微生物学美国IndianaUniversityPurdueUniversity授课02.ppt

Lecture Two微生物学美国IndianaUniversityPurdueUniversity授课02 Still waters run deep.流静水深流静水深,人静心深人静心深 Where there is life,there is hope。有生命必有希望。有生命必有希望Lecture TwoNormal FloraGeneral aspectsRemember definition:organisms frequently found on or within body of healthy individuals Most are bacteria,but some are viruses,fungi,and protozoaWe do not carry all of them all of the timeEach person has individualized normal floraBIOL 5332Lecture TwoNormal FloraSome are found only on body;others also found in environmentProblem:some people have transient normal flora(pathogens)Example:about 10%of population have meningococcus or pneumococcus as normal floraBIOL 5333Lecture TwoImportanceOpportunistic infections:normal flora in unusual sites;for example:Bacteriodes from intestine into deeper tissues as a result of trauma(or surgery)Staphylococci from skin and noseStreptococci and Gram cocci from throat and mouthBIOL 5334Lecture TwoImportanceDepends on pathogen and on defenses of host:Candida(yeast)causes pneumonia in people undergoing cancer chemotherapyPneumocystis carinii(common inhabitant of lung)causes pneumonia and death in AIDS patientsBIOL 5335Lecture TwoImmune StimulationAntigenic stimulation by normal flora do not have high antibody titersServe as defense mechanism even in low concentrationBacterial stimulation leads to production of IgA that is secreted through mucus membranesProbably interfere with colonization of deeper tissuesBIOL 5336Lecture TwoImmune StimulationSometimes antibodies elicited by normal flora cross-react with normal tissue:Antibodies against ABO blood group substances:A-make B antibodiesB-make A antibodiesO-make antibodies against bothWhy?Bacteria from intestinal flora contain Ag that cross-react with both A&B blood substancesBIOL 5337Lecture TwoImmune StimulationCross-reactivity does not normally cause diseasePossible for antibodies cross-reactive to microbial Ag to cause problemLupus erythematosusproduction of Ab against host DNASome evidence that Ag may be cross-reacting bacterial LPSMay cross-react with pathogen(meningococcus)BIOL 5338Lecture TwoPhysical&Chemical AspectsKeeps out invadersMechanisms:Physical advantage of previous occupancySome produce bacteriocins or antibioticsRelevance to lab work:E.coli K-12 cannot compete with intestinal floraBIOL 5339Lecture TwoPhysical&Chemical AspectsAntibiotic effects:wipes out normal floraBoth endogenous and exogenous organisms can cause diseaseInfecting dose of Salmonella decreases one million-fold when mice given streptomycinPatients treated with some potent antibiotics:Suffer from diarrhea due to overgrowth of yeasts,and staphylococciAdministration of clindmycin-Clostridium difficile(minor member of normal flora)causes pseudomembranous colitisBIOL 53310Lecture TwoPhysical&Chemical AspectsRole in human nutrition and metabolismE.coli and Bacteriodes synthesize vitamin KMetabolism of key compounds involves excretion from liver into intestine and their return to the liver BIOL 53311Lecture TwoPhysical&Chemical AspectsImportant for sex hormones and bile saltsExcreted through bile in conjugated form as glucuronides or sulfate,but cannot be reabsorbed in this formMembers of intestinal bacterial flora make glucuronidases and sulfatases that can deconjugate these compoundsPhysiological role not knownBIOL 53312Lecture TwoPhysical&Chemical AspectsSource of carcinogensLarge intestinal floraMany potential carcinogens are only active after being modifiedSome modifications are carried out by enzymes of intestinal bacteria;example:cyclamate converted to bladder carcinogen(cyclohexamine)by bacterial sulfatasesImportance of carcinogen production not clearBIOL 53313Lecture TwoEcology of Normal FloraUse of germ-free animalsImmune systems not well developedHave to be fed vitaminsBIOL 53314Lecture TwoEcology of Normal FloraParts of body colonizedContain large numbers:SkinRespiratory tract(nose and oropharynx)Digestive tract(mouth and large intestine)Urinary tract(anterior parts of urethra)Genital system(vagina)Most are strict anaerobesBIOL 53315Lecture TwoEcology of Normal FloraParts of body colonizedContain small numbers,many in transit:Rest of respiratory and digestive tractsBladderUterusFinding pathogens at these sites is suggestive of disease,but not proofBIOL 53316Lecture TwoEcology of Normal FloraSterile sitespathogens in these definitely indicate diseaseBloodCerebrospinal fluidSynovial fluidDeep tissuesBIOL 53317Lecture TwoStrategies for Studying Microbial PathogenesisBIOL 533Lecture 2Medical MicrobiologyBIOL 53318Lecture TwoIdentification of PathogensTraditional:associate disease with organismBIOL 53319Lecture TwoKochs Postulates1Bacterium found in all patients having disease and it or its products found in all body parts affected2The bacterium should be isolated and grown in pure cultureBIOL 53320Lecture TwoKochs Postulates3Pure culture inoculated into susceptible animal should produce disease4Same bacterium re-isolated in pure culture from experimental animalBIOL 53321Lecture TwoKochs PostulatesSome assumptions questioned in light of more modern approaches and new information about host-parasite interactionBIOL 53322Lecture TwoChallenge to Postulate#1Implies virulence resides only with pathogen and not at all with hostClearly,susceptibility of host is as importantImmuno-compromised individuals vs.healthy adults prove the pointMinor pathogen causes disease in immuno-compromised individuals onlyBIOL 53323Lecture TwoChallenge to Postulate#2Places considerable emphasis on culturing organisms in pure cultureSome organisms have not been cultured in laboratory mediaBIOL 53324Lecture TwoChallenge to Postulate#2For example,Treponema pallidium,Mycobacterium leprae clearly cause disease:Antibiotics cause both symptoms and organisms from tissues to disappearImmune response in infected patients to surface Ag of bacteria from infected tissueBIOL 53325Lecture TwoChallenge to Postulate#3Implies all members of a bacterial species are equally virulent and only a single species causes diseaseDifferent strains of species vary in virulenceDifferent strains can cause different diseasesSame symptoms caused by numerous organismsDisease caused by multiple organismsBIOL 53326Lecture TwoChallenge to Postulate#3Well known fact that cultivation of some pathogens can lead to loss of virulence factorsBIOL 53327Lecture TwoChallenge to Postulate#4Requires pathogen be reinoculated into an animal and produces symptoms of diseaseSome diseases dont affect animals,or cause different symptoms from human formTherefore,to be practical,Kochs Postulates require animal modelsBIOL 53328Lecture TwoIdentification of PathogensMolecular versionBIOL 53329Lecture TwoMolecular VersionEmphasis shifted from identification of pathogens to identification of virulence factorsNot complete agreement on requirements to prove a particular gene or product plays a role in disease,but criteria widely acceptedBIOL 53330Lecture TwoMolecular Version1Gene or product found in strains that cause disease and not in avirulent bacteriaIf gene found in organisms not known to cause disease,gene should be mutated to less active or inactive form,or not expressedBIOL 53331Lecture TwoMolecular Version2Disrupting gene in virulent strain reduces or eliminates its virulenceIntroduction of cloned gene into avirulent strain should make it virulent Systems with multiple genes:These other genes would also have to be modified or introducedBIOL 53332Lecture TwoMolecular Version3Gene is expressed in bacteria inside host sometime during disease process4Ab to gene product should be protective or in cases where cell-mediated immunity involved,gene product should elicit protective immunityBIOL 53333Lecture TwoIdentification without CulturingCombine PCR:Polymerase Chain Reaction with 16S r-RNA phylogeny16S r-RNA found in all bacteriaConserved(domain)and variable(particular organism)sequencesBIOL 53334Lecture TwoIdentification without CulturingSizable database and similarities in sequence correspond well to evolutionary relationshipsSequence will either identify it as member of known or unknown speciesBIOL 53335Lecture TwoIdentification without CulturingPCR primers that recognize two conserved regions of 16S rRNA flanking a variable region are used to amplify and clone a DNA segment from a clinical specimanIf amplified segment is obtained,indicates bacteria present in specimanIt can be sequenced to identify bacteriumBIOL 53336Lecture TwoIdentification without CulturingFluorescently labeled probe of sequence can then visualize bacterium in clinical specimanRules out PCR amplification of contaminating DNA from other sourcesBIOL 53337Lecture TwoLecture TwoQuestions?Comments?Assignments.BIOL 53338