First molecular characterization of Escherichia coli O157:H7 isolates from clinical samples in Paraguay using whole-genome sequencing Molecular characterization of Escherichia coli O157 isolates from Paraguay / Primera caracterización molecular de aislamientos de Escherichia coli O157:H7 provenientes de muestras clínicas en Paraguay utilizando secuenciación de genoma completo

Abstract Escherichia coli O157:H7 is a foodborne pathogen implicated in numerous outbreaks worldwide that has the ability to cause extra-intestinal complications in humans. The Enteropathogens Division of the Central Public Health Laboratory (CPHL) in Paraguay is working to improve the genomic characterization of Shiga toxin-producing E. coli (STEC) to enhance laboratory-based surveillance and investigation of foodborne disease outbreaks. Whole genome sequencing (WGS) is proposed worldwide to be used in the routine laboratory as a high-resolution tool that allows to have all the results in a single workflow. This study aimed to carry out for the first time, the genomic characterization by WGS of nine STEC O157:H7 strains isolated from human samples in Paraguay. We were able to identify virulence and resistance mechanisms, MLST subtype, and even establish the phylogenetic relationships between isolates. Furthermore, we detected the presence of strains belonging to hypervirulent clade 8 in most of the isolates studied.

Resumen Escherichia coli O157:H7 es un patógeno transmitido por alimentos implicado en numerosos brotes en todo el mundo y es capaz de causar complicaciones extraintestinales en humanos. La sección de «Enteropatógenos¼ del Laboratorio Central de Salud Pública trabaja en mejorar la caracterización genómica de STEC, de modo de potenciar la vigilancia laboratorial y la investigación de brotes de enfermedades transmitidas por alimentos. La secuenciación de genoma completo (WGS, por sus siglas en inglés) se propone a nivel mundial como una herramienta de alta resolución para ser utilizada en el laboratorio de rutina, ya que permite obtener todos los resultados en un único proceso. El objetivo de este trabajo fue llevar a cabo, por primera vez, la caracterización genómica por WGS de nueve cepas STEC O157:H7 aisladas en Paraguay a partir de muestras de origen humano. Pudimos identificar los factores de virulencia, los mecanismos de resistencia, el subtipo MLST, e incluso pudimos establecer la relación filogenética entre los aislamientos. Además, detectamos que la mayoría de las cepas pertenecían al clado hipervirulento 8.

Multi-enzyme isothermal rapid amplification assay for the detection of Escherichia coli O157∶H7 / 上海预防医学

ObjectiveA rapid enrichment and detection method for Escherichia coli O157∶H7 was developed by using multienzyme isothermal rapid amplification （MIRA） fluorescence method combined with metal organic frameworks immunomagnetic beads. MethodsUsing rfbE gene as the target， the primers， probes and reaction system were screened， and the specificity， sensitivity and practical application of this method were investigated. ResultsThe detection limit of Escherichia coli O157∶H7 was 1.18×105 CFU‧mL-1， and the detection limit of DNA concentration was 9 pg‧μL-1. The detection process was completed in 20 minutes. The test results of 47 strains （24 target strains and 23 non-target strains） were consistent with real-time PCR （RT-PCR）. ConclusionA method based on metal-organic framework immunomagnetic beads enrichment combined with MIRA assay is developed in this study. The method is simple， rapid and suitable for rapid enrichment and detection of Escherichia coli O157∶H7 in food.

Cambios en el perfil protéico de E. coli O157:H7 frente al tratamiento con Ib-M1 e IONP@Ib-M1 / Changes in the protein profile of E. coli O157: H7 against treatment with Ib-M1 and IONP@Ib-M1 / Identificação de alterações no perfil protetor de E. coli O157: H7 contra o tratamento com Ib-M1 e IONP@Ib-M1

Resumen Escherichia coli 0157:H7 es una bacteria patógena reconocida por su capacidad de resistencia a diversos antibióticos; razón por la cual, se generan complicaciones en el tratamiento de infecciones producidas por esta bacteria. El péptido Ib-M1 y el bioconjugado I0NP@Ib-M1 han surgido como una nueva alternativa antimicrobiana contra E. coli 0157:H7. El mecanismo de acción de Ib-Mi e I0NP@Ib-M1 contra esta bacteria aún es desconocido; por lo tanto, el objetivo de esta investigación fue identificar el cambio en el perfil de proteínas de E. coli 0157:H7 luego del tratamiento con Ib-M1 e I0NP@ Ib-M1 como primer paso para determinar su mecanismo de acción. Para esto, se llevó a cabo la obtención de proteínas, posteriormente se realizó una electroforesis bidimensional para finalmente realizar la determinación de la variabilidad de los perfiles proteicos. Una vez obtenidos estos perfiles, se llevó a cabo un análisis de varianza (AN0VA). Se identificaron 72 proteínas expresadas diferencialmente, las cuales pueden relacionarse con el efecto sobre el crecimiento de la bacteria en presencia de Ib-M1 e I0NP@Ib-M. Estas proteínas se encuentran involucradas en procesos de transferencia de grupos acilo (proteína Yhbs), translocación de lipoproteínas (proteína LolA) y transporte de aminoácidos (proteína GpmA), entre otros.

Abstract Escherichia coli 0157: H7 is a pathogenic bacterium which is recognized for the ability to resist multiple antibiotics; accordingly, complications occur in the treatment of infections caused by this bacterium. The Ib-M1 peptide and the I0NP @ Ib-M1 bioconjugate have emerged as a new antimicrobial alternatives against E. coli 0157: H7. The mechanism of action of Ib-M1 and I0NP @ Ib-M1 against this bacterium is still unknown; therefore, the goal of this research was to identify the change in the proteins profile of E. coli 0157: H7 after treatment with Ib-M1 and I0NP @ Ib-M1 as a first step to determine its mechanism of action. For this, the proteins were obtained first, and then a two-dimensional electrophoresis was performed to finally determine the variability of the protein profiles. 0nce the protein profiles were obtained, an analysis of variance (AN0VA) was carried out. 72 differentially expressed proteins were identified, which can be connected to the effect on the bacterium's growth in the presence of Ib-M1 and I0NP @ Ib-M. These proteins are involved in acyl groups transfer processes (Yhbs protein), lipoprotein translocation (LolA protein) and amino acid transport (GpmA protein), among others.

Resumo Escherichia coli O157: H7 é uma bactéria patogênica reconhecida por sua capacidade de resistir a vários antibióticos; razão pela qual, complicações são geradas no tratamento de infecções produzidas por essa bactéria. O peptídeo Ib-M1 livre e imobilizado em nanopartículas magnéticas de óxido de ferro (IONP @ Ib-M1) surgiu como uma nova alternativa antimicrobiana contra E. coli O157: H7 e isolados clínicos desta bactéria. O mecanismo de ação de Ib-M1 e IONP @ Ib-M1 contra E. coli O157: H7 ainda é desconhecido; Portanto, o objetivo desta pesquisa foi identificar a alteração no perfil proteico de E. coli O157: H7 após o tratamento com Ib-M1 e IONP @ Ib-M1 como um primeiro passo para determinar seu mecanismo de ação. Para isso, foi realizada a obtenção das proteínas, posteriormente foi realizada uma eletroforese bidimensional para finalmente determinar a variabilidade dos perfis protéicos. Uma vez obtidos os perfis de proteínas, foi realizada uma análise de variância (ANOVA). Os resultados mostram a identificação de proteínas expressas diferencialmente e que estão envolvidas em processos de transferência de grupos acila (proteína Yhbs), translocação de lipoproteínas (proteína LolA) e transporte de aminoácidos (proteína GpmA), entre outros.

Research progress on antibiotic resistance of Escherichia coli O157:H7 / 预防医学

@#Contamination of foodborne pathogens is the main cause of related diseases. Escherichia coli O157:H7 (E.coli O157:H7), as a representative of pathogenic E.coli, is one of the most severe and commonly reported E.coli in the world, but there is still no effective clinical treatment against the infection. Antibiotics show effective in the early infection of E.coli O157:H7. However, their extensive use has led to drug-resistant bacteria and genes in recent years, which becomes a great threat to public health. This article reviews the research progress of E.coli O157:H7 from the prevalence of antibiotic-resistant bacteria, the resistance mechanism, and the prevention and control methods, in order to provide a reference for its prevention, early clinical treatment and related research.

First detection of Shiga toxin producing Escherichia coli O157:H7 (HlyA gene) and enumeration of most probable number (MPN) of fecal coliforms and Escherichia coli in cage cultured oysters (Crassostrea iredalei) and water from southern Malaysia

Aims@#Escherichia coli O157:H7 is known to be transmitted via fecal-oral route, where water plays a role in the transmission process. Oysters as bivalves, bio accumulate pathogens from the water through filter feeding and are suspected to play a role as disease transmission vector. In Malaysia, the data on oyster’s microbiological quality are limited. Hence, it was vital to conduct oyster related studies in Malaysia. The main objectives of this study include the enumeration of most probable number (MPN) of fecal coliforms and E. coli and isolation of E. coli from oyster (Crassostrea iredalei) and water sample for the detection of 16S rRNA and HlyA (Hemolysin A) genes of E. coli O157:H7. @*Methodology and results@#A total of 120 oysters and water samples (n=6) were collected from a fisherman village located in southern Malaysia. Total fecal coliforms and E. coli were determined using the MPN procedure. Colonies of E. coli were identified based on Gram staining, biochemical test, and PCR detection for the presence of 16S rRNA and HlyA gene of E. coli O157:H7. The enumeration results showed that the MPN of the fecal coliforms and E. coli found in the collected oyster samples do not meet the standard to be directed for human consumption (0.72 ± 0.19 × 104 MPN/100 g and 0.13 ± 0.03 × 10 4 MPN/100 g, respectively). The PCR assays showed that 16 out of the 104 (15.38%) of E. coli isolated from water and oysters showed the presence of HlyA gene. The phylogenetic tree analysis showed there were genetic relationships between the HlyA gene of the E. coli isolated in this study with the ones isolated from calf and human faeces.@*Conclusion, significance and impact of study@#The detection of Shiga toxin producing E. coli O157:H7 (HlyA gene) in cage cultured oysters (C. iredalei) and water from southern Malaysia was first time reported here. In the future, more study can be conducted to study the expression of the HlyA gene and confirm of its identity as E. coli O157:H7 using different target genes such as eaeA (encodes a 94 kD outer membrane protein called intimin) and Stx1 (Shiga toxin, Shigella dysenteriae type 1).

Evaluación de Lactobacillus plantarum microencapsulado y su viabilidad bajo condiciones gastrointestinales simuladas e inhibición frente a Escherichia coli O157:H7 / Evaluation of microencapsulated Lactobacillus plantarum and its viability under simulated gastrointestinal conditions and inhibition against Escherichia coli O157: H7

RESUMEN Se evaluó la viabilidad de Lactobacillus plantarum microencapsulado, su efecto de inhibición sobre Escherichia coli O157:H7 y su crecimiento en condiciones gastrointestinales simuladas. Se utilizaron L. plantarum ATCC 8014® y E. coli ATCC 43888®. Se realizaron pruebas de inhibición de L. plantarum sobre E. coli y test de susceptibilidad a CTX (30 µg), P (10 IU), GN (10 µg), DCX (1 µg), CIP (5 µg) y KF (30 µg) para ambas cepas. En la bacteria láctica se determinó la cinética de fermentación y la presencia de péptidos y aminoácidos por HPLC en ambas cepas. Se evaluó el crecimiento a 37 y 45°C de L. plantarum y se valoró la viabilidad de su microencapsulación mediante condiciones gastrointestinales (bilis, sales biliares y pH ácido), al igual que la supervivencia y estabilidad de preparado y sus características físicas y morfológicas. Los resultados indicaron que L. plantarum inhibió a E. coli y el microencapsulado, resultados positivos con una viabilidad del 83,3%; eficiencia de 88,4%; humedad de 7,79%; actividad de agua 0,4; humectabilidad de 1 min, 56 s; solubilidad del 96%; morfología esférica y tamaño entre 15,18 a 35,68 pm. Finalmente, se observó un alto potencial de L. plantarum como agente inhibidor para E. coli O157:H7.

ABSTRACT The viability of microencapsulated Lactobacillus plantarum, its inhibition effect on Escherichia coli O157: H7 and growth in simulated gastrointestinal conditions was evaluated. L. plantarum ATCC 8014® and E. coli ATCC 43888® were used. Inhibition tests of L. plantarum on E. coli and susceptibility test to CTX (30 µg), P (10 IU), GN (10 µg) DCX (1 µg), CIP (5 µg) and KF (30) were performed µg) for both strains. In the lactic bacterium the fermentation kinetics were determined, and the presence of peptides and amino acids by HPLC in both strains. The growth at 37 ° C and 45 ° C of L. plantarum was evaluated and the viability of its microencapsulation was assessed by gastrointestinal conditions (Bile, Bile salts and acidic pH), as well as the survival and stability of the preparation and its physical characteristics and morphological. The results indicated that L. plantarum inhibited E. coli and the microencapsulated positive results with a viability of 83.3%, efficiency 88.4%, Humidity 7.79%, water activity 0.4, wettability of 1 min, 56 s, 96% solubility, spherical morphology and size between 15.18 to 35.68 pm. Finally, a high potential of L. plantarum was observed as an inhibitory agent for E. coli O157: H7.

Estandarización de una técnica de PCR múltiple para la detección de los serogrupos O157, O104 y "big six" de Escherichia coli productora de la toxina Shiga (STEC) / Standardization of a multiplex PCR technique for the detection of Shiga toxin-producing Escherichia coli (STEC) serogroups O157, O104 and "big six"

Los serogrupos O26, O45, O103, O104, O111, O121, O145 y O157 de STEC se relacionan con un elevado número de casos de SUH a nivel mundial, por lo que están incluidos dentro de las categorías de mayor riesgo para los humanos, según los criterios de autoridades alimentarias de Estados Unidos y Europa. El método convencional de identificación de antígenos O y H se realiza por aglutinación con antisueros de conejo. Este método además de ser muy costoso y laborioso, no se encuentra disponible en el país para empleo masivo. En este contexto, el objetivo de este estudio observacional descriptivo de corte transverso ha sido la estandarización de una técnica de PCR múltiple para la detección de estos 8 serogrupos, a fin de contar con un sistema de detección eficiente, sensible y con potencial de aplicación en la industria alimentaria. Se estandarizaron reacciones de PCR empleando como controles positivos cepas E. coli de referencia correspondientes a la totalidad de los serogrupos citados. Se obtuvieron productos de tamaños esperados para cada serogrupo, no se observaron amplificaciones cruzadas o falsos positivos. Esta técnica estandarizada podría representar una herramienta rápida y menos costosa que la técnica serológica, con la capacidad de ser aplicada a diferentes matrices, permitiendo la detección de estos serogrupos en aislados STEC de ganado en pie, fuentes de agua de consumo, alimentos e incluso en aislamientos clínicos asociados a enfermedades humanas(AU)

STEC serogroups O26, O45, O103, O104, O111, O121, O145, and O157, are related to a high number of cases of HUS worldwide, so they are included in the categories of greatest risk for humans, according to the food administration criteria of the United States and Europe. The conventional method of identifying antigens O and H is carried out by agglutination with rabbit antisera. This method is very expensive and laborious and is not available in the country for massive-scale use. In this context, the objective of this cross-sectional descriptive observational study has been the standardization of a multiplex PCR technique for the detection of these 8 serogroups, in order to have an efficient and sensitive detection system with the potential for application in the food industry. PCR reactions were standardized using as positive controls reference E. coli strains to correspond to all the mentioned serogroups. Products of expected sizes were obtained for each serogroup; no cross-amplification or false positives were observed. This standardized technique could represent a quick and less expensive tool than the serological technique, with the possibility to be applied to different kind of samples, allowing the detection of these serogroups in STEC isolates of live cattle, sources of drinking water, food and even in clinical isolates associated with human diseases(AU)

Síndrome hemolítico urémico asociado a Shigatoxina: ¿Cómo prevenirlo? / Shiga-toxin associated hemolytic uremic syndrome: how to prevent it?

Resumen: El síndrome hemolítico urémico (SHU) asociado a infección intestinal por bacterias productoras de Shigatoxina, que afecta principalmente a población infantil, puede causar morbilidad aguda grave, secuelas crónicas en varios órganos, y la muerte prematura en algunos de ellos. Dado su carácter zoonótico, adecuadas medidas de manejo agropecuario y correcta higiene de lo que consumimos es indispensable a la hora de prevenir la infección. Actualmente, una vez gatillado el SHU el manejo es médico y, principalmente, de soporte. En los últimos años diversas estrategias terapéuticas se han ido desarrollando para evitar que esta enfermedad ocurra, o, al menos, que pueda ser atenuada en sus consecuencias de morbi-mortalidad. El presente artículo describe acciones específicas a diferentes niveles de prevención de esta patología.

Abstract Hemolytic uremic syndrome (HUS) associated with intestinal infection by Shiga toxin-producing bacteria, which mainly affects children, can cause severe acute morbidity, chronic sequelae in seve ral organs, and premature death in some of them. Given its zoonotic nature, adequate measures of agricultural management and proper hygiene of what we consume are essential to prevent infection. Once the HUS is triggered, medical management is currently mainly supportive. In recent years, va rious therapeutic strategies have been developed to prevent this disease from occurring or, at least, to mitigate its morbidity and mortality consequences. This article describes specific actions at different levels of prevention of this pathology.

Primer reporte de escherichia coli productora de toxina shiga no O157 que codifica el gen de la enterohemolisina en carne cruda en colombia / First report of Shiga toxin-producing escherichia coli non-O157 that encodes the enterohemolysin gene in raw beef in colombia

Dentro de los patotipos de Escherichia coli, el grupo STEC puede producir en el ser humano desde diarrea hemorrágica hasta insuficiencia renal aguda e incluso la muerte; el ganado bovino es el principal reservorio de este agente patógeno y por ende la ingestión de alimentos derivados de estos animales de abasto son una fuente muy importante de infección para el hombre. El objetivo principal de este estudio fue determinar la prevalencia de STEC en muestras de carne cruda comercializada en Pamplona-Colombia y en cepas obtenidas a partir de las muestras. Se analizaron cien muestras de carne cruda aplicando la técnica de Reacción en Cadena de la Polimerasa para la detección de los siguientes genes en muestras y en cepas STEC: stx1, stx2, eae y hlyA. Adicionalmente, se estableció el patrón de resistencia-susceptibilidad antibiótica de cepas STEC aisladas empleando métodos regulados. En el 39% de las muestras fue posible detectar el gen stx2; en el 38%, de ellas, se detectaron los genes stx1 y stx2. Además, se aislaron cepas STEC en el 13% de las muestras analizadas, 85% de ellas portaban el gen hlyA. No se detectó la presencia del gen eae o del serogrupo O157. Las cepas aisladas demostraron resistencia frente a algunos antibióticos de primera y segunda generación. En conclusión, se detectó la presencia de genes que codifican factores de virulencia en las muestras de carne analizadas que representan un riesgo potencial para la salud de los consumidores. Este es el primer reporte de STEC no O157 que codifica el gen de la enterohemolisina en alimentos en Colombia(AU)

Within the Escherichia coli patotypes, the STEC group can produce in humans from hemorrhagic diarrhea to acute renal failure and even death; cattle are the main reservoir of this pathogen and therefore the ingestion of food derived from these stock animals are a very important source of infection for man. The main objective of this study was to determine the prevalence of STEC in raw meat samples marketed in Pamplona-Colombia and in strains obtained from those samples. One hundred raw meat samples were analyzed using the Polymerase Chain Reaction technique for the detection of the following genes in samples and in STEC strains: stx1, stx2, eae and hlyA. In addition, STEC strains were isolated in 13% of the analyzed samples, 85% of them carried the hlyA gene. The presence of the eae gene or serogroup O157 was not detected. The isolated strains demonstrated resistance against some first and second generation antibiotics. In conclusion, the presence of genes encoding virulence factors in the meat samples analyzed, that represent a potential health risk factor to consumers, was confirmed. This is the first report of STEC non-O157 that encodes the enterohemolysin gene in foods in Colombia(AU)

A fluorometric method for direct detection of inorganic polyphosphate in enterohemorrhagic O157:H7 / 南方医科大学学报

OBJECTIVE@#To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7.@*METHODS@#The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃, and freezing at -20 ℃, all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two mutant strains.@*RESULTS@#At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was =1849+127.5 (R=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with deletion.@*CONCLUSIONS@#We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.

Detección y caracterización de Escherichia coli productor de toxina Shiga en ninos atendidos en un hospital pediátrico interzonal de la ciudad de La Plata / Detection and characterization of Shiga toxin-producing Escherichia coli in children treated at an inter-zonal pediatric hospital in the city of La Plata

Escherichia coli productor de toxina Shiga (STEC) es un patógeno transmitido por alimentos que puede causar diarrea acuosa, diarrea sanguinolenta (DS) y síndrome urémico hemolítico (SUH). El objetivo de este estudio fue determinar las características fenotípicas y genotípicas de cepas STEC aisladas de niños con DS y SUH atendidos en un hospital pediátrico de la ciudad de La Plata en el período 2006-2012 y establecer la relación clonal de los aislamientos O157: H7 mediante electroforesis de campo pulsado. El porcentaje de muestras positivas fue de 4,9 y 39,2% en los pacientes que presentaron DS y SUH, respectivamente. Se aislaron 77 cepas STEC de 10 serotipos distintos, con el 100% de recuperación de colonias. El serotipo más frecuente fue O157: H7 (71,4%), seguido por O145: NM (15,6%). El 98,2% de los aislamientos O157: H7 correspondió al biotipo C y fue sensible a los antibióticos ensayados. Todos esos aislamientos presentaron el genotipo stx2, eae, fliC H7, ehxA, iha, efa, toxB, lpfA1-3 y lpfA2-2.Al estudiar la relación clonal de las cepas O157: H7, se identificaron un total de 42 patrones con al menos un 88% de similitud y se establecieron 6 clústeres que agruparon cepas con perfiles idénticos. Los aislamientos eae negativos pertenecieron a los serotipos O59: H19, O102: H6, O174: NM y O174: H21. Las cepas O59: H19 y O174: H21 fueron positivas para el gen aggR. Este estudio muestra que en la ciudad de La Plata y alrededores circulan STEC de diferentes serotipos y genotipos. A pesar de la diversidad genética observada entre los aislamientos O157: H7, algunos fueron indistinguibles por las técnicas de subtipificación utilizadas.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that can cause watery diarrhea, bloody diarrhea (BD), and hemolytic uremic syndrome (HUS). The objective of this study was to determine the phenotypic and genotypic profiles of STEC strains isolated from children with BD and HUS treated at a pediatric hospital in the city of La Plata in the period 2006-2012, and to establish the clonal relationship of O157: H7 isolates by pulsed field electrophoresis. The percentage of positive samples was 4.9% and 39.2% in patients with BD and HUS, respectively. Seventy-seven STEC strains from 10 different serotypes were isolated, with 100% colony recovery, O157: H7 being the most frequent (71.4%) serotype, followed by O145: NM (15.6%). An average of 98.2% of O157: H7 isolates belonged to biotype C and were sensitive to all the antibiotics tested. All of them (100%) carried genotype stx2, eae, fliC H7, ehxA, iha, efa, toxB, lpfA1-3 and lpfA2-2. When the clonal relationship of the O157: H7 strains was studied, a total of 42 patterns with at least 88% similarity were identified, and 6 clusters with identical profiles were established. The eae-negative isolates belonged to serotypes O59: H19, O102: H6, O174: NM and O174: H21. The strains O59: H19 and O174: H21 were positive for the aggR gene. This study shows that STEC of different serotypes and genotypes circulate in the city of La Plata and surroundings. Despite the genetic diversity observed between the O157: H7 isolates, some were indistinguishable by the subtyping techniques used.

Growth kinetics of Escherichia coli O157: H7 on the epicarp of fresh vegetables and fruits

ABSTRACT Despite the increasing reports on the incidence of fresh vegetables and fruits as a possible vehicle for human pathogens, there is currently limited knowledge on the growth potential of Escherichia coli O157:H7 on different plant substrates. This study analyzed the selective adhesion and growth of E. coli O157:H7 on chili habanero (Capsicum chinense L.), cucumber (Cucumis sativus), radish (Raphanus sativus), tomato (Lycopersicon esculentum), beet (Beta vulgaris subsp. vulgaris), and onion (Allium cepa L.) under laboratory conditions. The Gompertz parameters were used to determine the growth kinetics. Scanning electron microscopy was used to visualize the adhesion of E. coli O157:H7 on the epicarp of the samples. Predictive models were constructed to compare the growth of E. coli O157:H7 on the samples with different intrinsic factors and to demonstrate the low selectivity of the pathogen. No significant difference was observed in the lag-phase duration (LPD), generation time (GT), and exponential growth rate (EGR) of the pathogen adhered to the samples. The interaction between the microorganism and the substrate was less supportive to the growth of E. coli O157:H7 for onion, whereas for tomato and cucumber, the time for the microorganism to attain the maximum growth rate (M) was significantly longer than that recorded for other samples.

Immunogenicity of the nanovaccine containing intimin recombinant protein in the BALB/c mice

PURPOSE: Escherichia coli O157:H7 is one of the most important pathogens which create hemorrhagic colitis and hemolytic uremic syndrome in human. It is one of the most prevalent causes of diarrhea leading to death of many people every year. The first diagnosed gene in the locus of enterocyte effacement pathogenicity island is eae gene. The product of this gene is a binding protein called intimin belonging to the group of external membrane proteins regarded as a good stimulants of the immune system. Chitosan with its lipophilic property is an environmentally friendly agent able to return to the environment. MATERIALS AND METHODS: Intimin recombinant protein was expressed in pET28a vector with eae gene and purification was performed using Ni-NTA and finally the recombinant protein was approved through western blotting. This protein was encapsulated using chitosan nanoparticles and the size of nanoparticles was measured by Zetasizer. Intimin encapsulated was prescribed for three sessions among three groups of oral, injection, and oral-injection using Chitosan nanoparticles. Challenge was performed for all three groups with 108 E. coli O157:H7 bacteria. RESULTS: Intimin produced by chitosan nanoparticles improves immunological responses through the adjuvant nature of chitosan nanoparticles. Chitosan may be used as a carrier for transportation of the prescribed vaccine. Among the mice, encapsulated intimin could be able to provide suitable titers of IgG and IgA by the aid of chitosan nanoparticles. Results of mice challenge showed that decreased the bacterial shedding significantly. CONCLUSION: Results showed that the chitosan nanovaccine with intimin protein may be used as a suitable candidate vaccine against E. coli O157:H7.

Microbiological and serological control of Escherichia coli O157: H7 in kindergarten staff in Buenos Aires city and suburban areas

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening hemolytic-uremic syndrome (HUS). Despite the magnitude of the social and economic problems caused by HUS, no licensed vaccine or effective therapy is currently available for human use. Prevention of STEC infections continues being the most important measure to reduce HUS incidence. This is especially true for Argentina where HUS incidence among children is extremely high and shows an endemic pattern. The aim of this work was to investigate serologically adult staff of kindergartens in Buenos Aires city and suburban areas in order to detect possible carriers, and to educate personnel about good practices to reduce HUS transmission. We also assessed the microbiological quality of water and meal samples from the same kindergartens. We tested 67 healthy adults, 13 water supplies and 6 meals belonging to 6 public kindergartens. We analysed hand swabs for isolation of STEC and serum samples for the presence of antibodies against Stx and lipopolysaccharide (LPS) of O157 serogroup. We identified 46 Stx2-positive individuals, but only 7 for O157 LPS. No presence of STEC pathogens was detected in hands of staff, water or meal samples.

Las infecciones bacterianas con Escherichia coli productor de toxina Shiga (Stx) (STEC) están implicadas en el desarrollo del síndrome urémico hemolítico (SUH). A pesar de la magnitud del problema social y económico causado por el SUH, actualmente no existe un tratamiento específico o una vacuna eficaz para uso humano. Por lo tanto, la prevención de las infecciones por STEC es la tarea central para reducir la incidencia del SUH. Esto es especialmente cierto para Argentina en donde el SUH muestra un comportamiento endémico y presenta una incidencia extremadamente alta entre los niños. En efecto, la mediana de casos notificados en menores de 5 años para el periodo 2010-2015 fue 306, mientras que la tasa de notificación fue 8.5 casos cada 100 000 menores/año (http://www.msal.gob.ar/images/stories/boletines/boletin_integrado_vigilancia_N335-SE45.pdf). El objetivo de este trabajo fue analizar serológicamente al personal adulto de jardines de infantes de la ciudad de Buenos Aires y el área suburbana con el fin de detectar portadores, y brindarles formación sobre las buenas prácticas para reducir la transmisión de infecciones con STEC y así evitar el SUH. También se evaluó la calidad microbiológica de las muestras de agua y de la comida elaborada en los mismos jardines. Hemos estudiado 67 adultos, a través del hisopado de manos para la búsqueda de STEC y suero para la presencia de anticuerpos contra Stx y el lipopolisacárido (LPS) de serogrupo O157. También se analizaron 13 suministros de agua y 6 muestras de comida pertenecientes a 6 jardines de infantes públicos. Se identificaron 46 individuos positivos para Stx2, pero solo 7 para LPS-O157. No se detectó presencia de patógenos STEC en las muestras de las manos del personal, ni en los reservorios de agua o muestras de comida.

Preparation and evaluation of fluorescent carbon dots (CDs) and CDs based immunofluorescence probe / 医疗卫生装备

Objective To fabricate an immunofluorescence probe system of carbon dots conjugated antibody based on antigen-antibody reaction principles.Methods A green one-step microwave assisted pyrolysis method was applied to preparing fluorescent carbon dots (CDs) using aminoglucose as carbon source and the obtained CDs were conjugated with antibody via EDC/NHS reactions to build CDs based fluorescent probe.Furthermore,the properties of CDs and CDs based probe system were evaluated by Fourier transform infrared (FTIR) spectra,transmission electron microscopy (TEM),UV-vis absorption and so on.Results The as-prepared CDs showed excellent fluorescence and hydrophilicity and CDs based immunofluorescence probe exhibited the capability of quick detection of E.coli O157:H7.Cinclusion Fluorescent CDs as one new emerging environment-friendly nanomaterial has great potential in biosensors.

Molecular characteristics of human-derived non-O157 Shiga toxin-producing Escherichia coli strains isolated in five regions of China / 中华微生物学和免疫学杂志

Objective To understand the molecular characteristics of human-derived non-O157 Shiga toxin-producing Escherichia coil (STEC) strains circulating in five regions of China.Methods Twenty-seven non-O157 STEC strains isolated in five geographic regions were investigated by serotyping, stx1/stx2 subtyping and PCR screening for adhesion and other virulence genes.A multilocus sequence typing (MLST) scheme provided by E.coil MLST database were performed to amplify and sequence seven housekeeping genes (adk, icd, fumC, rgyrB, purA, mdh and recA) in those strains.Results Twenty-seven non-O157 STEC strains were typed into 16 O∶H serotypes.Among those strains, 11 harbored stx1a, 12 harbored stx1c, two harbored stx2e and the other three strains respectively harbored stx1a+stx2b, stx2d and stx2g.Positive rates of eae, efa1, saa, paa, toxB, astA and ehxA genes were 18.5%, 18.5%, 29.6%, 22.2%, 11.1%, 11.1% and 25.9%, respectively.The 27 strains were typed into 16 different sequence types (STs) based upon MLST.Conclusion Human-derived non-O157 STEC strains circulating in five regions of China are heterogeneous in their serotypes, stx1/stx2 subtypes and virulence gene profiles.

Inhibiting effect of Lactobacillus acidophilus on intestinal colonization of enterohaemorrhagic E.coli O157:H7 in vitro and vivo / 实用医学杂志

Objective To test the inhabiting effect of Lactobacillus acidophilus on E.coli O157: H7 in intestinal colonization and explore its mechanism. Methods The suppressive effects of Lactobacillus acidophilus against E.coli O157:H7 adhering to Ht29 cells were carried out by competition , exclusion and replacement as-says. Furthermore, we evaluated the cytokine levels of IL-4, IL-12, and INF-γ in serum of mice. In addition, E.coli O157:H7 fecal shedding was monitored and the pathological changes of intestines were observed in mice. Results The competition, exclusion and replacement assays showed Lactobacillus acidophilus inhibited E.coli O157:H7 adhering to Ht29 cells. In vivo, the mice of treatment group were induced significantly higher level of IL-4, IL-12, and INF-γ, though prevention group induced IL-12 only. Fifteen days after E.coli O157:H7 infec-tion, there were 8 mice (80%) in prevention group and 5 mice (50%) in treatment group stopped shedding. Moreover, the pathological changes of intestines of both prevention group and treatment group appeared normal , but control groups showed seriously damaged in intestinal villus. Conclusion Lactobacillus acidophilus inhibits E.coli O157:H7 in intestinal colonization and the preventative effect was better than treatment effect. Thus , Lac-tobacillus acidophilus can be used for E.coli O157:H7 in prevention and treatment infection as probiotics.

Immunofluorescence probe based on fluorescent carbon dots and its application to the special recognition of Escherichia coli / 军事医学

Objective Carbon dots (CDs) are an emerging carbon nano-material which is environmentally-friendly, economical , efficient and stable .Their fluorescence properties can match the semiconductor quantum dot .Moreover , CDs have more excellent biocompatibilities .The purpose of this experiment is to apply CDs to the fluorescent immune probe to make them a new label , which can replace the traditional fluorescent dyes .Methods Using microwave heating method , the high strength fluorescent carbon dots were prepared .Wtih the EDC coupling method , the high strength fluorescent car-bon dots could bond with Escherichia coli antibodies to form a complex immune fluorescent probe .Specific recognition exper-iments were carried out in the model of E.coli O157∶H7.Results CDs were successfully applied to immune recognition of E.coli O157∶H7 and multicolor fluorescence was observed .Conclusion CDs can serve as a label of the fluorescent im-mune probe , and are expected to become a new type of low toxicity biosensor with independent intellectual property rights .

Prevalence of Escherichia Coli O157:H7 and Enterobacteriaceae on Hands of Workers in Halal Cattle Abattoirs in Peninsular Malaysia

Background: Several occupational diseases of multiple origins are encountered among abattoir workers. Presence of indicator microorganisms (coliforms) on hands of workers can be used a gauge for hygienic practices. Methods: A cross-sectional study was performed to assess the prevalence of E.coli and enterobacteriaceae among Halal abattoir workers in some government halal abattoirs of Malaysia. A total of one hundred and sixty-five hand swab samples were collected from workers of Halal abattoirs in Malaysia. The samples were subjected to microbiological analysis for characterisation and serotyping. Results: The results have shown that no Escherichia coli O157:H7 was isolated on the hands of abattoir workers before and after work. However, a total prevalence of 9.7% was recorded for all samples during work. For non-O157:H7, total prevalence of 33.3% during work and 13% after work were obtained. High prevalence was recorded in sample taken during work from Tampin, Jasin and Kemaman (100% each) while low prevalence where observed in Shah Alam, Banting and Ipoh (20% each). Conclusions: Based on the findings the hygienic practices of hand washing among the workers in few locations was found to be low especially after work.

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.