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Unformatted text preview: Salmonella Salmonella Laboratorio de Microbiología Aplicada Laboratorio de Microbiolog Dra. Esther Z. Vega Bermúdez Salmonella sp. S almonella Salmonella Salmonella Salmonella enterica Subs. enterica Subs. salamae Subs. arizonae Salmonella bongori 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. 186:5883­5898. Incidencia de Salmonella I ncidencia de Salmonella Caracter í sticas C aracter Enterobacterias Bacilos Gram (­) M ó tiles con flagelaci ó n per í trica No esporulan No fermentan lactosa 2, 300 especies Crece entre 40­140 o C Variantes Gen é ticas V ariantes Gen 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 Reservorios / Alimentos R eservorios / Alimentos Relacionados Carnes crudas Aves Huevos Leche y productos l á cteos Pescado Camarones Salsas Mezclas para bizcocho Postres de crema otros Patog é nesis P atog Dosis infecciosa tan bajo como 15­20 c é lulas (depediendo de hospedero) Ant í genos estructurales (pared celular o ant í geno O, H­ flagelar y el IV­ant í geno capsular termol á bil ­poder frente a los á cidos) Sustancias t ó xicas Endotoxinas Exotoxinas (efecto irritante y citot ó xico para el tracto intestinal) Ruta oral­fecal Sintomatolog í a y Enfermedades S intomatolog Septicemias Per í odo de incubaci ó n de Bacteremias Cistitis, endocarditis y 6­48hrs Diarreas Dolor abdominal Fiebre entre 8­72hrs Dolor de cabeza y en el cuerpo Escalofr í os Na ú seas V ó mitos osteomilitis S í ndrome de Reiters que puede terminar en artritis cr ó nica Fiebre tifoidea ó paratifoidea Diagn ó stico y Tratamiento D iagn Pruebas de serolog í a y de heces fecales P ruebas de serolog Aislar el paciente Tratamiento para todos en el hogar Antib í oticos Muestreo en Alimentos M uestreo en Alimentos M é todos convencionales de aislaci ó n que requieren de cinco d í as para confirmar resultados. Cultivo 37­42 0 C o en nevera . Salmonella Salmonella Metodología Metodolog Pre­Enriquecimiento – Caldo de lactosa Permite la recuperación de células “heridas” Aumenta la razón Salmonella:Non­Salmonella – Agua de dilución peptonada Productos Animales Dilución 1:10 – I.e.. 25 g muesra a 225 ml de caldo 24 horas a 35 oC Enriquecimiento Selectivo 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 Placas Selectivas Placas Selectivas Tres tipos de medio selectivo – Bismuth Sulfite Marrón, gris, negro con “metallic sheen” – Hektoen Enteric Agar Azul­verdoso con o sin centro negro o “glossy black” – Xylose Lysine Desoxycholate Rosado con o sin centro negro Estriado de “Selective Enrichments” Incubación de 18­24 h a 35oC Resultados – PRESUMPTIVE POSITIVE Medio Diferencial – Discernimiento 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 Triple Sugar Iron Agar Triple Sugar Iron Agar Reacción de Salmonella – Red Slant (Alkalino) – Acid Butt (Amarillo) – Precipitado Negro Lysine Iron Agar­ Discernimiento bioquímico Lysine Iron Agar­ Discernimiento bioqu Stab Butt and Streak Slant – Se quiere condiciones anaeróbicas en el fondo para que ocurra la decarboxilación Decarboxilación a partir de L­Lysina Sulfato Ferroso Bromo Cresol Purple – Violeta – Alkalino, Amarillo ­ Acido Glucosa Tiosulfato Lysine Iron Agar Lysine Iron Agar Reacción de Salmonella – Alkaline Butt – Purple – Precipitado Negro Sólo es negativo si hay condiciones ácidicas, pueden obtenerse otras reacciones no típicas Confirmación Bioquímica Confirmaci Aglutinación de antígenos superficiales con anticuerpos específicos para Salmonella Antígenos de Salmonella – – – O – Somático – Lipopolisacárido H – Flagellar – Proteína K – Capsular ­ Polisacáridos Aglutinación Aglutinaci Mezcle anticuerpos con un poco de cultivo Aglutinación es un resultado positivo Aislamiento vs. Enumeración Aislamiento vs. Enumeraci Usualmente sólo se aisla Se enumera para propósitos de investigación – – Método MPN Método de filtración por membranas No “Accurate” Otros métodos­ kits bioquímicos Otros m Table 1. Partial list of miniaturized biochemical kits and automated systems for identifying foodborne bystem 8, 15, 16 Sacteria* ( 5, Format , 21, 35, 36 ). Manufacturer Organisms APIb biochemic bioMerieux Enterobacteriaceae, Listeria, Staphylococcus, Campylobacter, Nonal fermenters, anaerobes Cobas IDA biochemic Hoffmann Enterobacteriaceae b al LaRoche Micro-ID biochemic REMEL Enterobacteriaceae, Listeria EnterotubeI al biochemic Roche Enterobacteriaceae I pectrum al S biochemic Austin Enterobacteriaceae 10 al Biological RapID biochemic Innovative Enterobacteriaceae Decton BBL Crystal al biochemic B iag. Enterobacteriaceae, Vibrionaceae, Non-fermenters, anaerobes al Decton Minitek biochemic B ickinson Enterobacteriaceae al Dickinson Microbact biochemic Microgen Enterobacteriaceae, Gram negatives, Non-fermenters, Listeria b al Vitek biochemic bioMerieux Enterobacteriaceae, Gram negatives, Gram positives a al Microlog C Biolog Enterobacteriaceae, Gram negatives, Gram positives oxidationaa Microbial-ID MISb Fatty acid Enterobacteriaceae, Listeria, Bacillus, Staphylococcus, Campylobacter Walk/Away biochemic MicroScan Enterobacteriaceae, Listeria, Bacillus , Staphylococcus, Campylobacter a Replianalyz al biochemic Oxoid Enterobacteriaceae, Listeria, Bacillus , Staphylococcus, Campylobacter a er al Riboprinter nucleic Qualicon Salmonella, Staphylococcus, Listeria, Escherichia coli acida Cobas biochemic Becton Enterobacteriaceae, Gram negatives, Non-fermenters a b Micro-ID al Dickinson Malthus conductan Malthus Salmonella, Listeria, Campylobacter, E. coli, Pseudomonas, coliforms a ce Bactometer impedanc bioMerieux Salmonella a e * Table modified from: Feng, P., App.I., FDA Bacteriological Analytical Manual, 8A ed. a Automated systems NOTE: This table isadopted AOAC Official First or Final and lists known available methods. Presence on this list does b Selected systems intended for general reference only Action. not indicate verification, endorsement, or approval by FDA for use in food analysis. Identificaci ó n basada DNA­DNA I dentificaci 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 reaction b Bacterial Ice Nucleation Diagnostics c 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. Identificación basada en detección de antígenos Identificaci 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 Salmonella Trade Name Assay Formata Manufacturer 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. **Automated ELISA b 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 strainsEthat are not - enterotoxigenic These O157, non-H7 strains, generally do not produce Shiga toxins and are regarded as c EHEC - Enterohemorrhagic . coli; ETEC of H7 serotype. E. coli NOTE: This tablefor intended for general reference only and lists known available methods. Presence on this list ,does not indicate verification, endorsement, or is nAlso detects E. coli humans. Furthermore, some antibodies to O157 can also cross react with Citrobacter E. hermanii and other enteric organisms. d ot pathogenic LT enterotoxin approval by FDA for use in food analysis. e Adopted AOAC Official First or Final Action Identificación basada en la utilización de sustratos Identificaci 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. NOTE: This table is intended for general reference only and lists known available methods. Presence on this c Application for water analysis list does not indicate verification, endorsement, or approval by FDA for use in food analysis. d EHEC - enterohemorrhagic Escherichia coli ...
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  • Fall '11
  • Dr.Aslam
  • Escherichia coli, Bacteriological Analytical Manual, Salmonella Salmonella, FDA Bacteriological Analytical
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