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.

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.

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.

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.

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.

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.

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.

A total of 153 Shiga-toxin-producing Escherichia coli (STEC) isolates from feces of cattle and beef products (hamburgers and ground beef) in Argentina were characterized in this study. PCR showed that 22 (14%) isolates carried stx1 genes, 113 (74%) possessed stx2 genes and 18 (12%) both stx1 and stx2. Intimin (eae), enterohemolysin (ehxA), and STEC autoagglutinating adhesin (saa) virulence genes were detected in 36 (24%), 70 (46%) and in 34 (22%) of the isolates, respectively. None of 34 saa-positive isolates carried the gene eae, and 31 were ehxA-positive. Fourteen (7 of serotype O26:H11 and 4 of serotype O5:H-) isolates had intimin b1, 16 isolates possessed intimin g1 (11 of serotype O145:H- and 5 of serotype O157:H7), 5 isolates had intimin type ε1 (4 of serotypes O103:H- and O103:H2), and one isolate O111:H- showed intimin type θ/g2. Although the 153 STEC isolates belonged to 63 different seropathotypes, only 12 accounted for 58% of isolates. Seropathotype ONT:H- stx2 (18 isolates) was the most common, followed by O171:H2 stx2 (12 isolates), etc. The majority (84%) of STEC isolates belonged to serotypes previously found in human STEC and 56% to serotypes associated with STEC isolated from patients with hemolytic uremic syndrome (HUS). Thus, this study confirms that cattle are a major reservoir of STEC pathogenic for humans. To our knowledge, this is the first study that described the presence of saa gene in STEC of serotypes O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, and ONT:H21. The serotypes O120:H19 and O185:H7 were not previously reported in bovine STEC.

Neste estudo, um total de 153 Escherichia coli produtoras de toxinas Shiga (STEC) isoladas de fezes de gado bovino e de carne moida e hamburgues de carne bovina na Argentina foram caracterizados. Os ensaios de PCR mostraram que 22 (14%) dos isolados possuiam o gene stx1, 113 (74%) o gene stx2 e que 18 (12%) tinham ambos os genes. Os genes de virulência para a intimina (eae), a enterohemolisina (ehxA) e a adesina autoaglutinante de STEC (saa) foram detectados em 36 (24%), 70 (46%) e 34 (22%) dos isolados, respectivamente. Nenhum dos 34 isolados saa-positivos tinham o gene eae, no entanto 31 eram ehxA-positivos. Catorze isolados (7 do serótipo O 26:H11 e 4 do serótipo O5:H-) tinham a intimina b1, 16 possuiam a intimina g1 (11 do serótipo O145:H- e 5 do serótipo O157:H7), 5 isolados foram positivos para a intimina tipo ε1 (4 dos serótipos O103:H- e O103:H2), e um isolado O111:H- apresentou a intimina tipo θ/g2. Enquanto os 153 isolados de STEC pertenciam a 63 seropatótipos, somente 12 representaram 58% dos isolados. O seropatótipo ONT:H- stx2 (18 isolados) foi o mais comum, seguido pelo O171:H2 stx2 (12), etc. A maioria dos isolados (84%) de STEC pertenciam à serótipos encontrados previamente em seres humanos e cêrca de 56% à serótipos associados com STEC isolados de pacientes com a síndrome urémica hemolítica (HUS). Esses dados confirmam que o gado bovino é um importante reservatório de STEC patogênicos para humanos. Segundo nossa informação, este é o primeiro estudo que descreve a presença do gene saa em STEC dos serótipos O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, e ONT:H21. Os serótipos O120:H19 e O185:H7 também não haviam sido descritos previamente em STEC de origem bovina.

En este estudio hemos caracterizado un total de 153 Escherichia coli productores de toxinas Shiga (STEC) aisladas de las heces de ganado bovino y de carne picada y hamburguesas de vacuno en Argentina. Los ensayos de PCR mostraron que 22 (14%) aislamientos llevaban el gen stx1, 113 (74%) presentaban el gen stx2 y que 18 (12%) tenían ambos genes. Los genes de virulencia para la intimina (eae), la enterohemolisina (ehxA) y la adhesina autoaglutinante de STEC (saa) fueron detectados en 36 (24%), 70 (46%) y 34 (22%) de los aislamientos, respectivamente. Ninguno de los 34 aislamientos saa-positivos llevaba el gen eae, pero 31 eran ehxA-positivos. Catorce aislamientos (7 del serotipo O26:H11 y 4 del serotipo O5:H-) tenían la intimina b1, 16 poseían la intimina g1 (11 del serotipo O145:H- y 5 del serotipo O157:H7), 5 aislamientos eran positivos para la intimina tipo ε1 (4 de los serotipos O103:H- y O103:H2), y un aislamiento O111:H- mostró la intimina tipo θ/g2. Aunque los 153 aislamientos de STEC pertenecían a 63 seropatotipos, sólo 12 constituían el 58% de los aislamientos. El seropatotipo ONT:H- stx2 (18 aislamientos) fue el más común, seguido por el O171:H2 stx2 (12 aislamientos), etc. La mayoría de los aislamientos (84%) de STEC pertenecían a serotipos encontrados previamente en seres humanos y el 56% a serotipos asociados con STEC aislados de pacientes con el síndrome urémico hemolítico (HUS). Por tanto, este estudio confirma que el ganado bovino es un importante reservorio de STEC patógenos para humanos. Según nuestra información, este es el primer estudio que describe la presencia del gen saa en STEC de los serotipos O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, y ONT:H21. Los serotipos O120:H19 y O185:H7 tampoco habían sido descritos previamente en STEC de origen bovino.

The hemolytic-uremic syndrome (HUS) is a multisystemic disorder that is characterized by the onset of acute renal failure, microangiopathic hemolytic anemia and thrombocytopenia. It is the most common cause of acute renal failure and the second cause of chronic renal failure and renal transplantation in children in Argentina. Our country has the highest incidence of HUS in the world, with approximately 420 new cases observed each year with an incidence of 12.2 cases per 100 000 children in the age group 0-5 years. Numerous etiologic factors have been associated with HUS but the infection with enterohemorrhagic Escherichia coli (EHEC) is considered the most common cause. The majority of outbreaks and sporadic cases in humans have been associated with serotype O157:H7, although other O:H serotypes have been isolated, and they are a subgroup of Verocytotoxin-producing Escherichia coli (VTEC). Cattle are the principal reservoir of VTEC. Infections in humans are a consequence of consumption of undercooked meat, raw milk and other contaminated food or water. Direct contact with animals or people infected is another source of infection.

El síndrome urémico hemolítico (SUH).es un desorden multisistémico caracterizado por presentar insuficiencia renal aguda, anemia hemolítica microangiopática y trombocitopenia. 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 O157:H7, aunque otros serotipos han sido también aislados, y éstos son un subgrupo de E. coli verocitotoxigénico (VTEC)..El bovino es considerado el principal reservorio 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.

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% 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.

In the present study PCR was applied to detect leptospires in human urine. Several approaches for sample processing were evaluated to optimize the detection of leptospires in urine mixed with this bacterium. Furthermore, some changes in the composition of the reaction mix were studied. No amplification was observed in acidic urine, therefore neutralization of the sample immediately after collection is strongly recommended. PBS gave better results than Tris or NaOH as neutralizing reagents. Freezing and thawing of samples before processing yielded negative results. Elimination of epithelial cells, leukocytes and crystals by centrifugation at 3,000 rpm at room temperature increased sensitivity. In addition, both the washing step after collecting leptospires by centrifugation and the inclusion of 0.1% bovine serum albumin in the reaction mix minimized the interference of other inhibitory compounds. These modifications were useful to improve the detection of Leptospira in urine by PCR.