1、Enterobacteriaceae1Family EnterobacteriaceaeEscherichia Shigella Salmonella Enterobacter Klebsiella SerratiaProteus.2Yersinia pestis Self regulated learningLife-long learning 3Family Enterobacteriaceae Facultative gram-negative rods 27 genera and 102 species.4General structures & characteristicsMode

2、rate in size O=LPSK=polysaccharide capsule H=flagella protein菌毛 (Pilus/ Pili)5Cell wall 67Flagella(H antigen) Facultative :growth is rapid Ferment glucose reduce nitrate Catalase(+) oxidase(-)Growth & biochemical characteristics8Biochemical identification Lactose fermentation Resistance to bile salt

3、s2志賀氏菌3沙門氏菌4大腸桿菌9Classification O antigen somatic polysaccharide K antigen(Vi antigen) capsular substance H antigen flagellaBiochemical characteristics: establish genus and species Escherichia, Shigella， Salmonella, Klebsiella and Yersinia Antigenic characters: define serotypes within species 10Path

4、ogenesis and clinical Findings (E.coli)Urinary tract infectionE coli-associated diarrheal diseases Sepsis Meningitis Enterpathogenic E coli (EPEC) Enterotoxigenic E coli (ETEC) Shiga toxin-producing E coli (STEC) Enteroinvasive E coli (EIEC) Enteroaggregative E coli (EAEC)11Toxins LPS Protein extoxi

5、ns (E.coli, Shigella,Yersinia) Cytotoxin Enterotoxins cause secretion and diarrhea 12Diseases caused by Enterobacteriaceae Epidemiology Present in intestinal tract and the natureShigella and S Typhi are found only in humans 13Enterobacteriaceae diseases 14Pathogenesis Opportunistic infections Intest

6、inal infections dysentery, watery diarrhea, enteric fever ( Cytotoxin, enterotoxins , LPS)Regulation of Virulence Immunity15Regulation : Contact secretion system16Enterobacteriaceae: clinical aspects Manifestations: UTI and acute diarrheaDiagnosis : Culture :routine media; special indicator media; s

7、elective media Gene probesTreatment Sensitivity to antimicrobials is highly variable 17Anatomy of the gastrointestinal tract 18Micro-organisms in the GI TractMicrobes:First living creatures on earthAppeared over 3.7 billion years agoMore than one million species of bacteria existOnly 4200 species ar

8、e describedFecal Bacteria:First observed microscopically some 300 years ago 500 bacterial species30-40 species = 99% of the total populationMost cannot be cultured: 60% unknown 19Intestinal MicrobiotaComplex ecosystem 500 species1014 bacteria, colon (99%)Composition varies with age Functions:Functio

9、nal barrierPlays an important role in normal nutrition and metabolism Help in the development of the intestines mucosal immune system1g of large intestinal contents: 150 times more bacteria than people on the planet20Intestinal Microbiota Composition21Barrier Effect of the MicrobiotaProposed Mechani

10、smsCompetition for nutrients/blockade of epithelial accessProduction of antibiotic substancesStimulation of innate and/or adaptive host immune defenses22The Intestine: An Unknown OrganGerm free animals have got a poorly developed intestinal tract:Thin and less cellular tract, reduced surface area, e

11、tcetera60 to 70% of our immune cellsSurface of approximately 300m2100 million neurons100.000 billion bacteria23The Intestine: A Huge Playing Field24It all starts as a sterile surface25But, after some first colonizers26A complex ecosystem 27Prebiotics must stimulate one or a limited number of benefic

12、ial bacteria, like bifidobacteria or lactobacilli, within the colonic microbiotaNon-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon that can improve host health Prebiotics must sur

13、vive acidic conditionsPrebiotics must be selectively fermented in the colonPrebiotics must evade digestion in the small intestinePrebiotic ConceptFuller & Gibson, 199728Importance of the “Bifidus flora”Linked to an enhanced resistance to enteric infections (Koletzko et al., 1998; Salminen et al., 19

14、98)Play a role in the development of a healthy immune system (Kalliomki et al., 2001; Bjrksten et al., 2001)29Molecular Approaches to Study the Intestinal MicrobiotaIsolateRNA/DNADNAMolecular fingerprinting(PFGE, ARDRA, RAPD, T-RFLP, ribotyping)T/DGGECloning andsequenceanalysis(Selective)cultivation

15、ColonyhybridisationFISHDot blothybridisationqPCREnumerationSpecific detection/identification with probes and primersRelative quantificationDiversity assessment30FISH: MicroscopeFISH: An Example31Enterobacteriaceae with clinical significance32Enterobacteriaceae with clinical significanceGenusNo. of s

16、peciesCitrobacter4Edwardsiella4Enterobacter13Escherichia5Shigella4Ewingella1Hafnia2Klebsiella7Morganella2Salmonella7 subgroupsSerratia10Yersinia1133Distribution of Enterobacteriaceae 34 Escherichia coli : antigenic structure Escherichia35Escherichia Serotypes determined by O,H, K antigens Pili Type

17、I pili bind mannose P pili bind uroepithelial cells Pili of diarrhea strains bind enterocytes 3637Urinary tract infection due to Escherichia coli 38Toxins - Hemolysin ： pore-forming cytotoxinCNF (cytotoxic necrotizing factor )Shiga toxin： Shigella and E coli Labile toxin (LT) : inhibit protein synth

18、esis by ribosomal modification Stable toxin( ST)： stimulates guanylate cyclase 39Stx (Shiga toxin )A-B type toxin40E coli opportunistic infections Urinary tract infection (UTI) Cystitis P pili prominent in pyelonephritis Other opportunistic infections Septicemia, meningitis, Gastroenteritis41Factors

19、 involved in E coli opportunistic infections Common Reservoir : perineal flora Water, soil, vegetationMinor trauma admits E coli to the bladderNumerous virulence factors Uropathic E coli (UPEC)4243Urinary tract infection due to Escherichia coli Uropathic E coli (UPEC) play prominent role in UTI44Sou

20、rces of infection? Endogenous spread in susceptible patientsIngestion of contaminated food & waterNosocomial infections (procedure-related)45Ecoli intestinal infections Enterotoxigenic E coli (ETEC) Plasmid-mediated adherence ,small intestine (bloody stool)Enteropathogenic E coli (EPEC) Pathogenic E

21、. ColiEnterohemorrhagic E coli (EHEC) Cytotoxic “verotoxin”-large intestineEnteroinvasive E coli （EIEC） Plasmid-mediated invasion and destruction of epithelial cells lining colonEnteroaggregative E coli (EAEC) Plasmid-mediated adherence and destruction of epithelial cells in small intestine (nonbloo

22、dy stool) 46Pathogenesis and spectrum of disease Organism Virulence factorsSpectrum of disease and infections Escherichia coli Endotoxin, capsule, pili，exotoxinsUrinary tract infections, bacteremia, nosocomial infections of various body sites. Enterotoxigenic E.coli(ETEC)Pili (gastrointestinal colon

23、ization)Heat-labile(LT) and heat-stable(ST) enterotoxinsTravelers and childhood diarrheaEnteroinvasive E.coli (EIEC)Virulence factors uncertainDysentery usually in young children with poor sanitationEnteropathogenic E coli(EPEC)Bundle forming pilus, intimin, and other factors that mediate organism a

24、ttachementDiarrhea in infantsChronic diarrheaEnterohemorrhagic E.coli(EHEC)Toxin similar to Shiga toxin associated with certain serotypesInflammation and bleeding of the mucosa of the large intestine hemolytic-uremic syndromeEnteroaggregative E.coli (EAEC)pili, ST-like, and hemolysin-like toxins; ac

25、tual pathogenic mechanism not knownWatery diarrhea47Pathogenesis and spectrum of diseaseOrganism Virulence factorsSpectrum of disease and infections Shigella spp.?Salmonella spp.?Yersinia pestis?Yersinia enterocolitica subsp.enterocolitica?48OrganismIncubation PeriodClinical FeaturesDiagnostic Featu

26、resEHEC/STEC (O157:H7 and other serotypes)1-8 daysDiarrhea : often grossly bloody, without blood Abdominal pain and vomiting may occurFever is usually absentAbout 8% develop HUS (more common in children)Thrombocytopenic purpura (TTP), more common in adults, Stool culture requires special mediaTestin

27、g of stool must be requestedETEC1-3 daysWatery diarrhea: lasts 3-7 days, but can be protracted (months with relapses)Stool does not contain leukocytes, mucus, or bloodFever usually absentAbdominal cramps, sometimes with nausea and headacheDetection of toxin-producingE coliin stoolRequires special te

28、chniques (most laboratories do not perform testing)Testing performed by reference or research laboratoriesEPECVariableSevere acute diarrheaLow-grade feverVomitingMay be persistent and protracted, resulting in weight loss, malnutrition, and deathDetection of organism by PCR or DNA probes forbfporeaegenes or tissue culture assay for local adherence (typical strains only)Testing performed by reference or research laboratoriesEIECVariablewatery diarrheaDysentery with bloody diarrhea occurs in 150,000). The sto