Characterization of Shiga toxigenic Escherichia coli isolated from foods.

The aim of this study was to characterize Shiga toxigenic Escherichia coli (STEC) by PCR using strains isolated from ham, beef, and cattle in Colombia. A total of 189 E. coli strains were tested for the presence of the uidA, stx1, and stx2 genes, and identification was confirmed by the automated PCR BAX system for E. coli O157:H7. Genes encoding Shiga-like toxins (stx) were found in eight (6.06%) of 132 strains previously isolated from minced beef; four (50%) of these strains yielded amplification products for both toxin genes (stx1 and stx2), and four (50%) yielded products only for the stx2 toxin. None of the strains analyzed were positive by PCR for the presence of the single base-pair mutation in the uidA gene from E. coli O157:H7; these results were confirmed by the BAX system analysis. A multiplex PCR assay was standardized for the three genes. Results from this study confirmed previous data about the low prevalence of E. coli O157:H7 and Shiga-like toxins in Colombia and is the first known report of the prevalence of non-O157 enterohemorrhagic E. coli in this country.

[Molecular typing of Listeria monocytogenes isolated from clinical and food samples]. / Tipificación molecular de Listeria monocytogenes aisladas de muestras clínicas y alimentos.

INTRODUCTION: Listeria monocytogenes is an emergent foodborne pathogen acquired by the ingestion of contaminated food. This bacterium causes a disease called listeriosis, whose mortality rate world wide is around 20% to 30%, reaching up to 80% in cases of neonatal infections. The random amplified polymorphic DNA technique allows different isolates to be distinguished and characterized at the molecular level, which can provide useful information about the diversity of this pathogen in Colombia. OBJECTIVE: To molecularly characterize different L. monocytogenes isolates from food and clinical samples using this technique to determine possible relationships among these two origins. MATERIALS AND METHODS: Thirty eight L. monocytogenes isolates were analyzed; 22 from human clinical samples and 16 from food processing plants and food using two 10bp primers (HLW74, Arbitrary). The data were analyzed using Quantity One and SYN-TAX software. RESULTS: A high percentage of polymorphism was detected with both primers (HLWL-74, 81.81%; Arbitrary, 85.71%). Two major lineages were found, which were divided into four major clusters (A, B C and D) and great genetic diversity was observed. Most of the clinical isolates were grouped within the same cluster, and were more distantly related to the food isolates. CONCLUSION: The results of this study demonstrate a high degree of genetic diversity of DNA polymorphisms among the L. monocytogenes isolates circulating in Colombia, which could reflect phenotypic and pathogenic differences in these isolates.

Molecular characterization of diarrheagenic Escherichia coli strains from stools samples and food products in Colombia.

The prevalence of diarrheagenic Escherichia coli in childhood diarrhea and the role of contaminated food products in disease transmission in Colombia are largely unknown. The aim of this study is to identify E. coli pathotypes, including E. coli O157:H7, from 108 stool samples from children with acute diarrhea, 38 meat samples and 38 vegetable samples. Multiplex PCR and Bax Dupont systems were used for E. coli pathotype detection. Eighteen (9.8%) E. coli diarrheagenic pathotypes were detected among all clinical and food product samples tested. Four different pathotypes were identified from clinical samples, including enteroaggregative E. coli, enterotoxigenic E. coli, shiga-toxin producing E. coli, and enteropathogenic E. coli. Food product samples were positive for enteroaggregative and shiga-toxin producing E. coli, suggesting that meat and vegetables may be involved in transmission of these E. coli pathotypes in the community. Most E. coli strains identified belong to the phylogenetic groups A and B1, known to be associated with intestinal rather than extraintestinal E. coli clones. Our data is the first molecular E. coli report that confirms the presence of E. coli pathotypes circulating in Colombia among children with diarrhea and food products for human consumption. Implementation of multiplex PCR technology in Latin America and other countries with limited resources may provide an important epidemiological tool for the surveillance of E. coli pathotypes from clinical isolates as well as from water and food product samples.

Serotipificación molecular e identificación del fragmento 85M deaislamientos colombianos de Listeria monocytogenes: Un estudio descriptivo / Molecular serotyping and identification of the 85M gragment in different Colombian isolates of Listeria monocytogenes strains: A descriptive study

Introduction: Listeria monocytogenes is a pathogen acquired through the consumption of contaminated foods.Thirteen serotypes have been reported, of which 1/2a, 1/2b, and 4b are responsible for 98% of human listeriosis cases. This study examines the association between serotypes and virulent clones, offering greater information and providingtools to prevent and control diseases caused by L. monocytogenes serotype 4b. Objective: To identify the serotypes from L. monocytogene strains isolated from different samples by performingthe molecular subtyping technique; to determine the 85M fragment that codifies for epidemic clone I.Methods : 108 strains of L. monocytogenes were used, isolated from samples of animals, body fluids, foods, and food processing plant equipment and spaces. The samples were identified by following the Bacteriological AnalyticalManual protocol described by the Food and Drug Administration (FDA). The strains were identified by PolymeraseChain Reaction (PCR) using primers and a standardized protocol from a previous research project. Serotypeidentification was performed by multiplex PCR. The determination of the 85M fragment of the SSCS cassette was done by following the protocol by Yildrim et al. Results : Of the 108 L. monocytogenes strains analyzed, 60.2% (65 strains) belonged to the 4b-4d-4e serotype, 17.6%(19 strains) were identified as 1/2a-3a serotype, 14.8% (16 strains) were 4a-4c serotype, 3.7% (4 strains) belonged to the 1/2c-3c serotype, and (3.7%) corresponded to the 1/2b-3b-7 serotype. It was determined that the L. monocytogenes strains serotype 4b-4d-4e and 1/2a-3b have the 85M fragment of the SSCS cassette.Conclusion : This study reports the predominant existence of L. monocytogenes strains serotype 4b-4d-4e in food, environmental, and clinical samples. The presence of an epidemic clone I region was also found in L. monocytogenes strains.

Aislamiento e identificación de Enterobacter sakazakii en lactarios de Bogotá / Isolation and identification of Enterobacter sakazakii in milk feeders of Bogotá, D. C

Introducción. Enterobacter sakazakii es un patógeno oportunista emergente de alto riesgo, responsable de meningitis grave y enterocolitis necrosante. El principal vehículo de transmisión de esta bacteria son los productos infantiles deshidratados, debido a su contaminación después del tratamiento térmico. Objetivo. Identificar cepas de E. sakazakii en muestras de lactarios recolectadas en la ciudad de Bogotá, D.C. Materiales y métodos. Se analizaron 222 muestras de 9 lactarios, de superficies estériles y no estériles, utensilios empleados para la preparación de biberones y operarios. Se realizó recuento de coliformes totales y detección de E. sakazakii utilizando el protocolo propuesto por la Food and Drug Administration y por el Sistema Automático Bax® Dupont Qualicon. Resultados. De las 222 muestras recolectadas en las clínicas de Bogotá, se reportó que 27,4% (61) de las muestras analizadas presentaban coliformes totales; se detectó la presencia de E. sakazakii en 3,6% por el método automatizado de PCR BAX Dupont a partir de muestras de biberones y superficies. Conclusiones. Se demostró la presencia de E. sakazakii en lactarios en Colombia. Debido a que este microorganismo es un patógeno oportunista de alto riesgo para neonatos y que está asociado a las prácticas higiénicas en los lactarios, la información de este estudio puede ser útil para la toma de medidas profilácticas que reduzcan el riesgo de contaminación con este patógeno para la población infantil y, también, aporta información importante para la salud pública.

Introduction: Enterobacter sakazakii is an emergent opportunistic pathogen of high risk responsible of severe meningitis and necrotizing enterocolitis in neonates and infants with underlying medical conditions. One of the principal transmission vehicles for the transmission of these bacteria, is the infant dehydrated formula after exposing them to the heating treatment. Objective: To identify strains of E. sakazakii in milk feeders’ samples from Bogotá. Materials and methods: 222 samples from 9 milk feeders including sterile and non sterile surfaces, utensils used for the formula preparation and food handlers were analyzed. Total coliforms counts and identification of E. sakazakii was done using the FDA protocol and the automatic system Bax ® Dupont Qualicon. Results: From de 222 samples collected from hospitals in Bogotá, it was reported that 27.4% (61) had total coliforms, and the presence of E. sakazakii was detected in 3.6% (8) from one feeding bottle and surfaces. Conclusion: The presence of E. sakazakii strains was reported in Colombian milk feeders. Because this microorganism is a high risk opportunistic pathogen for newborn infants, usually associated with hygiene practices in milk feeders, the information of this research could be useful to develop preventive measurements to reduce the risk of contamination in the infant population and provides important public health information.

Tipificación molecular de Listeria monocytogenes aisladas de muestras clínicas y alimentos / Molecular typing of Listeria monocytogenes isolated from clinical and food samples

Introducción. Listeria monocytogenes es un patógeno emergente adquirido por el consumo de alimentos contaminados. Causa una enfermedad llamada listeriosis, cuya tasa de mortalidad a nivel mundial varía entre 20 por ciento y 30 por ciento, alcanzando hasta un 80 por ciento en casos de infecciones neonatales. La técnica del ADN polimorfo amplificado aleatorio permite distinguir entre diferentes aislamientos y caracterizarlos molecularmente, lo cual aporta información útil acerca de la diversidad de este patógeno en Colombia. Objetivo. Caracterizar molecularmente diferentes aislamientos de L. monocytogenes aisladas de muestras clínicas y alimentos utilizando ésta técnica para determinar posibles relaciones entre estos dos orígenes. Materiales y métodos. Se analizaron 38 aislamientos de L. monocytogenes; 22 de muestras clínicas y 16 de alimentos y plantas procesadoras de alimentos utilizando dos oligonucléotidos de 10pb (HLWL-74 y Arbitrario). Los datos se analizaron utilizando los programas Quantity One y SYN-TAX. Resultados. Se detectó un alto porcentaje de polimorfismo mediante los oligonucleótidos HLWL-74 (81,81 por ciento) y Arbitrario (85,71 por ciento). Se pudieron describir dos linajes superiores luego del análisis, los cuales se dividieron a su vez en cuatro grupos mayores (A, B C y D) donde se observó una gran diversidad genética. La mayoría de aislamientos clínicos se agruparon bajo el mismo grupo y se encontraron alejados de los aislamientos de alimentos. Conclusión. Los resultados de este estudio demuestran que existe una gran diversidad de polimorfismos de ADN entre los aislamientos de L. monocytogenes que circulan en Colombia, lo que podría reflejar diferencias a nivel fenotípico y patogénico en estos aislamientos.

Introduction. Listeria monocytogenes is an emergent foodborne pathogen acquired by the ingestion of contaminated food. This bacterium causes a disease called listeriosis, whose mortality rate world wide is around 20% to 30%, reaching up to 80% in cases of neonatal infections. The random amplified polymorphic DNA technique allows different isolates to be distinguished and characterized at the molecular level, which can provide useful information about the diversity of this pathogen in Colombia. Objective. To molecularly characterize different L. monocytogenes isolates from food and clinical samples using this technique to determine possible relationships among these two origins. Materials and methods. Thirty eight L. monocytogenes isolates were analyzed; 22 from human clinical samples and 16 from food processing plants and food using two 10bp primers (HLW74, Arbitrary). The data were analyzed using Quantity One and SYN-TAX software. Results. A high percentage of polymorphism was detected with both primers (HLWL-74, 81,81%; Arbitrary, 85,71%). Two major lineages were found, which were divided into four major clusters (A, B C y D) and great genetic diversity was observed. Most of the clinical isolates were grouped within the same cluster, and were more distantly related to the food isolates. Conclusion. The results of this study demonstrate a high degree of genetic diversity of DNA polymorphisms among the L. monocytogenes isolates circulating in Colombia, which could reflect phenotypic and pathogenic differences in these isolates.

Caracterización molecular de cepas toxigénicas de Staphylococci aisladas de operarios de plantas de alimentos / Molecular characterization of toxigenic Staphylococci strains isolated from food handlers

Resumen Objetivo. Utilizar la técnica de amplificación aleatoria de ADN polimórfico ( random amplification of polymorphic DNA, RAPD para caracterizar molecularmente cepas de Staphylococci productoras de toxinas, aisladas de operarios de plantas de producción de alimentos. Materiales y métodos. Se utilizaron 31 aislamientos enterotoxigénicos de Staphylococci para la extracción y cuantificación de ADN. Posteriormente, se realizó un ciclo general de amplificación utilizando los oligonucleótidos HLWL-74 y arbitrario, seguido por la visualización de los productos de RAPD por electroforesis en gel de agarosa. Resultados. Para los dos oligonucleótidos utilizados, se observaron de 1 a 15 bandas, dos linajes, divididos en tres conglomerados (A, B y C). El oligonucleótido arbitrario generó 10 bandas polimórficas (66,66%) y el oligonucleótido HLWL-74 arrojó 13 bandas altamente polimórficas (86,66%). Cada oligonucleótido mostró un agrupamiento diferente de cada una de las cepas, lo cual muestra una alta diversidad de aislamientos de Staphylococci presentes en humanos. Conclusiones. Se presentó una alta diversidad molecular en cuanto a aislamientos de garganta, nariz y manos de un mismo individuo, así como en todos los aislamientos analizados, lo cual demuestra que las cepas enterotoxigénicas de Staphylococci encontradas en los operarios analizados tienen una alta diversidad molecular.

Objective. To use Randomly Amplified Polymorphic DNA (RAPD) to compare molecularly enterotoxigenic Staphylococci strains isolated from people working in food processing plants. Materials and methods. 31 Staphylococci enterotoxigenic isolates were used for extraction and quantification of DNA, followed by a general amplification cycle with the HLWL-74 and arbitrary oligonucleotides with visualization of the RAPD products by agarose gel electrophoresis. Results. The two oligonucleotides used generated 1 to 15 bands, two mayor lineages divided in three clusters (A, B, and C). The arbitrary oligonucleotide generated 10 polymorphic bands (66.66%), the HLWL-74 oligonucleotide generated 13 polymorphic bands (86.66%). Each oligonucleotide generated a different type of grouping with respect to each of the strains analyzed. This shows a high diversity between the human isolates of Staphylococci. Conclusion. A high molecular diversity was present amongst throat, nose and hands isolates from the same person, and among the analyzed isolates; this demonstrates a high molecular diversity in Staphylococci enterotoxigenic isolates.

Capacidad bactericida de Bifidobacterium sp. aislada de leche materna y de heces de neonatos, frente a los principales causantes de enfermedades transmitidas por alimentos / Antibiotic activity of Bifidobacterium sp. isolated from breast milk and newborn faeces, against the main causes for foodborne illnesses

Introducción. La microbiota del tubo digestivo humano contiene bacterias benéficas para la salud que regulan el funcionamiento del colon e inhiben algunos microorganismos patógenos intestinales. Las bifidobacterias aisladas de neonatos y de leche materna se usan como microorganismos probióticos para prevenir enfermedades infecciosas, incluidas las transmitidas por alimentos. Objetivo. Aislar e identificar Bifidobacterium sp. en humanos y determinar su capacidad bactericida frente a patógenos causantes de enfermedades transmitidas por alimentos, importantes en Colombia y en el mundo. Materiales y métodos. Se recolectaron 17 muestras de leche materna, y 19 muestras de meconio y heces de neonatos, en diferentes hospitales de Bogotá. Los 26 aislamientos sospechosos se identificaron a nivel de género mediante PCR 16-23S; para la identificación de especie, se secuenciaron algunos de los aislamientos. La capacidad antagonista de las 26 cepas de Bifidobacterium sp. fue evaluada contra Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076, Listeria monocytogenes ATCC 7644 y E. coli O157:H7 ATCC 35150. Para las cepas que presentaron mayor actividad antagonista, se analizó el extracto inhibidor con ensayos de difusión en placa. Resultados. Todas las cepas amplificaron la banda esperada para la confirmación de género; asimismo, las cepas Bif 013 y Bif 023 se identificaron por secuenciación como Bifidobacterium breve, con una homología del 97%. Del total de cepas, 17 mostraron capacidad de inhibir, al menos, uno de los patógenos evaluados. E. coli ATCC 25922 fue el patógeno más inhibido. Se determinó que la cepa Bif 023 es eficiente como antagonista, ya que inhibió todos los patógenos evaluados. Los halos de inhibición presentaron diámetros mayores a lo esperado, lo que indica una muy buena capacidad antagonista de las cepas nativas. Se aisló Bifidobacterium sp. de leche materna, meconio y heces de neonatos, por lo cual se confirmó que este microorganismo es microbiota humana (2). Conclusión. Se concluye que, debido a la gran capacidad antagonista de la mayoría de las bacterias aisladas, éstas pueden estar cumpliendo una importante función protectora en el recién nacido, en particular las cepas Bif 013 y Bif 023 aisladas de materia fecal. Estos microorganismos deben continuar siendo estudiados para definir su potencial probiótico. También, se pueden evaluar para bioconservación en la industria y contra patógenos transmitidos por alimentos.

Introduction. The microbiota in the human gastrointestinal tract contains beneficial microorganisms for human health, which contribute to the regulation of colonic function and inhibition of some intestinal pathogens growth. Bifidobacterium sp. isolated from newborns and breast milk are used as probiotic microorganisms, which are useful in the prevention of infectious diseases including foodborne illnesses. Objective: To isolate and identify human Bifidobacterium sp. and to determine its antibiotic activity against important pathogens which cause foodborne illnesses in Colombia and the world. Materials and methods. 17 breast milk samples and 19 meconium and newborn faeces samples were collected from different hospitals in Bogotá. 26 presumptive Bifidobacterium strains were identified at genus level by PCR 16-23S; some strains were identified at species level by nucleic acid sequencing. The antagonistic activity of 26 Bifidobacterium sp. strains was tested against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076, Listeria monocytogenes ATCC 7644 and E. coli O157:H7 ATCC 35150. For the strains that showed a greater antibiotic activity, the inhibitory compound was analyzed using disk diffusion tests. Results. All strains amplified the expected band for genus confirmation. Strains Bif 023 and Bif 013 were identified by DNA-sequencing as Bifidobacterium breve, with 97% homology. 17 strains were able to inhibit at least one of the pathogens tested. Escherichia coli ATCC 25922 was the most inhibited. It was determined that the strain Bif 023 is highly efficient as an antagonist strain due its ability to inhibit all the evaluated pathogens. Inhibition areas showed higher diameters than expected, suggesting an enhanced antagonist capacity of native strains. Bifidobacterium sp. was isolated from breast milk, meconium and newborn faeces which confirmed that this microorganism is human microbiota. Conclusion. Due to the high antagonist activity of most isolated bacteria, they could be playing an important protective function in the newborn, in particular strains Bif 013 and Bif 023, isolated from faeces. Other studies must be performed with these organisms to determine their probiotic potential as well as their use in the biocontrol industry due their activity against foodborne pathogens.