Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32MUTATIONS in SPI-1 genes abrogate Salmonella invasionSlide 34Slide 35Salmonella Pathogenicity Island I (SPI-1)Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Slide 49Slide 50Slide 51Slide 52Slide 53Slide 54Bacterial pathogensof the GI tractDr. Terry D. [email protected] gastrointestinal tractContains a variety of commensal bacteriaBrian K. DieckgraefeStomach 1.0 x 102 bacteriaDuodenum 1.0 x 102 bacteriaJejunum 1.0 x 102 bacteriaProximal Ileum 1.0 x 103 bacteriaDistal Ilium 1.0 x 108 bacteriaColon 1.0 x 1011 bacteriaHuman body 6.0 x 1013 cellsTotal bacteria 1.0 x 1014 bacteriaFrom 60x-100x more bacterial cells thaneukaryotic cells in our body!!Bacterial pathogensof the GI tractSalmonella spp.Shigella spp.Yersinia spp.Vibrio spp.Campylobacter spp.Helicobacter spp.Escherichia coliClostridium perfringensBacillus spp.http://www.digestionhelp.org/Salmonella spp.Salmonella spp.Five prior ‘species’ were reclassified as a single species: S. entericawith FIVE major pathogenic serovarsSerovar: A group of closely related microorganisms distinguished by acharacteristic set of molecules that evoke an antibody responseS. enterica Serovar typhimurium - mouse typhoid fever S. enterica S. choleraesuis - swine gastroenteritis S. enterica S.. enteritidis - gastroenteritisS. enterica S. typhi - typhoid fever (human)S. enterica S. paratyphi - typhoid-like (human)Salmonella spp. Gram-negative, motile (flagellated) bacillusFacultative anaerobe (aerobic or anaerobic)Non-lactose fermenter (utilizes other sugars)Oxidase-negative (reduction of DPD)DPD = N,N-dimethyl-p-phenylenediamineSulfur-reduction (alternative energy production)LPS: consists of O polysaccharide side-chains- lipopolysaccharide- in outer membrane- used for serotyping of strainsJ.P. Duguid and J.F. Wilkinsonx7800Amanda L. HensleyVan London – pHoenix CompanyHuman gastrointestinal tractHow do you determine if Salmonella has colonized a G.I. tract in a sick patient?By culturing?Fecal sample → Hundreds of different types (Gm+, Gm-) of bacteria!Brian K. DieckgraefeTake advantage of the bacterium’s PHENOTYPES and GROWTH REQUIREMENTSSalmonella spp. Gram-negative, motile (flagellated) bacillusFacultative anaerobe (aerobic or anaerobic)Non-lactose fermenter (utilizes other sugars)Oxidase-negative (reduction of DPD)DPD = N,N-dimethyl-p-phenylenediamineSulfur-reduction (alternative energy production)LPS: consists of O polysaccharide side-chains- lipopolysaccharide- in outer membrane- used for serotyping of strainsJ.P. Duguid and J.F. Wilkinsonx7800Amanda L. HensleyVan London – pHoenix CompanySalmonella-Shigella AgarSelective growth medium for Gram-negative bacteria-Bile salts (inhibits Gm+)Sugar utilization, fermentation- Lactose- Neutral red (pH)- Use of lactose AcidSulfur reduction- Sodium thiosulfate Thiosulfate H2S H2S + Fe = BLACK (Sulfur reduction)ShigellaSalmonellaE. coliBlack precipitantLactose fermentationKlebsiellaE. coliSalmonellaProteusPseudomonasSalmonella spp.: Gram-negative (bile salt resistant)lactose non-fermenter (no pink color)reduces thiosulfate to H2S (black)LactoseNeutral red dyeBile saltsThiosulfateHow do we classify different strains of Salmonella?Why would we want to have the abilityto classify different strains?Salmonella spp. Gram-negative, motile (FLAGELLATED) bacillusFacultative anaerobe (aerobic or anaerobic)Non-lactose fermenter (utilizes other sugars)Oxidase-negativeSulfur-reducing (alternative energy production)LPS: lipopolysaccharide - consists of various O-polysaccharide side-chains - located in outer membrane - different strains have different side chains; antigenically diverseFlagellae - composed of arrays of flagellar proteinsFlagellar proteins are very antigenically diverse Antibodies againstthe FLAGELLAE of onestrain of Salmonella will NOT bind to the FLAGELLAE expressed by a different strain ofSalmonellaSalmonella spp. Gram-negative, motile (FLAGELLATED) bacillusFacultative anaerobe (aerobic or anaerobic)Non-lactose fermenter (utilizes other sugars)Oxidase-negativeSulfur-reducing (alternative energy production)LPS: LIPOPOLYSACCHARIDE - Located in outer membrane - LipidA decorated with various O-polysaccharide side-chains - Different strains have different side chains - Antigenically diverseAbs against one type of LPS will NOT recognize another type of LPSSerological targets used to differentiate Salmonella strains/species>2400 O serotypes (Ab-specific)Examples: O7:Ha; O11:Hc; etc. >119 H (Flagellar)SerotypesSalmonella serovarsFive major pathogensSerovar S. typhimurium - mouse typhoid fever Serovar S. choleraesuis - swine gastroenteritis Serovar S. enteritidis - GASTROENTERITIS(Humans, other mammals, birds, etc.)Serovar S. typhi - typhoid fever (human)Serovar S. paratyphi - typhoid-like (human)EggsTurtlesLizardsChickensTurkeysOver 2,000,000 cases annually in the U.S.Recent outbreak: contaminated peanut butterSalmonella food poisoning“The first two days, I thought that I was going to die…….”Salmonella food poisoning“The first two days, I thought that I was going to die…….”On the last two days, I was afraid that I wasn’t!!!!A really, really nasty infection!!!Serovar enteritidis - Oral entry of infection - Usually limited to intestine (Non-disseminated – remains in GI) - INVASIVE bacterium ! - Enters into cytoplasm of gut cells - Fever, nausea, cramps, diarrhea - Diarrhea- electrolyte imbalance - dehydration - Self-limiting disease (2-4 days) - Rarely can be fatal !!Stimulates its own uptake into gut cells.Induces rearrangement of the actin cytoskeleton of the host cell.Plasma membrane “ruffling” observed prior to invasion by Salmonella into the host cell.(Salmonella)Serovar enteritidis - Oral entry of infection - Usually limited to intestine(non-disseminated) - INVASIVE bacterium ! - enters into cytoplasm of cells - Fever, nausea, cramps, diarrhea - Diarrhea- electrolyte imbalance - dehydration - Self-limiting disease (2-4 days) - Rarely is fatal !!Salmonella serovarsFive major pathogensSerovar typhimurium - mouse typhoid fever Serovar choleraesuis - swine