Caracterización molecular de aislamientos de Escherichia coli productores de toxina Shiga obtenidos en 2 establecimientos ganaderos del Paraguay Caracterización molecular de aislamientos de STEC de ganado bovino paraguayo / Molecular characterization of Shiga toxin producing Escherichia coli isolated from 2 livestock establishments of Paraguay

Resumen Escherichia coli productora de toxina Shiga (STEC) es un patógeno de importancia alimentaria en los humanos, el bovino es su principal reservorio. El objetivo de este estudio fue determinar la portación de STEC en bovinos del Paraguay y analizar el perfil de virulencia y los serotipos de los aislados reunidos. Se estudiaron 197 muestras de hisopado rectal de bovinos y un promedio de 5 a 50 colonias por bovino positivo a genes stx 1 /stx 2. Se amplificaron por PCR los genes stx 1, stx 2, saa, ehxA y eae. El 84,8% de los bovinos resultaron portadores de STEC. Los perfiles de virulencia predominantes fueron stx 2 y stx 2 /saa/ehxA. La serotipificación se realizó por reacciones de aglutinación en 60 aislamientos seleccionados, se encontró un aislamiento del serogrupo O103, capaz de producir infecciones en humanos. Este trabajo muestra los primeros datos de portación de STEC de ganado bovino paraguayo y señala la necesidad de efectuar otros estudios con mayor cobertura territorial, para lograr una visión completa de este fenómeno.

Abstract Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen in humans, with cattle being the main reservoir. The objective of this study was to determine the carrying of STEC in Paraguayan bovines and to analyze the virulence profile and serotypes of these isolates. A total of 197 samples of bovine fecal samples and an average of 5 to 50 colonies from stx 1 /stx 2 positive samples were studied. The stx 1 , stx 2 , saa, ehxA and eae genes were amplified by PCR. 84.8% of the cattle were carriers of STEC. The predominant virulence profiles were stx 2 and stx 2 /saa/ehxA. The serotyping was performed by agglutination reactions for 60 selected isolates, resulting in isolation of serogroup O103, which could produce infections in humans. This work shows the first data of STEC carriers in Paraguayan cattle, and indicates the need for other studies with greater territorial coverage for a complete vision of this phenomenon.

Efectos del medio de cultivo y de la metodología aplicada sobre la formación de biopelículas de 2 cepas de Escherichia coli diarreagénicas / Effects of the culture medium and the methodology applied on the biofilm formation of 2 diarrheagenic Escherichia coli strains

La capacidad de formar biopelículas de los microorganismos patógenos en gran variedad de ambientes, superficies y condiciones trae consigo un importante riesgo, tanto para la industria alimentaria como para la salud pública. Este trabajo tuvo como objetivo evaluar y comparar los efectos de la metodología empleada y de los medios de cultivo utilizados, sobre la capacidad de una cepa de Escherichia coli verotoxigénica no O157 y una enteropatogénica de formar biopelículas sobre una superficie de poliestireno. Se ensayaron 2 variantes metodológicas en cultivo estático y se utilizaron medios de cultivo con diferente composición. Los resultados mostraron que ambas cepas formaron una mayor cantidad de biopelícula en cultivo en LB suplementado con glucosa, con recambio del medio a las 24 h y la cuantificación de la biopelícula realizada a las 48 h de incubación. Dichas condiciones podrían ser utilizadas en futuros estudios sobre formación de biopelícula.

The ability to form biofilms of pathogenic microorganisms in a wide variety of environments, surfaces and conditions constitute an important risk, both for the food industry and for public health. The aim of this work was to evaluate and to compare the effects of the methodology applied and the culture medium used on the ability of a non-O157 verotoxigenic Escherichia coli strain and an enteropathogenic strain to form biofilm on polystyrene surface. Two methodological variants were tested in static culture and culture mediums with different composition were used. The results showed that both strains were able to form a greater biofilm under culture in LB supplemented with glucose, with medium replacement at 24 h and the quantification of the biofilm carried out at 48 h of incubation. These conditions could be used in future studies on biofilm formation.

Efecto inhibitorio de Lactobacillus spp. sobre bacterias implicadas en enfermedades transmitidas por alimentos / Inhibitory capacity of Lactobacillus spp. against pathogens involved in foodborne diseases

El género Lactobacillus despierta día a día un creciente interés entre microbiólogos y tecnólogos, quienes intentan descubrir nuevas aplicaciones biotecnológicas y propiedades probióticas. El objetivo de este trabajo fue evaluar la capacidad inhibitoria de Lactobacillus spp. frente a patógenos implicados en enfermedades de transmisión alimentaria (ETA), como Escherichia coli O157:H7, Salmonella spp. y Staphylococcus aureus. Para ello se tomaron muestras de las distintas etapas de la cadena productiva porcina. De dichas muestras se aislaron en total 78 cepas bacterianas, de las cuales 27 (34,61%) tuvieron características fenotípicas y genotípicas correspondientes al género Lactobacillus spp.; el 85,18% de ellas presentó capacidad inhibitoria frente a por lo menos una de las cepas patógenas evaluadas. Estos resultados indican que los microorganismos aislados representan una potencial alternativa para inactivar a los patógenos presentes en los alimentos y así brindar alimentos más seguros a los consumidores.

The genus Lactobacillus daily generates a growing interest among microbiologists and technologists, who try to discover new biotechnological applications and probiotic properties. The main goal of this study was to evaluate the inhibitory capacity of Lactobacillus spp. against pathogens (Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus) involved in foodborne diseases. For this purpose, samples were collected at different stages of the pork production chain. Seventy eight bacterial strains were isolated. Twenty seven (27) of these strains (37.18%) had genotypic and phenotypic characteristics corresponding to Lactobacillus spp. whereas 85.18% of them showed inhibitory capacity. These data showed that the studied strains represent a potential alternative to inactivate foodborne pathogens and thus provide safe food to consumers.

Antibiotic resistance and integrons in Shiga toxin-producing Escherichia coli (STEC)

Shiga toxin-producing Escherichia coli (STEC) cause hemorrhagic colitis (HC) and hemolytic-uremic syndrome in humans (HUS). Cattle are the main reservoir of STEC and transmission to humans occurs through contaminated food and water. Antibiotics are used in pig production systems to combat disease and improve productivity and play a key role in the dissemination of antibiotic resistance genes to the bacteria. Integrons have been identified in resistant bacteria allowing for the acquisition and dissemination of antibiotic resistance genes. STEC strains isolated from humans and animals have developed antibiotic resistance. In our laboratory, 21 non-157 STEC strains isolated from pigs were analyzed to detect class 1 and 2 integrons by PCR. Eight carried integrons, 7 of them harbored intl2. In another study 545 STEC strains were also analyzed for the presence of intl1 and intl2. Strains carrying intl1 belonged to isolates from environment (n = 1), chicken hamburger (n = 2), dairy calves (n = 4) and pigs (n = 8). Two strains isolated from pigs harbored intl2 and only one intl1/intl2, highlighting the presence of intl2 in pigs. The selection for multiresistant strains may contribute to the emergence of antibiotic resistant pathogens and facilitate the spreading of the mobile resistance elements to other bacteria.

Supervivencia de VTEC O157 y no-O157 en agua de bebederos y materia fecal de bovinos / Survival of VTEC O157 and non-O157 in water troughs and bovine feces

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Verotoxin-producing Escherichia coli (VTEC) is the etiologic agent of hemolytic-uremic syndrome (HUS), which typically affects children ranging in age from six months to five years old. Transmission is produced by consumption of contaminated food, by direct contact with animals or the environment and from person to person. In previous studies we determined that the environment of a dairy farm is a non-animal reservoir; thus, we proposed to study the survival of 4 VTEC isolates (O20:H19; O91:H21; O157:H7 and O178:H19) in sterile water troughs and bovine feces by viable bacteria count and detection of virulence genes by PCR. It was demonstrated that the survival of different VTEC isolates (O157 and non-O157) varied in terms of their own characteristics as well as of the environmental conditions where they were found. The main differences between isolates were their survival time and the maximal counts reached. The competitive and adaptive characteristics of some isolates increase the infection risk for people that are visiting or working on a farm, as well as the risk for reinfection of the animals and food contamination.

Recommendations for the detection of Leptospira in urine by PCR

No presente estudo, a PCR foi utilizada para detectar leptospiras em urina humana. Diversas abordagens para processamento de amostra foram avaliadas para otimizar a detecção de leptospiras em urina misturada com esta bactéria. Além disso, algumas mudanças na composição da mistura de reação foram analisadas. Não se observou amplificação em urina ácida, conseqüentemente, a neutralização da amostra imediatamente após a coleta é fortemente recomendada. PBS apresentou melhores resultados que Tris ou NaOH como reagentes neutralizadores. Congelamento e descongelamento de amostras antes do processamento produziram resultados negativos. Eliminação de células epiteliais, leucócitos e cristais por centrifugação a 3.000rpm, à temperatura ambiente, aumentou a sensibilidade. Ademais, ambas, a etapa de lavagem após a coleta de leptospiras por centrifugação e a inclusão de albumina de soro bovino a 0,1 por cento na mistura de reação minimizaram a interferência de outros compostos inibidores. Essas modificações contribuíram para melhorar a detecção de Leptospira em urina através da PCR.

Escherichia coli enterohemorragica y sindrome uremico hemolitico en Argentina / Enterohemorrhagic escherichia coli and hemolytic-uremic syndrome in Argentina

El síndrome urémico hemolítico (SUH) es un desorden multisistémico caracterizado por presentar insuficiencia renal aguda, anemia hemolítica microangiopática y trobocitopenia. Constituye la principal causa de insuficiencia renal aguda y la segunda causa de insuficiencia renal crónica y de transplante renal en niños en la Argentina. Actualmente, nuestro país presenta el registro más alto de SUH en todo el mundo, con aproximadamente 420 casos nuevos declarados anualmente y una incidencia de 12.2/100 000 niños menores de 5 años de edad. Se reconocen múltiples agentes etiológicos, aunque se considera a la infección por Escherichia coli enterohemorrágica (EHEC) como la principal etiología de SUH. La gran mayoría de brotes epidémicos y casos esporádicos en humanos se han asociado con el serotipo 0157:H7, aunque otros serotipos han sido también aislados, y éstos son un subgrupo de E.coli verocitotoxigénico (VTEC). El bovino es considerado el principal reservario de VTEC. El contagio al hombre frecuentemente se debe al consumo de alimentos cárneos y lácteos contaminados, deficientemente cocidos o sin pasteurizar, o al contacto directo con los animales o con sus heces, consumo de agua, frutas o verduras contaminadas. También puede producirse contagio mediante el contacto interhumano.