RESUMO
The aims of the present study were: a) to estimate the minimal dose of gamma irradiation required to reduce 5 log CFU/g of native O157 and non-O157 Shiga toxin-producing Escherichia coli population in ground beef samples inoculated with high inoculum; b) to assess its effectiveness in samples with low inoculum and 3) to evaluate consumer acceptance. Based on the results, 1 kGy was estimated as the minimal dose of gamma irradiation required to reduce 5 log CFU/g of STEC in ground beef. However, when samples with low inoculum level were subjected to 1 kGy, 3.9% of the samples were positive for stx and eae genes after an enrichment step. Consumer acceptance analysis was carried out with samples subjected to 2.5 kGy and no significant differences were found between irradiated and control samples. Therefore, 2.5 kGy was identified as the gama irradiation dose that reduces STEC but has no impact on consumer acceptance of ground beef.
Assuntos
Irradiação de Alimentos/métodos , Produtos da Carne/microbiologia , Escherichia coli Shiga Toxigênica/efeitos da radiação , Animais , Argentina , Bovinos , Comportamento do Consumidor , Raios gama , Humanos , Escherichia coli Shiga Toxigênica/genéticaRESUMO
The non-O157 Shiga toxin-producing Escherichia coli (STEC) contamination in carcasses and feces of 811 bovines in nine beef abattoirs from Argentina was analyzed during a period of 17 months. The feces of 181 (22.3%) bovines were positive for non-O157 STEC, while 73 (9.0%) of the carcasses showed non-O157 STEC contamination. Non-O157 STEC strains isolated from feces (227) and carcasses (80) were characterized. The main serotypes identified were O178:H19, O8:H19, O130:H11, and O113:H21, all of which have produced sporadic cases of hemolytic-uremic syndrome in Argentina and worldwide. Twenty-two (7.2%) strains carried a fully virulent stx/eae/ehxA genotype. Among them, strains of serotypes O103:[H2], O145:NM, and O111:NM represented 4.8% of the isolates. Xba I pulsed-field gel electrophoresis pattern analysis showed 234 different patterns, with 76 strains grouped in 30 clusters. Nine of the clusters grouped strains isolated from feces and from carcasses of the same or different bovines in a lot, while three clusters were comprised of strains distributed in more than one abattoir. Patterns AREXSX01.0157, AREXBX01.0015, and AREXPX01.0013 were identified as 100% compatible with the patterns of one strain isolated from a hemolytic-uremic syndrome case and two strains previously isolated from beef medallions, included in the Argentine PulseNet Database. In this survey, 4.8% (39 of 811) of the bovine carcasses appeared to be contaminated with nonO157 STEC strains potentially capable of producing sporadic human disease, and a lower proportion (0.25%) with strains able to produce outbreaks of severe disease.
Assuntos
Matadouros , Bovinos/microbiologia , Qualidade de Produtos para o Consumidor , Contaminação de Alimentos/análise , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Argentina/epidemiologia , DNA Bacteriano/análise , Surtos de Doenças/prevenção & controle , Fezes/microbiologia , Feminino , Contaminação de Alimentos/prevenção & controle , Genótipo , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/prevenção & controle , Humanos , Masculino , Prevalência , Medição de Risco , Sorotipagem , Escherichia coli Shiga Toxigênica/classificação , Pele/microbiologiaRESUMO
Multiplex PCR for the detection of sea, seb, sec, sed and see genes of Staphylococcus aureus. Characterization of isolates from food. The presence of Staphylococcus aureus in food represents a potential risk to public health, being its enterotoxins the major virulence factor. Enterotoxin detection can be determined by ELISA, but only for the pool of enterotoxins SEA, SEB, SEC, SED and SEE. The main aims of this study were to optimize two PCR techniques for detection of S. aureus sea, seb, sec, sed and see, and to characterize Staphylococcus spp. isolates associated with food intoxication. Two PCR techniques were optimized and 115 Staphylococcus spp. isolates from Ciudad Aut6noma de Buenos Aires, and Buenos Aires, Córdoba, and Neuquén provinces were characterized. The characterization was performed by biochemical tests, ELISA and PCR. Sixty-eight isolates (59.1%) were positive by ELISA, while 61 (53%) were positive by PCR. Out of the positive PCR isolates, 34 (55.7%) carried the sea gene, 9 (14.8%) the seb gene, 5 (8.1%) the see gene, 4 (6.5%) the sec gene, 6 (9.9%) were positive for sea and seb genes, 2 (3.3%) for sea and sec genes, and 1 (1.7%) for sea and sedgenes. This is the first study of genotypic characterization of S. aureus isolates associated with food intoxication from different provinces of Argentina.
Assuntos
Genes Bacterianos/genética , Staphylococcus aureus/genética , Microbiologia de Alimentos , Reação em Cadeia da Polimerase/métodos , Staphylococcus aureus/isolamento & purificaçãoRESUMO
In Argentina, Escherichia coli O157:H7/NM (STEC O157) is the prevalent serotype associated with hemolytic uremic syndrome (HUS), which is endemic in the country with more than 400 cases per year. In order to estimate the prevalence and characteristics of STEC O157 in beef cattle at slaughter, a survey of 1,622 fecal and carcass samples was conducted in nine beef exporting abattoirs from November 2006 to April 2008. A total of 54 samples were found positive for STEC O157, with an average prevalence of 4.1% in fecal content and 2.6% in carcasses. Calves and heifers presented higher percentages of prevalence in feces, 10.5 and 8.5%, respectively. All STEC O157 isolates harbored stx(2) (Shiga toxin 2), eae (intimin), ehxA (enterohemolysin), and fliC(H7) (H7 flagellin) genes, while stx(1) (Shiga toxin 1) was present in 16.7% of the strains. The prevalent (56%) stx genotype identified was stx(2) combined with variant stx(2c (vh-a)), the combination of which is also prevalent (>90%) in STEC O157 post-enteric HUS cases in Argentina. The clonal relatedness of STEC O157 strains was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 54 STEC isolates were categorized into 12 different phage types and in 29 XbaI-PFGE patterns distributed in 27 different lots. STEC O157 strains isolated from 5 of 21 carcasses were identical by PFGE (100% similarity) to strains of the fecal content of the same or a contiguous bovine in the lot. Five phage type-PFGE-stx profiles of 10 strains isolated in this study matched with the profiles of the strains recovered from 18 of 122 HUS cases that occurred in the same period.
Assuntos
Matadouros , Bovinos/microbiologia , Escherichia coli O157 , Fezes/microbiologia , Toxinas Shiga/biossíntese , Animais , Argentina/epidemiologia , Tipagem de Bacteriófagos , Análise por Conglomerados , Contagem de Colônia Microbiana , Qualidade de Produtos para o Consumidor , DNA Bacteriano/análise , Escherichia coli O157/classificação , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/metabolismo , Genótipo , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Epidemiologia Molecular , Fenótipo , Prevalência , Toxinas Shiga/genéticaRESUMO
Shiga toxin-producing Escherichia coli is an emerging foodborne pathogen. There are many STEC serotypes associated with human diseases, being the O157:H7 serotype the most prevalent. Ground beef is the main transmission vehicle. In Concepción city, Tucumán Province, between September and December 2004, two hemolytic uremic syndrome (HUS) cases were diagnosed. The main objective of this work was to detect, isolate and characterize STEC O157 and non-O157 strains in fresh ground beef. Between September and December 2004, 53 fresh ground beef samples were collected from butcher shops in Concepción city. The USDA-FSIS (2002) methodology was used for detection, isolation and characterization of STEC O157:H7. Two PCR techniques for E. coli non-O157 detection and a previous intra-laboratory validated methodology for the isolation and characterization of these strains were used. The stx2 gen was identified in seven samples and the rfbO157 gene also in four of them. However, only one E. coli O157:H7 strain, biotype C, carrying the eae, stx2 and ehxA genes, was isolated. The present study shows the importance of implementing techniques for the detection of this emerging pathogen in meat samples.
Assuntos
Escherichia coli O157/isolamento & purificação , Contaminação de Alimentos , Microbiologia de Alimentos , Carne/microbiologia , Escherichia coli Shiga Toxigênica/isolamento & purificação , Adesinas Bacterianas/genética , Animais , Argentina/epidemiologia , Bovinos , Doenças Endêmicas , Proteínas de Escherichia coli/genética , Proteínas Hemolisinas/genética , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Sorotipagem , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/genéticaRESUMO
En febrero de 2006 ocurrió un brote epidémico de gastroenteritis aguda de origen alimentario, en ocasión de un festejo popular en una pequeña localidad de la provincia de Neuquén, Argentina. Aproximadamente 800 personas participaron de un almuerzo en las instalaciones del Gimnasio Municipal, y unas tres horas después de finalizado, cerca de 150 asistentes consultaron al hospital local, afectados por síndrome gastroentérico agudo. Se realizó una investigación epidemiológica caso-control a través de un muestreo representativo no probabilístico. Los resultados epidemiológicos establecieron un brote de ETA a fuente común, con una relación caso-control de 1:1,8. Los principales síntomas fueron cólicos abdominales (88%), vómitos (73,5%) y diarrea (60%). La torta que se sirvió en ese evento fue identificada como el alimento causal (OR 9,79; IC 95%; 2,66-36,00; valor p = 0,0001), sujeto a condiciones higiénico-sanitarias insatisfactorias en los diferentes procesos de elaboración, conservación y manipulación. De una porción de la torta se aisló una cepa de Staphylococcus aureus subespecie aureus, coagulasa positiva, enterotoxigénica, con un recuento de 2,4x10(6) UFC/g, y también se aisló este microorganismo de tres muestras de manos y narinas de personas involucradas en la preparación y el servicio. Las cepas aisladas de un operador y de la torta portaron el gen sea y presentaron el mismo patrón de SmaI-PFGE. Se atribuyó el brote de ETA a la contaminación durante el proceso de preparación de la torta consumida durante ese almuerzo popular, lo que podría estar relacionado con deficiencias en aspectos higiénicos y con la falta de refrigeración y de mantenimiento de la cadena de frío.
In the summer of 2006, an epidemic outbreak of acute gastrointestinal illness related to food consumption occurred in a small town in the province of Neuquén, Argentina. During a popular feast, approximately 800 local residents attended lunch held in the facilities of the Municipal Gymnasium. About three hours later, nearly 150 attendees sought medical assistance at the local hospital due to acute gastroenteritis. A case-control epidemiological investigation was conducted using representative non-probability sampling. The epidemiological investigation showed a common-source foodborne disease outbreak with a case-control ratio of 1:1.8. The main symptoms were abdominal cramps (88%), vomiting (73.5%) and diarrhea (60%). The cake was identified as the source of infection (OR 9.79; IC 95%, 2.66-36.00; p = 0.0001), and unsatisfactory hygienic conditions in food production, conservation and handling steps were identified. Coagulase positive, enterotoxigenic Staphylococcus aureus, subspecies aureus was detected in a piece of cake, with a count of 2.4x10(6) CFU/g, and in samples from the hands and nostrils of three people involved in food preparation and service. The strains isolated from both the cake and one of the food handlers carried the sea gene, and presented the same SmaI-PFGE pattern. The foodborne disease outbreak was considered to be due to contamination in the preparation process of the cake consumed at the feast, which was related to inadequate hygienic conditions, lack of refrigeration and cold chain disruption.
Assuntos
Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Surtos de Doenças , Contaminação de Alimentos , Gastroenterite/epidemiologia , Intoxicação Alimentar Estafilocócica/epidemiologia , Argentina/epidemiologia , Técnicas de Tipagem Bacteriana , Estudos de Casos e Controles , Portador Sadio/metabolismo , Manipulação de Alimentos , Gastroenterite/etiologia , Gastroenterite/microbiologia , Refrigeração , Intoxicação Alimentar Estafilocócica/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Shiga toxin-producing Escherichia coli is an emergent pathogen, being the Shiga toxin (Stx) the main virulence factor. These toxins are classified into 6 types (1,2, 2c, 2d, 2e and 2f) and 22 variants. In Argentina, two PCR for stx gene detection, PCR-MK and multiplex-PCR, were validated. The aim of this work was to analyze, by using bioinformatic tools, the stx variants that could be amplified by these PCRs, and to experimentally show the amplification of 8 stx variants. Twenty-five nucleotide sequences were collected from GenBank corresponding to 21 stx variants. The BLAST 2 sequences program was used to analyze the complementarities between the nucleotide sequence of the variants and the primers corresponding to the PCR studied. PCR-MK could detect types stx1, stx2, stx2c, stx2d and stx2f, but not type stx2e and three type stx2c variants. On the other hand, the multiplex-PCR could detect types stx1, stx2, stx2c, stx2d, but not stx2e and stx2f types. It was experimentally determined that both PCRs can detect those variants that cause severe disease in humans.
Assuntos
Reação em Cadeia da Polimerase/métodos , Toxina Shiga/genética , Toxina Shiga/isolamento & purificação , Biologia Computacional , Toxina Shiga/classificaçãoRESUMO
Escherichia coli productor de toxina Shiga es un patógeno emergente cuyo principal factor de virulencia son las toxinas Shiga (Stx), codificadas por los genes stx. Estas toxinas se clasifican en 6 tipos (1, 2, 2c, 2d, 2e y 2f) que agrupan a 22 variantes. En Argentina se validaron dos técnicas de PCR para la detección de los genes stx, PCR-MK y PCR múltiple. Los objetivos del trabajo fueron analizar mediante el uso de herramientas bioinformáticas la capacidad de dichas técnicas para detectar las variantes del gen stx y demostrar experimentalmente la amplificación de 8 variantes stx. Se recopilaron 25 secuencias nucleotídicas de la base de datos GenBank correspondientes a 21 variantes de stx. Se utilizó el programa BLAST 2 sequences para analizar la complementariedad de las bases nucleotídicas entre las secuencias de las variantes y las secuencias de los cebadores utilizados en las PCR estudiadas. La técnica de PCR-MK permite detectar los tipos stx1, stx2, stx2c, stx2d y stx2f, aunque no permite detectar el tipo stx2e y tres variantes del tipo stx2c. La PCR múltiple permite detectar los tipos stx1, stx2, stx2c, stx2d, pero no los tipos stx2e y stx2f. Se demostró experimentalmente que ambas técnicas de PCR son apropiadas para la detección de las variantes que están asociadas a enfermedad grave en el hombre.
Shiga toxin-producing Escherichia coli is an emergent pathogen, being the Shiga toxin (Stx) the main virulence factor. These toxins are classified into 6 types (1, 2, 2c, 2d, 2e and 2f) and 22 variants. In Argentina, two PCR for stx gene detection, PCR-MK and multiplex-PCR, were validated. The aim of this work was to analyze, by using bioinformatic tools, the stx variants that could be amplified by these PCRs, and to experimentally show the amplification of 8 stx variants. Twentyfive nucleotide sequences were collected from GenBank corresponding to 21 stx variants. The BLAST 2 sequences program was used to analyze the complementarities between the nucleotide sequence of the variants and the primers corresponding to the PCR studied. PCR-MK could detect types stx1, stx2, stx2c, stx2d and stx2f, but not type stx2e and three type stx2c variants. On the other hand, the multiplex-PCR could detect types stx1, stx2, stx2c, stx2d, but not stx2e and stx2f types. It was experimentally determined that both PCRs can detect those variants that cause severe disease in humans.
Assuntos
Reação em Cadeia da Polimerase/métodos , Toxina Shiga/genética , Toxina Shiga/isolamento & purificação , Biologia Computacional , Toxina Shiga/classificaçãoRESUMO
In the summer of 2006, an epidemic outbreak of acute gastrointestinal illness related to food consumption occurred in a small town in the province of Neuquén, Argentina. During a popular feast, approximately 800 local residents attended lunch held in the facilities of the Municipal Gymnasium. About three hours later, nearly 150 attendees sought medical assistance at the local hospital due to acute gastroenteritis. A case-control epidemiological investigation was conducted using representative non-probability sampling. The epidemiological investigation showed a common-source foodborne disease outbreak with a case-control ratio of 1:1.8. The main symptoms were abdominal cramps (88%), vomiting (73.5%) and diarrhea (60%). The cake was identified as the source of infection (OR 9.79; IC 95%, 2.66-36.00; p = 0.0001), and unsatisfactory hygienic conditions in food production, conservation and handling steps were identified. Coagulase positive, enterotoxigenic Staphylococcus aureus, subspecies aureus was detected in a piece of cake, with a count of 2.4 x 10(6) CFU/g, and in samples from the hands and nostrils of three people involved in food preparation and service. The strains isolated from both the cake and one of the food handlers carried the sea gene, and presented the same Smal-PFGE pattern. The foodborne disease outbreak was considered to be due to contamination in the preparation process of the cake consumed at the feast, which was related to inadequate hygienic conditions, lack of refrigeration and cold chain disruption.