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Salmonella

Salmonella. Laboratorio de Microbiología Aplicada Dra. Esther Z. Vega Bermúdez. Salmonella sp. Salmonella. Salmonella enterica. Salmonella bongori. Subs. enterica. Subs. salamae. Subs. arizonae. Subs. diarizonae. Subs. indica. Subs. houtenae.

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Salmonella

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  1. Salmonella Laboratorio de Microbiología Aplicada Dra. Esther Z. Vega Bermúdez

  2. Salmonellasp.

  3. Salmonella Salmonella enterica Salmonella bongori Subs. enterica Subs. salamae Subs. arizonae Subs. diarizonae Subs. indica Subs. houtenae From: Porwollik, S., et al., 2004. Characterization of Salmonella enterica subspecies I genovars by Use Of Microarrays. J. of Bacteriol. 186:5883-5898.

  4. Incidencia de Salmonella

  5. Características • Enterobacterias • Bacilos Gram (-) • Mótiles con flagelación perítrica • No esporulan • No fermentan lactosa • 2, 300 especies • Crece entre 40-140oC

  6. Variantes Genéticas • Mediada por Plasmidios • Utilización de lactosa y sucrosa • Microorganismos atípicos pueden “escapar”a la detección en situaciones clínicas • Las especies de Salmonella no son un grupo homogéneo de organismos

  7. Reservorios / Alimentos Relacionados • Carnes crudas • Aves • Huevos • Leche y productos lácteos • Pescado • Camarones • Salsas • Mezclas para bizcocho • Postres de crema • otros

  8. Patogénesis • Dosis infecciosa tan bajo como 15-20 células (depediendo de hospedero) • Antígenos estructurales (pared celular o antígeno O, H- flagelar y el IV-antígeno capsular termolábil -poder frente a los ácidos) • Sustancias tóxicas • Endotoxinas • Exotoxinas (efecto irritante y citotóxico para el tracto intestinal) • Ruta oral-fecal

  9. Período de incubación de 6-48hrs Diarreas Dolor abdominal Fiebre entre 8-72hrs Dolor de cabeza y en el cuerpo Escalofríos Naúseas Vómitos Septicemias Bacteremias Cistitis, endocarditis y osteomilitis Síndrome de Reiters que puede terminar en artritis crónica Fiebre tifoidea ó paratifoidea Sintomatología y Enfermedades

  10. Diagnóstico y Tratamiento • Pruebas de serología y de heces fecales • Aislar el paciente • Tratamiento para todos en el hogar • Antibíoticos

  11. Muestreo en Alimentos • Métodos convencionales de aislación que requieren de cinco días para confirmar resultados. • Cultivo 37-420C o en nevera.

  12. Salmonella

  13. Metodología • Pre-Enriquecimiento • Caldo de lactosa • Permite la recuperación de células “heridas” • Aumenta la razón Salmonella:Non-Salmonella • Agua de dilución peptonada Productos Animales • Dilución 1:10 • I.e.. 25 g muesra a 225 ml de caldo • 24 horas a 35 oC

  14. Enriquecimiento Selectivo • 1 ml de caldo de Lactosa • 10 ml Selenite Cystine • 10 ml tetrathionate broth • Permite el crecimiento de Salmonella • No hay resultados despúes de este paso • 24 horas a 35oC

  15. Placas Selectivas • Tres tipos de medio selectivo • Bismuth Sulfite • Marrón, gris, negro con “metallic sheen” • Hektoen Enteric Agar • Azul-verdoso con o sin centro negro o “glossy black” • Xylose Lysine Desoxycholate • Rosado con o sin centro negro • Estriado de “Selective Enrichments” • Incubación de 18-24 h a 35oC • Resultados – PRESUMPTIVE POSITIVE

  16. Medio Diferencial – Discernimiento bioquímico • Triple Sugar Iron Agar • Streak Slant and Stab Butt • 1% Lactosa • 0.1% Glucosa • Sulfato Ferroso • Indicador de H2S • Tiosulfato • Producción de H2S • Rojo fenol • Alkalina – Rojo, Acido – Amarillo • Incubación a 35 oC por 24 horas

  17. Triple Sugar Iron Agar • Reacción de Salmonella • Red Slant (Alkalino) • Acid Butt (Amarillo) • Precipitado Negro

  18. Lysine Iron Agar- Discernimiento bioquímico • Stab Butt and Streak Slant • Se quiere condiciones anaeróbicas en el fondo para que ocurra la decarboxilación • Decarboxilación a partir de L-Lysina • Sulfato Ferroso • Bromo Cresol Purple • Violeta – Alkalino, Amarillo - Acido • Glucosa • Tiosulfato

  19. Lysine Iron Agar • Reacción de Salmonella • Alkaline Butt – Purple • Precipitado Negro • Sólo es negativo si hay condiciones ácidicas, pueden obtenerse otras reacciones no típicas

  20. Confirmación Bioquímica • Aglutinación de antígenos superficiales con anticuerpos específicos para Salmonella • Antígenos de Salmonella • O – Somático – Lipopolisacárido • H – Flagellar – Proteína • K – Capsular - Polisacáridos

  21. Aglutinación • Mezcle anticuerpos con un poco de cultivo • Aglutinación es un resultado positivo

  22. Aislamiento vs. Enumeración • Usualmente sólo se aisla • Se enumera para propósitos de investigación • Método MPN • Método de filtración por membranas • No “Accurate”

  23. Table 1. Partial list of miniaturized biochemical kits and automated systems for identifying foodborne bacteria* ( 5, 8, 15, 16, 21, 35, 36 ). System Format Manufacturer Organisms APIb biochemical bioMerieux Enterobacteriaceae, Listeria, Staphylococcus, Campylobacter, Non-fermenters, anaerobes Cobas IDA biochemical Hoffmann LaRoche Enterobacteriaceae Micro-IDb biochemical REMEL Enterobacteriaceae, Listeria EnterotubeII biochemical Roche Enterobacteriaceae Spectrum 10 biochemical Austin Biological Enterobacteriaceae RapID biochemical Innovative Diag. Enterobacteriaceae BBL Crystal biochemical Becton Dickinson Enterobacteriaceae, Vibrionaceae, Non-fermenters, anaerobes Minitek biochemical Becton Dickinson Enterobacteriaceae Microbact biochemical Microgen Enterobacteriaceae, Gram negatives, Non-fermenters, Listeria Vitekb biochemicala bioMerieux Enterobacteriaceae, Gram negatives, Gram positives Microlog C oxidationa Biolog Enterobacteriaceae, Gram negatives, Gram positives MISb Fatty acida Microbial-ID Enterobacteriaceae, Listeria, Bacillus, Staphylococcus, Campylobacter Walk/Away biochemicala MicroScan Enterobacteriaceae, Listeria, Bacillus, Staphylococcus, Campylobacter Replianalyzer biochemicala Oxoid Enterobacteriaceae, Listeria, Bacillus, Staphylococcus, Campylobacter Riboprinter nucleic acida Qualicon Salmonella, Staphylococcus, Listeria,Escherichia coli Cobas Micro-ID biochemicala Becton Dickinson Enterobacteriaceae, Gram negatives, Non-fermenters Malthusb conductancea Malthus Salmonella, Listeria, Campylobacter, E. coli, Pseudomonas, coliforms Bactometer impedancea bioMerieux Salmonella * Table modified from: Feng, P., App.I., FDA Bacteriological Analytical Manual, 8A ed.a Automated systems b Selected systems adopted AOAC Official First or Final Action. NOTE: This table is intended for general reference only and lists known available methods. Presence on this list does not indicate verification, endorsement, or approval by FDA for use in food analysis. Otros métodos- kits bioquímicos

  24. Identificación basada DNA-DNA Table 2. Partial list of commercially-available, nucleic acid-based assays used in the detection of foodborne bacterial pathogens* (2, 5, 8, 12, 14, 22, 25, 36, 37). Organism Trade Name Format Manufacturer Salmonella GENE-TRAKc probe GENE-TRAK BAX PCR Qualicon BINDb phage BioControl Probelia PCR BioControl * Table modified from: Feng, P., App.I, FDA Bacteriological Analytical Manual, 8A ed.a Polymerase chain reactionb Bacterial Ice Nucleation Diagnosticsc Adopted AOAC Official First or Final Action NOTE: This table is intended for general reference only and lists known available methods. Presence on this list does not indicate verification, endorsement, or approval by FDA for use in food analysis.

  25. Table 3. Partial list of commercially-available, antibody-based assays for the detection of foodborne pathogens and toxins* (3, 5, 8, 12, 33, 36). Organism/toxin Trade Name Assay Formata Manufacturer Salmonella Bactigen LA Wampole Labs Spectate LA Rhone-Poulenc Microscreen LA Mercia Wellcolex LA Laboratoire Wellcome Serobact LA REMEL RAPIDTEST LA Unipath Dynabeads Ab-beads Dynal Screen Ab-beads VICAM CHECKPOINT Ab-blot KPL 1-2 Teste diffusion BioControl SalmonellaTEKe ELISA Organon Teknika TECRAe ELISA TECRA EQUATE ELISA Binax BacTrace ELISA KPL LOCATE ELISA Rhone-Poulenc Assurancee ELISA BioControl Salmonella ELISA GEM Biomedical Transia ELISA Transia Bioline ELISA Bioline VIDASe ELFAb bioMerieux OPUS ELISAb TECRA PATH-STIK Ab-ppt LUMAC Reveal Ab-ppt Neogen Clearview Ab-ppt Unipath UNIQUEe Capture-EIA TECRA * Table modified from: Feng, P., App.I, FDA Bacteriological Analytical Manual, 8A ed.a Abbreviations: ELISA, enzyme linked immunosorbent assay; ELFA, enzyme linked fluorescent assay; RPLA, reverse passive latex agglutination; LA, latex agglutination; Ab-ppt, immunoprecipitation. b Automated ELISA c EHEC - Enterohemorrhagic E. coli; ETEC - enterotoxigenic E. colid Also detects E. coli LT enterotoxin e Adopted AOAC Official First or Final Action ** CAUTION: unless the assays claim that they are specific for the O157:H7 serotype, most of these tests detect only the O157 antigen; hence will also react with O157 strains that are not of H7 serotype. These O157, non-H7 strains, generally do not produce Shiga toxins and are regarded as not pathogenic for humans. Furthermore, some antibodies to O157 can also cross react with Citrobacter, E. hermanii and other enteric organisms. NOTE: This table is intended for general reference only and lists known available methods. Presence on this list does not indicate verification, endorsement, or approval by FDA for use in food analysis. Identificación basada en detección de antígenos

  26. Table 4. Partial list of other commercially available rapid methods and specialty substrate media for detection of foodborne bacteria* (4, 8, 13, 20, 27, 36). Organism Trade Name Formata Assay Manufacturer Salmonella Isogridb HGMF QA Labs OSRT Medium/ motility Unipath (Oxoid) Rambach Medium CHROMagar MUCAP C8esterase Biolife XLT-4 Medium Difco MSRVb Medium * Table modified from: Feng, P., App.I, FDA Bacteriological Analytical Manual, 8A ed.a Abbreviations: APC, aerobic plate count; HGMF, hydrophobic grid membrane filter; ATP, adenosine triphosphate; MUG, 4-methylumbelliferyl-ß-D-glucuronide; ONPG, O-nitrophenyl ß-D-galactoside; MPN, most probable number.b Adopted AOAC Official First or Final Action.c Application for water analysisd EHEC - enterohemorrhagic Escherichia coli NOTE: This table is intended for general reference only and lists known available methods. Presence on this list does not indicate verification, endorsement, or approval by FDA for use in food analysis. Identificación basada en la utilización de sustratos

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