细菌感染诊断防治消毒灭菌课件.ppt

1、Bacterial InfectionNormal flora Opportunistic pathogenPathogenic bacteria(virulent bacteria)DysbacteriosisExotoxins EndotoxinsSeparation&Culture of bacteriaBacteriemiaSepticemiaPyemiaToxemiaEndotoxemiaCarrier Colony Toxoids AntitoxinNot all bacteria cause disease Normal flora The normal flora are ba

2、cteria which are found in or on our bodies on a semi-permanent basis without causing disease.There are more bacteria living in or on our bodies,than we have cells of our own.A human body contains around 1013 cells.The human body is home to around 1014 bacteria.One fourth of fecal weight is made of b

3、acteria!The normal flora are particularly important in the large intestine.Normal flora are also found in the nose,mouth,throat,skin.Opportunistic bacteria can be part of the normal flora but not pathogenic for normal persons,cause disease only in immunocompromised persons,or under specific conditio

4、ns.Pathogenic bacteria(virulent bacteria)Normally not members of the normal flora,have mechanisms to promote their own growth in the expense of hosts tissue or organs functionThe normal flora protect us from disease by:1.Competing with invaders for space and nutrients.2.Producing compounds(bacterioc

5、ins)which kill other bacteria.3.Lowering the pH so that other bacteria cant grow.In addition to the above ways of protecting us from disease our normal flora help us in other ways.Of course,there is a down side as well.The Good SideProduce vitamins we are unable to produce such as vitamin B12.Boost

6、our immune system Germ free animals(born and raised in a germ free plastic tent)are very susceptible to disease when removed from the germ free environment because their immune system is underdeveloped.Help digest food.The Bad SideIf the normal flora escape from their normal location,they can cause

7、disease.For example,Escherichia coli,commonly found in the intestine,can cause urinary tract infections if introduced into the bladder.Immunosuppression can allow otherwise harmless bacteria to cause disease.AIDS,some cancer treatments and transplant rejection drugs all suppress the immune system an

8、d allow the normal flora to cause occasionally serious disease.Three conditions correlate with opportunistic infection:1.Immune System Compromise2.Dysbacteriosis3.Change of Inhabiting Places:Pathogenesis is a multi-factorial process which depends on the nature of the species or strain(virulence fact

9、ors),the Pathway of Bacterial Entrance(gateway)and the immune status of the host,as well as the number of organisms in the initial exposure and the Environmental conditions.Bacterial Pathogenesis1.Adherence Factors 2.Surface hydrophobicity,3.pili,4.Capsule&glycocalyx 5.Bacterial Biofilms 6.Invasion

10、of Host Cells&Tissues:Enzymes7.immune evasion:8.Capsule&glycocalyx,9.IgA1 Proteases,10.Antigenic variation,11.Interference of complement activity 12.Intracellular pathogenicity.Bacterial Virulence.Bacterial Virulence FactorsFactors4.Toxins Exotoxins EndotoxinsExotoxins proteins usually enzymes destr

11、oy cellular structures destroy extracellular matrix Antibodies(anti-toxins)neutralize -vaccinationCholera toxin and E.coli labile toxinADP-ribosylation of regulator adenylate cyclase activation cyclic AMP active ion and water secretion diarrheaNormal condition(Acetylcholine)15Endotoxinsn Lipopolysac

12、charide(LPS):n a toxic lipid A n a core polysaccharide n O antigen polysaccharide side chains n Cell wall lysis required n Formaldehyde and heat resistant n Poor antigen as free molecule n Endotoxin effects Endotoxin effects Fever-pyrogen 1 microgram/kg Leukopenia and leukocytosis necrosis Endotoxem

13、ia and shock Disseminated intravascular coagulation(DIC).Exotoxins Toxins produced by bacteria and secreted to the outside of the bacteria cell(different from the endotoxin)acting on cell surface By binding to certain receptors Possess some degree of host cell specificity denatured by formaldehyde t

14、reatment to generate toxoids which lack toxic activity but still induce protective immunity when used as vaccines.Many exotoxins have an A-B subunit structure A subunit-provides the toxic activity B subunit-mediates adherence of the toxin complex to a host cell SummaryEndotoxins(LPS)n derived from G

15、cell walls and are often liberated when the bacteria lyse.n heat-stable,n three main regions n a toxic lipid A n a core polysaccharide n O antigen polysaccharide side chains n The pathophysiologic effects of LPS are similar.Pathway of Bacterial EntrancenContact:direct or indirect(Sexual contact)nInh

16、alation:transmitted by the respiratory route(Airborne droplets)nIngestion:ingested and transmitted through intestinal tract to the outside and get new infections by contaminating of food and water,which is called fecal-oral spread(Food,Water)nInoculation&blood transfusionnAnimal vectors:transmitted

17、by animal vectors(insect bitting)Response to InfectioninfectionxdiseaseInnate immunityno diseaserecoveryadaptive immunityre-infectionno diseasex Immunity of extracellular bacterial infection:antibodies(IgG,IgM,SIgA);phagocytes(neutrophils);complement;humoral immunity mainly.Immunity of intracellular

18、 bacterial infection:cell-mediated immunity(delayed-type hypersensitivity,DTH response(DTH)involving TH1and macrophages)mainly.No diseaseHost defenseBacterial infectionBalance between Infection and Immunity(immunity)Disease develops only in the right host and under the right conditionsDiseaseHostPat

19、hogenEnvironmentDiseaseHost defenseBacterial infectionEnvironmental conditions can help tilt the balance Environmental signals often control the expression of the virulence genes.Common signals,include:Temperrature/Iron availability:C diphtheriae/low ion/Osmolality/Growth phase/pH/Specific ionsSourc

20、es of InfectionB.Endogenous Infection:opportunistic pathogen infections A.Exogenous Infection PatientsCarriersAnimalsTypes of Bacterial InfectionAccording to infectious state:Inapparent infection:without clear clinic symptoms.Apparent Infection:have evident clinic symptoms.According to infectious si

21、tes(Local infection and Generalized or systemic infectionBacteriemia:bacteria circulate but not multiply in the blood.Septicemia:Bacteria circulate and multiply in the blood.Pyemia:produce septicemia with multiple abscesses in internal organs.Toxemia:Bacteia multiply at invading location and do not

22、enter blood stream,but the exotoxins enter blood and cause corresponding toxic symptoms Endotoxemia:multiply at location or in blood stream,release a lot of amount endotoxin released from bacterial cell rupture Carrier:Principles of Diagnosis and Prevention of Bacterial InfectionnManifestations of I

23、nfection:Signs and symptoms vary according to the site and severity of infection.Diagnosis requires a composite of information,including history,physical examination,radiographic findings,and laboratory data.nMicrobial Causes of Infection:Infections may be caused by bacteria,viruses,fungi,and parasi

24、tes.The pathogen may be exogenous(acquired from environmental or animal sources or from other persons)or endogenous(from the normal flora).1.The quantity material must be adequate.2.Collect from appropriate site.3.Prepare site to minimize contamination.4.Whenever possible,collect specimens prior to

25、antibiotics.5.Transport system maximized for pathogen survival should be used.General guidelines for specimen collectionBody fluid for specimen collectionnBlood septicemia nCerebrospinal fluid bacterial meningitisnPeritoneal(abdominal)nPleural(chest)nSynovial(joint)nPericardial(heart)nUrine Sample c

26、ollection devicesMicroscopy and Stains:cellular morphology and stain may permit preliminary identification.Separation&Culture of bacteria:Isolation of infectious agents frequently requires specialized media.Nonselective(noninhibitory)media permit the growth of many microorganisms.Selective media con

27、tain inhibitory substances that permit the isolation of specific types of microorganisms.Colony morphology can sometimes be useful in bacterial identificationIdentification of bacteria:Cultural characteristics:Growth characteristics under various conditions,Biochemical characterization:utilization o

28、f carbohydrates and other substrates,enzymatic activitySerology Identification A high or rising titer of specific IgG antibodies or the presence of specific IgM antibodies may suggest or confirm a diagnosis.Genomic MethodsAntimicrobial Susceptibility:Microorganisms,particularly bacteria,are tested i

29、n vitro to determine whether they are susceptible to antimicrobial agents.Microbiologic ExaminationMicroscopy and Stains Direct examination of stained or unstained preparations by light(bright field)microscopy is a relatively simple:with a 100 oil immersion objective,a 5 to 10 eyepiece,optimal light

30、ing.The two most important methods,the Gram and acid-fast techniques,to classify as well as stain the organism.employ staining,decolorization,counterstaining Examples of Gram StainsGram Positive Rods and CocciGram Negative Rods and CocciExamples of Acid-Fast StainsFully Acid-Fast Rods(Mycobacterium

31、species)Partially Acid-Fast Rods(Nocardia species)types of media for routine culture:The standard medium for specimens:blood agar,-Usually made with 5%sheep blood.-Most aerobic and facultatively anaerobic organisms will grow on blood agar A second necessary medium:Chocolate agar,-a medium containing

32、 heated blood with or without supplements,-Some organisms that do not grow on blood agar,including pathogenic neisseria and haemophilus,will grow on chocolate agar.CultureSelective medianSelective media is one that grows only certain microorganisms while inhibiting(or preventing)others from growing,

33、that is to say,the media has certain chemicals that allow one organism to grow but another organism cannot grow with those ingredients in the media,thus,is selective for the organisms that can grow in that media.Therefore,most commonly grows only one type of organism.Differential medianDifferential

34、media is one that distinguishes one microorganism from another,it can grow more than one microorganism,but depending on how each organism reacts to the media(like turns red)it differentiates from another microorganism.In this type of media if you are trying to distinguish between 2 types of microorg

35、anisms,both should grow but they will have different reactions to the media,and thru their visible reactions you can tell them apart(differentiate them).qGrowth on selective-differential media,such as Salmonella-Shigella(SS)medium,eosin-methylene-blue(EMB)and MacConkey agarqThe select effect of the

36、media in suppressing unwanted gram-positive organisms is exerted by bile salts or bacteriostatic dyes in the agar.qThe differential ability of these media is based on lactose fermentation:normal flora positive(colored colonies)and pathogens negative(colorlesscolonies).Separation of bacteria:plate st

37、reaking.Bacteriologic plate streaking.Colony:the visible growth of bacteria on solid growth media.Ideally,the colony is the progeny of one,or at most,a few bacteria.A colony will usually contain millions of bacterial cells.Colony morphology can sometimes be useful in bacterial identification.Colonie

38、s are described as to such properties as size,shape,texture,elevation,pigmentation,effect on growth medium.ColonyGrowth on blood agar to test for hemolytic properties-hemolytic:-hemolytic:incomplete lysis of red incomplete lysis of red blood cells,resulting blood cells,resulting in a greenish halo i

39、n a greenish halo around the colonyaround the colony-hemolytic:-hemolytic:complete lysis of red complete lysis of red blood cells,blood cells,resulting in a clear resulting in a clear halo around the halo around the colonycolony-hemolytic:non-hemolytic1.Cultural characteristics:lunique nutritional r

40、equirements,lpigment production,lhemolytic properties Identification of BacteriaMacConkeys AgarContains lactose and a pH indicator,E.coli ferment lactose to produce acid,which turns the pH dye red.So,E.coli colonies appear red.2.Biochemical characterizationIdentification of Bacterial the ability to

41、attack various substrates l or to produce particular metabolic products 2.Biochemical characterizationTriple Sugar Iron AgarIdentification of BacteriaA simple approach to rapid diagnosis(as an example of antigen detection)is used in many doctors offices for the group A streptococcus.The patients thr

42、oat is swabbed and streptococcal antigen extracted directly from the swab(without prior bacteriological culture).The bacterial antigen is detected by aggregation(agglutination)of antibody coated latex beads.Serologic identification of an antibody response(in patients serum)to the infecting agent can

43、 only be successful several weeks after an infection has occurred.3.Serology Identificationuse of antibodies of known specificity to detect antigens present on whole bacteria or free in bacterial extracts Identification of BacteriaSerological methods used to detect both antigen and antibody in speci

44、mensnThe fastest and most specific waynImmunofluorescence microscopy,FACSnEnzyme-immunoassay ELISA,Western blotnRadioimmunoassay quantitate antigen-antibody complexFACS(fluorescence activated cell sorter)4.Genomic Methodsn16S DNA sequencingnLabeled probes specific for the 16S rRNA of a species are a

45、dded,and the amount of label on the double-stranded hybrid is measured.n This technique is widely used for the rapid identification of many organisms.nFluorescence in situ hybridization(FISH)nPolymerase Chain Reaction(PCR)nNucleic Acid Sequence Analysis nChecker board DNA hybridization(DNA microarra

46、y)Identification of BacteriaELISA(Enzyme-Linked Immunosorbent Assay)E.coli 16S RNAl The 16S rRNA of each species of bacteria has stable(conserved)portions of the sequence.l Many copies are present in each organism.FISH PCR(polymerase chain reaction)BacteriamRNAcDNADNA microarrays37oC cDNA labeledw/f

47、luorescein tag25oC cDNA labeledw/rhodamine tagDNA array 6000 genes“Transcriptome”SummarynDirect microscopynFastnGive some hints on the type of bacterianLow sensitivitynCulturenHigh sensitivitynCan make definitive IDnSlownOnly works on culturable bacterianSerological assaynFastnHigh specificitynCan d

48、etect both antigen and antibodynEasy(can be used at chairside or bedside)nLow sensitivitynGenomic based assaynFastnHigh sensitivity and specificitynWorks on both culturable and non-culturable bacterianEspecially useful for detecting slow growing bacteria such as T.b.nRequires technical expertisenFal

49、se positive or negativePrevention of Bacterial InfectionArtificial active immunitynToxoids:a modified form of the toxin that preserves its antigenicity but has lost its toxicity.This has been spectacularly successful with tetanus and diphtheria.nInactivated vaccines:nAttenuated live vaccines:nSpecia

50、l vaccines:polysaccharide vaccine,subunit vaccine,(conjugate vaccine,bio-engineered vaccine,chemical vaccine,synthetic vaccine),nucleic acid vaccine,idiotype vaccine,autovaccine,etc.Artificial active immunityArtificial passive immunity nAntitoxin:e.g.Tetanus antitoxin and diphtheria antitoxin.It is