia cerebral o puede ser totalmente normal incluso si el paciente tiene un compromiso neurológico severo. El electroencefalograma generalmente muestra una disminución generalizada o focal de la actividad de fondo, picos u ondas agudas, a pesar de ser normal en alrededor del 20% de los pacientes. Presentamos un paciente adulto de sexo masculino, derivado a nuestro centro para ser tratado con hemodiálisis debido a SUH asociado a diarrea, complicado por insuficiencia renal aguda. Luego, durante el transcurso de la terapia de intercambio de plasma, el paciente desarrolló una parálisis ocular aislada del sexto par craneal. Se logró una recuperación renal completa por medio de la terapia de intercambio plasmático; no obstante, la parálisis del nervio motor ocular externo remedió la disminución de la inmunoglobulina G (IgG) mediante el tratamiento de rescate de inmunoadsorción

Pathogenic and phylogenetic characteristics of non-O157 Shiga toxin-producing Escherichia coli isolates from retail meats in South Korea

Herein, we report the pathogenic and phylogenetic characteristics of seven Shiga toxin (Stx)-producing Escherichia coli (STEC) isolates from 434 retail meats collected in Korea during 2006 to 2012. The experimental analyses revealed that all isolates (i) were identified as non-O157 STEC, including O91:H14 (3 isolates), O121:H10 (2 isolates), O91:H21 (1 isolate), and O18:H20 (1 isolate), (ii) carried diverse Stx subtype genes (stx₁, stx(2c), stx(2e), or stx₁ + stx(2b)) whose expression levels varied strain by strain, and (iii) lacked the locus of enterocyte effacement (LEE) pathogenicity island, a major virulence factor of STEC, but they possessed one or more alternative virulence genes encoding cytotoxins (Cdt and SubAB) and/or adhesins (Saa, Iha, and EcpA). Notably, a significant heterogeneity in glutamate-induced acid resistance was observed among the STEC isolates (p < 0.05). In addition, phylogenetic analyses demonstrated that all three STEC O91:H14 isolates were categorized into sequence type (ST) 33, of which two beef isolates were identical in their pulsotypes. Similar results were observed with two O121:H10 pork isolates (ST641; 88.2% similarity). Interestingly, 96.0% of the 100 human STEC isolates collected in Korea during 2003 to 2014 were serotyped as O91:H14, and the ST33 lineage was confirmed in approximately 72.2% (13/18 isolates) of human STEC O91:H14 isolates from diarrheal patients.

Piglet colibacillosis diagnosis based on multiplex polymerase chain reaction and immunohistochemistry of paraffin-embedded tissues

Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in pigs, referred to as colibacillosis. The aim of this study was to optimize multiplex polymerase chain reaction (PCR) and immunohistochemistry (IHC) analyses of paraffin-embedded material to detect pathogenic E. coli strains causing colibacillosis in pigs. Multiplex PCR was optimized for fimbriae (F18, F4, F6, F5, and F41) and toxins (types A and B heat-stable toxins [STaP and STb], heat-labile toxin [LT], and type 2 Shiga toxin [ST(x2e)]), and IHC was optimized for an anti-E. coli polyclonal antibody. Samples (132) from pigs received between 2006 and 2014 with clinical and histopathological diagnoses of colibacillosis were analyzed. E. coli was detected by IHC in 78.7%, and at least one virulence factor gene was detected in 71.2%. Pathogenic strains of ETEC with at least one fimbria and one toxin were detected in 40% of the samples in multiplex PCR. The most frequent virulence types were F18-STaP (7.5%), F18-STaP-STb (5.7%), and F4-STaP (3.8%). A statistically significant association was noted between virulence factors F4, F18, STaP, and STb and positive immunostaining results. Colibacillosis diagnosis through multiplex PCR and IHC of paraffin-embedded tissues is a practical approach, as samples can be fixed and stored for long periods before analysis.

Serotipos, perfiles de virulencia y subtipos de stx en Escherichia coli productor de toxina Shiga aislados de productos de pollo / Serotypes, virulence profiles and stx subtypes of Shigatoxigenic Escherichia coli isolated from chicken derived products

Shigatoxigenic Escherichia coli (STEC) is a foodborne pathogen that causes hemolytic uremic syndrome (HUS) and the consumption of chicken products has been related to some HUS cases. We performed a non-selective isolation and characterization of STEC strains from retail chicken products. STEC isolates were characterized according to the presence of stx1, stx2, eae, saa and ehxA; stx subtypes and serotypes. Most of them carried stx2, showing subtypes associated with severe human disease. Although reported in other avian species, the stx2f subtype was not detected. The isolates corresponded to different serotypes and some of them, such as O22:H8, O113:H21, O130:H11, O171:H2 and O178:H19, have also been identified among STEC isolated from patients suffering from diarrhea, hemorrhagic colitis, HUS, as well as from cattle. Considering the virulence profiles and serotypes identified, our results indicate that raw chicken products, especially hamburgers sold at butcheries, can be vehicles for high-risk STEC strains.

Escherichia coli productor de toxina de Shiga (STEC) es un patógeno transmitido por alimentos que causa el síndrome urémico hemolítico (SUH). Algunos casos de SUH están relacionados con el consumo de productos de pollo. Se realizó el aislamiento no selectivo y la caracterización de cepas STEC provenientes de productos de pollo atendiendo a la presencia de stx1, stx2, eae, saa y ehxA, subtipos de stx y serotipos. La mayoría de los aislamientos portaba stx2 y subtipos de stx asociados con enfermedades graves en humanos. Aunque se ha detectado en otras especies aviares, el subtipo stx2f no se encontró. Se detectaron diferentes serotipos, entre ellos O22:H8, O113:H21, O130:H11, O171:H2 y O178:H19, también identificados como STEC aislados de pacientes con diarrea, colitis hemorrágica y SUH, y de ganado bovino. Teniendo en cuenta los perfiles de virulencia y los serotipos identificados, nuestros resultados indican que los productos de pollo crudos, especialmente las hamburguesas que se venden en las carnicerías, pueden ser vehículos de cepas STEC de alto riesgo.

Isolation of atypical enteropathogenic and shiga toxin encoding Escherichia coli strains from poultry in Tehran, Iran

Aim: The purpose of this study was to investigate the prevalence of enteropathogenic Escherichia coli [EPEC] and shiga toxin producing E. coli [STEC] strains in healthy broilers in Iran

Background: STEC and EPEC strains as diarrheagenic E. coli are among the most prevalent causative agents in acute diarrhea. Domestic animals, mainly cattle and sheep, have been implicated as the principal reservoirs of these pathotypes; however their prevalence among the broilers is varied among different countries

Patients and methods: A total of 500 cloacal swab samples from broilers of five different poultry houses [A-E] were collected to investigate the presence of stx1, stx2, hly, eae, and bfp virulence genes among the E. coli isolates by polymerase chain reaction. The shiga toxin encoding strains were evaluated serologically to detect their interaction with a commercial antiserum against O157 antigen

Results: Out of the 500 collected samples, 444 E. coli strains were isolated. Three strains [0.67%] presented at least one of the studied virulence genes [stx2, hly and eae], two strains were identified as STEC [stx2+, O157:nonH7] and one as an atypical EPEC strains [eae[+]bfp[-]]

Conclusion: The study established the presence of STEC and atypical EPEC in healthy broilers in Iran. Poultry might serve as vectors for transmission of pathogenic E. coli to human populations

Formação de biofilmes de escherichia coli produtora de toxina Shiga sorotipos O153: H25, O113:H21 e O111: H8 em superfície de aço inoxidável e eficácia de sanitizante / Biofilm formation of shiga toxin-producing escherichia coli O153: H25, O113:H21 and O111: H8 on the stainless steel surface and the effectiveness of sanitizer

Neste estudo as cepas de Escherichia coli produtora de toxina Shiga (STEC) O153:H25, O113:H21 e O111:H8, isoladas de rebanhos do país, foram avaliadas quanto à capacidade de formar biofilmes em superfície de aço inoxidável utilizada na indústria de alimentos, bem como a eficácia de diferentes concentrações de hipoclorito de sódio na inativação desses biofilmes. A capacidade de formação de biofilme foi detectada em todas as cepas de E. coli produtoras de toxina Shiga. Na avaliação da eficáciado sanitizante hipoclorito de sódio, nas concentrações de 100 mg.L-1 e 200 mg.L-1, observou-se a redução a <1 log UFC/cm2 em todas as cepas e nos tempos avaliados. Estes dados reforçam aimportância do correto procedimento de higienização, uma vez que biofilmes podem tornar-se importantes fontes de contaminação no ambiente de produção de alimentos.

This study aimed at evaluating the biofilm formation of Shiga toxin-producing Escherichia coli O153:H25, O113:H21 and O111:H8 (STEC non-O157), isolated from Brazilian cattle, on the stainless steel surface, and also the efficacy of different s concentrations of sodium hypochlorite for inactivating these biofilms. The ability to form biofilm was demonstrated in all of Shiga toxin-producing E. coli strains. In assessing the effectiveness of sodium hypochlorite sanitizer, a reduction to <1 log CFU/cm2 was observed in all of the evaluated strains and times. These data strengthen the relevance of the correct cleaning procedure, considering that biofilms formations might be an important source of food contamination.

[Prevalence of Shiga toxin-producing genes in Escherichia coli isolated from patients with urinary tract infections in Khorramabad]

Background and Objectives: Shiga toxins [verotoxin] is one of the most important bacterial toxin produced by the Escherichia coli [O157: H7 and nonO157: H7], and is responsible for various diseases in humans and animals. Hemolytic uremic syndrome [HUS] is serious human diseases that occur because of these toxins. There is no comprehensive information in this regard, here will studied the prevalence of these genes among the E- coli isolated from urine samples in Khorramabad

Material and Method: The study is based on 146 Escherichia coli isolates of Patients with symptoms of urinary tract infection referred to clinical laboratories in Khorramabad during 90-1389. We used we identified the isolates by biochemical tests .The Multiplex PCR Multiplex PCR method were used to presence of genes

Results: A total of 117 [13/80%] of the women and 29 [86/19] of the men and none of them carried both stx1and stx2

Conclusion: Shiga toxin-producing E. coli isn't responsible for urinary tract infection in KhorramabadAlthough the frequency of these genes in urine samples is low, but evaluation of the other human and animal samples in different parts of the country is essential

Comparative possession of Shiga toxin, intimin, enterohaemolysin and major extended spectrum beta lactamase [ESBL] genes in Escherichia coli isolated from backyard and farmed poultry

The present work was conducted to compare the occurrence of Escherichia coli possessing virulence and ESBL genes in backyard and farmed poultry. Three hundred and sixty samples from the poultry kept in backyard system and 120 samples from the farmed birds were collected from West Bengal, India. Among the E. coli isolates of backyard poultry [O2, O10, O25, O55, O60, O106, UT], none of them possessed any of the Shiga toxin genes and eight E. coli isolates [8/272; 2.9%] harboured eaeA gene alone. Whereas among the E. coli isolated from the farmed poultry [O17, O20, O22, O102, O114, O119, rough, UT], four isolates [4/78, 5.1%] harboured stx[1]/stx[2] gene and 11 isolates [11/78, 14.1%] possessed eaeA gene. None of the E. coli isolates from the backyard poultry harboured any studied ESBL gene. Whereas 29.4% of E. coli isolates from the farmed poultry were found to possess the ESBL genes

Excretion product of shigella dysenteriae (sdyep) induced cell death in early larval stage of zebrafish (danio rerio): acridine orange and ethidium bromide (AO/EB) in vivo staining / Producto de excreción de shigella dysenteriae (pesdy) induce muerte celular en larvas de pez cebra (danio rerio): marcaje in vivo con naranja de acridina y bromuro de etidio (NA/BE)

In the field of studies of acute toxicity induced by bacterial agents, Shiga toxins have been relevant due to the severity of the extra-intestinal diseases they cause. Numerous studies have shown that Shiga toxin induced apoptosis in different cell types; however, this important process has been little studied in vivo experimental models. In this study, the effects of excretion products of Shigella dysenteriae, in which Shiga toxin is present, were investigated on early larval stages of Zebrafish, an animal model with many advantages over other in vivo experimental models traditionally used. Both the collection of eggs and larvae of Zebrafish, and the product from excretion from Shigella dysenteriae (SdyEP) were performed according to laboratory standards. Also, toxicity bioassay, larvae treatment with pure and diluted solution, 10-1, 10-2, 10-3 , 10-4 and 10-5, v/v SdyEP and cell death in vivo using Acridine Orange (AO) and Ethidium Bromide (EB) were applied. The excretion product of Shigella dysenteriae (SdyEP) effect was expressed in terms of larval mortality and dependent dilution rather than incubation time. The larval population surviving treatment with Shigella excretion product presents severe morphological effects. The larval population generally presents notable severe morphological damage, the necrosis state is represented by the opacity of the larvae after being treated for 24 h (b) compared to control. Other changes associated with larval anatomy were also observed; particularly the caudal end curvature was significant into 10%. The use of AO/EB revealed a distribution pattern from fluorescence into green and orange in surviving larvae SdyEP poisoning, there was a large population of dead cells around the anal and caudal region as evidenced by the presence of orange nuclei in greater numbers as controls in the larvae. The results support the application of coloring AO/EB in Zebrafish experimental models for the evaluation of the toxic action of new molecules and new products with therapeutic potential.

En el campo de los estudios de toxicidad aguda inducida por agentes bacterianos, las toxinas Shiga resultan relevantes debido a la severidad de las enfermedades extra-intestinales que causan. Numerosos estudios han demostrado que la toxina Shiga induce la apoptosis en diferentes tipos de células, sin embargo, este importante proceso ha sido poco estudiado en modelos experimentales in vivo. En este estudio, fueron evaluados los efectos del productos de excreción de Shigella dysenteriae (PESdy), sobre estadios larvarios de pez cebra (Danio rerio), un modelo animal con muchas ventajas sobre otros modelos experimentales in vivo utilizados tradicionalmente. Tanto la recolección de los huevos y larvas de pez cebra, así como la obtención del producto de la excreción, se realizaron de acuerdo a los estándares de laboratorio. Poblaciones larvarias, fueron tratadas con distintas soluciones; pura y diluidas, 101, 10-2, 10-3, 10-4 y 10-5, v/v de PESdy. La muerte celular in vivo, usando naranja de acridina (NA) y bromuro de etidio (BE) fue evaluada. El efecto del SdyEP, se expresó como dependiente de la concentracion y del tiempo de exposición. La población de larvas sobrevivientes, presentaron curvatura troncal en un 10%, en relación a los controles. La necrosis se puso en evidencia a través de la opacidad de las larvas después de 24 h. El uso de NA/BE reveló un patrón de distribución de la fluorescencia en verde y naranja en larvas sobrevivientes al tratamiento. Una granpoblación de células muertas, alrededor de la región anal y caudal, se ponen en evidencia por la presencia de núcleos de naranja en mayor número que en los controles. Los resultados apoyan la aplicación de la coloración NA/BE en modelo experimental de pez cebra, para la evaluación de la acción tóxica de nuevas moléculas y nuevos productos de excreción bacterial con potencial terapéutico.

Shiga toxin-associated hemolytic uremic syndrome complicated by intestinal perforation in a child with typical hemolytic uremic syndrome / 소아과

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in childhood and is primarily diagnosed in up to 4.5% of children who undergo chronic renal replacement therapy. Escherichia coli serotype O157:H7 is the predominant bacterial strain identified in patients with HUS; more than 100 types of Shiga toxin-producing enterohemorrhagic E. coli (EHEC) subtypes have also been isolated. The typical HUS manifestations are microangiopathic hemolytic anemia, thrombocytopenia, and renal insufficiency. In typical HUS cases, more serious EHEC manifestations include severe hemorrhagic colitis, bowel necrosis and perforation, rectal prolapse, peritonitis, and intussusceptions. Colonic perforation, which has an incidence of 1%-2%, can be a fatal complication. In this study, we report a typical Shiga toxin-associated HUS case complicated by small intestinal perforation with refractory peritonitis that was possibly because of ischemic enteritis. Although the degree of renal damage is the main concern in HUS, extrarenal complications should also be considered in severe cases, as presented in our case.

Componentes alimentarios de potencial aplicación en la dietoterapia de enfermedades provocadas por cepas de escherichia coli productoras de toxina tipo shiga / Food components of potential application in the dietoterapia of diseases provoked by vine-stocks of escherichia coli producers of toxin type shiga

En Argentina, Escherichia coli O157:H7 es el serotipo prevalente asociado a grandes brotes y casos esporádicos de colitis hemorrágica y síndrome urémico hemolítico (SUH), siendo el país con mayor tasa de incidencia en el mundo con alrededor de 500 nuevos casos por año. El SUH representa la principal causa de falla renal aguda en la infancia, la segunda causa de falla renal crónica y el 20% de los casos de transplante renal durante la infancia y la adolescencia. Se ha demostrado que la habilidad de la cepa para causar la enfermedad está relacionada a la acción de factores de virulencia implicados en el proceso de colonización del epitelio intestinal con posterior reacción inflamatoria y por la capacidad de secretar toxinas VT1 y VT2 responsables del daño del endotelio vascular con el consecuente desarrollo de SUH. En la presente tesis doctoral se investigó desde la dietoterapia, extractos de origen vegetal y componentes de cepas probióticas potencialmente útiles en la inhibición de la colonización de E. coli O157:H7 a nivel intestinal como medida preventiva, e inhibición de la acción citotóxica de VT sobre células eucariotas. Se determinaron componentes fenólicos obtenidos a partir de la maceración de harina de algarroba (Prosopis alba) y café de mistol (Ziziphus mistol) en distintos solventes (alcohol, agua destilada, acetona y hexano). Los mismos fueron nalizados mediante marcha analítica fitoquímica, cromatografía en capa delgada y cuantificados por espectrofotometría. Por otro lado se obtuvieron componentes de leche fermentada con granos de kéfir.

SUMMARY: In Argentina, Escherichia coli O157:H7 is prevalent serotype associated with large outbreaks and sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome (HUS), being the country with the highest incidence rate in the world with about 500 new cases per year. HUS is the leading cause of acute renal failure in infancy, the second leading cause of chronic renal failure and 20% of cases of renal transplantation during childhood and adolescence. It has been shown that the ability of the strain to cause disease is linked by the action of virulence factors required in the colonization of intestinal epithelium and subsequent inflammatory response and their ability to secrete toxins, VT1 and VT2 responsible for damage to the vascular endothelium with the subsequent development of HUS. In this thesis we investigated plant extracts and components of probiotic strains potentially useful in diet therapy to inhibit the colonization of E. coli O157:H7 in the intestine as a preventive measure, able to interfere the cytotoxic action of VT on eukaryotic cells. Phenolic compounds were identified, they were obtained from plant extracts of P. alba and Z. mistol macerated in different solvents (alcohol, distilled water, acetone and hexane). They were analyzed using phytochemical methods, thin layer chromatography and quantified by spectrophotometry. Furthermore components were obtained from fermented milk kefir grains.

Excretion product of shigella dysenteriae (sdyep) induced cell death in early larval stage of zebrafish (danio rerio): acridine orange and ethidium bromide (ao/eb) in vivo staining / El producto de excreción de la shigella dysenteriae (pesdy) Induce muerte celular en larvas de pez cebra (danio rerio): marcaje in vivo con naranja de acridina y bromuro de etidio (na/be)

In the field of studies of acute toxicity induced by bacterial agents, Shiga toxins have been relevant due to the severity of the extra-intestinal diseases they cause. Numerous studies have shown that Shiga toxin induced apoptosis in different cell types; however, this important process has been little studied in vivo experimental models. In this study, the effects of excretion products of Shigella dysenteriae, in which Shiga toxin is present, we investigated on early larval stages of Zebrafish, an animal model with many advantages over other in vivo experimental models traditionally used. Both the collection of eggs and larvae of Zebrafish, and the product from excretion from Shigella dysenteriae (SdyEP) were performed according to laboratory standards. Also, toxicity bioassay, larvae treatment with pure and diluted solution, 10-1, 10-2, 10-3 , 10-4 and 10-5 v/v SdyEP and cell death in vivo using Acridine Orange (AO) and Ethidium Bromide (EB) were applied. The excretion product of Shigella dysenteriae (SdyEP) effect was expressed in terms of larval mortality and dependent dilution rather than incubation time. The larval population surviving treatment with Shigella excretion product presents severe morphological effects. The larval population generally presents notable severe morphological damage, the necrosis state is represented by the opacity of the larvae after being treated for 24 h (b) compared to control. Other changes associated with larval anatomy were also observed; particularly the caudal end curvature was significant into 10%. The use of AO/EB revealed a distribution pattern from fluorescence into green and orange in surviving larvae SdyEP poisoning, there was a large population of dead cells around the anal and caudal region as evidenced by the presence of orange nuclei in greater numbers as controls in the larvae. The results support the application of coloring AO/EB in Zebrafish experimental...

En el campo de los estudios de toxicidad aguda inducida por agentes bacterianos, las toxinas Shiga resultan relevantes debido a la severidad de las enfermedades extra-intestinales que causan. Numerosos estudios han demostrado que la toxina Shiga induce la apoptosis en diferentes tipos de células, sin embargo, este importante proceso ha sido poco estudiado en modelos experimentales in vivo. En este estudio, fueron evaluados los efectos del productos de excreción de Shigella dysenteriae (PESdy), sobre estadios larvarios de pez cebra (Danio rerio), un modelo animal con muchas ventajas sobre otros modelos experimentales in vivo utilizados tradicionalmente. Tanto la recolección de los huevos y larvas de pez cebra, así como la obtención del producto de la excreción, se realizaron de acuerdo a los estándares de laboratorio. Poblaciones larvarias, fueron tratadas con distintas soluciones; pura y diluidas, 10-1, 10-2, 10-3 , 10-4 and 10-5 v/v de PESdy. La muerte celular in vivo, usando naranja de acridina (NA) y bromuro de etidio (BE) fue evaluada. El efecto del PESdy, se expresó como dependiente de la concentración y del tiempo de exposición. La población de larvas sobrevivientes, presentaron curvatura troncal en un 10 por ciento, en relación a los controles. La necrosis se puso en evidencia a través de la opacidad de las larvas después de 24 h. El uso de NA/BE reveló un patrón de distribución de la fluorescencia en verde y naranja en larvas sobrevivientes al tratamiento. Una gran población de células muertas, alrededor de la región anal y caudal, se ponen en evidencia por la presencia de núcleos de naranja en mayor número que en los controles. Los resultados apoyan la aplicación de la coloración NA/BE en modelo experimental...

Conservação de espinafre (Tetragonia expansa) pelo emprego de radiação gama: aspectos físico-químicos, microbiológicos e sensoriais / Preservation of spinach (Tetragonia expansa) using gamma radiation: physical chemical, microbiological and sensorial characteristics

Nos últimos anos, vegetais têm sido responsáveis por surtos de enfermidades transmitidas por alimentos (ETA) em diversas regiões do mundo por serem veículos dos mais diferentes micro-organismos patogênicos, entre eles Salmonella spp, Listeria monocytogenes e Escherichia coli produtora de toxina de Shiga (STEC). O uso de sanitizantes nem sempre reduz de maneira significativa a população de micro-organismos presentes nos vegetais, sendo necessária a aplicação de técnicas mais eficientes, entre elas, a radiação gama. Assim, os objetivos do presente estudo foram avaliar o efeito da irradiação na redução de STEC, Salmonella spp. e L. monocytogenes inoculadas em espinafre minimamente processado, bem como sobre os atributos físico-químicos e sensoriais do vegetal. Amostras de espinafre (Tetragonia expansa) foram inoculadas com um "pool" de cepas de Salmonella spp, um "pool" de cepas de L. monocytogenes e um "pool" de cepas de STEC, separadamente, e expostas às doses de 0; 0,2; 0,4; 0,6; 0,8 e 1,0 kGy. Os valores de D10 para Salmonella spp, L. monocytogenes e STEC foram, respectivamente, 0,19 a 0,20 kGy, 0,20 a 0,21 kGy e 0,17 kGy. Foram avaliados os comportamentos de Salmonella spp, L. monocytogenes e STEC em amostras de espinafre expostas à doses cinco vezes maiores do que o valor D10 obtido para cada micro-organismo: 1,0; 1,05 e 0,85 kGy, respectivamente, e em amostras não irradiadas armazenadas por 12 dias a (4±1) °C e a (10±1) °C. Os resultados mostram que as doses empregadas reduziram a população de Salmonella e de STEC em aproximadamente 6 ciclos log no dia zero tendo permanecido abaixo do limite de detecção (<10 UFC/g), mesmo após 12 dias de armazenamento em ambas as temperaturas. A dose de 1,05 kGy reduziu a população de L. monocytogenes em, aproximadamente, 5 log imediatamente após a irradiação, porém com recuperação de 2,62 log nas amostras armazenadas a (10±1) °C ao final do período de armazenamento. Amostras de espinafre expostas às doses de 1 e 1,5 kGy e a amostra-controle, mantidas sob refrigeração (4±1) ºC, foram utilizadas para a avaliação da vida de prateleira (VP), análise sensorial, análise de cor, determinação de ácido ascórbico, flavonoides, compostos fenólicos e capacidade antioxidante. A VP da amostra exposta à dose de 1 kGy foi de 15 dias, dois dias a mais que a da amostra-controle, enquanto a exposta a 1,5 kGy apresentou VP de 12 dias. Todas as amostras expostas à radiação foram aceitas pelos provadores. A irradiação não provocou alterações significativas na concentração de compostos fenólicos e atividade antioxidante, porém houve alteração na cor e na concentração de flavonoides. As estações do ano, por sua vez, tiveram influência sobre a coloração, concentração de compostos fenólicos e atividade antioxidante. Apesar da alteração na coloração ter sido observada na análise instrumental, esta não foi percebida pelos provadores durante a análise sensorial. O processo de irradiação mostrou ser uma boa alternativa para aumentar a segurança microbiológica de espinafre sem alterar as características sensoriais. No entanto, o uso das Boas Práticas de Fabricação nunca deve ser negligenciado

In recent years, fresh produce have been responsible for foodborne disease outbreaks worldwide, due to their contamination by different pathogenic microorganisms such as Salmonella spp., Listeria monocytogenes and Shiga toxin-producing Escherichia coli (STEC). The use of sanitizers does not always significantly reduce the microbial populations present in vegetables, and thus, the application of more efficient techniques such as gamma radiation, is required. The objectives of this study were to evaluate the effect of irradiation on the reduction of the populations of STEC, Salmonella spp. and L. monocytogenes, inoculated on minimally processed spinach, as well as to assess its effect on the sensory and physicochemical characteristics of the vegetable. Spinach (Tetragonia expansa) samples were individually inoculated, with a cocktail of three strains of Salmonella spp, three strains of L. monocytogenes and three strains of STEC and exposed to doses of 0, 0.2; 0.4; 0.6; 0.8 and 1.0 kGy. The D10 values determined in this study ranged from 0.19 to 0.20 kGy for Salmonella spp, 0.20 to 0.21 kGy for L. monocytogenes, and 0.17 kGy for STEC. The behavior of Salmonella spp., L. monocytogenes and STEC were evaluated in spinach samples exposed to doses of 1.0, 1.05 and 0.85 kGy, respectively, and in non-irradiated samples, stored for 12 days at (4±1) °C and (10±1) °C. The results showed that the populations of Salmonella and STEC were reduced at about 6 log, on day zero, and remained below the detection limit (<10 CFU/g) even after 12 days of storage at both temperatures tested. The 1.05 kGy dose reduced the population of L. monocytogenes in approximately 5 log, but in the samples stored at (10±1) °C, the growth of the microorganism (2,62 log) was observed at the end of the storage time. Spinach samples exposed to 1 and 1.5 kGy, as well as the control sample, all kept under refrigeration (4±1) °C were used for the evaluation of the product shelf life, sensory analysis, color analysis, determination of ascorbic acid, flavonoids, phenolic compounds and the antioxidant capacity. The samples exposed to 1 kGy displayed a shelf life of 15 days, two days longer than that observed for the control sample, while those exposed to 1.5 kGy showed a shelf life of 12 days. All samples exposed to radiation were accepted by the sensorial panel. The irradiation had no significant effect either on the concentration of phenolic compounds or on the antioxidant activity. Nevertheless, there was a reduction in the concentration of flavonoids and change on the color. The color, phenolic compounds concentration and antioxidant activity were influenced by the seasons of the year. Although the change in color was observed by instrumental analysis, this was not perceived by the panelists during sensory analysis. The irradiation process is a great alternative for microbiological safety purpose together with Good Manufacturing Practices