4細(xì)菌的感染,診斷,防治,消毒,滅菌

1、Bacterial Infection1Normal flora Opportunistic pathogenPathogenic bacteria (virulent bacteria)DysbacteriosisExotoxins EndotoxinsSeparation & Culture of bacteriaBacteriemiaSepticemiaPyemiaToxemiaEndotoxemiaCarrier Colony Toxoids AntitoxinKey Words2Not all bacteria cause diseaseNormal flora The normal

2、 flora are bacteria 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 1013cells. The human body is home to around 1014 bacteria. One fourth of fecal we

3、ight is made of bacteria!The normal flora are particularly important in the large intestine.Normal flora are also found in thenose, 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 un

4、der specific conditions.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 function3The normal flora protect us from disease by:1. Competing with invaders for space and nutrients.2. Pr

5、oducing compounds (bacteriocins) which kill other bacteria.3. Lowering the pH so that other bacteria cant grow.4In 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 p

6、roduce such as vitamin B12.Boost 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.5The Bad SideIf the normal flora escape from t

7、heir normal location, they can cause 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 rejecti

8、on drugs all suppress the immune system and allow the normal flora to cause occasionally serious disease.6 Three conditions correlate with opportunistic infection: 1. Immune System Compromise2. Dysbacteriosis3. Change of Inhabiting Places:7Pathogenesis is a multi-factorial process which depends on t

9、he nature of the species or strain (virulence factors), 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 Pathogenesis8BACTERIAL VIRULENCE FACTORS 9Adherence Factors S

10、urface hydrophobicity, pili, Capsule & glycocalyx Bacterial Biofilms Invasion of Host Cells & Tissues: Enzymesimmune evasion: Capsule & glycocalyx, IgA1 Proteases, Antigenic variation, Interference of complement activity Intracellular pathogenicity.Bacterial Virulence Factors104. ToxinsExotoxinsEndo

11、toxinsExotoxins proteins usually enzymes destroy cellular structures destroy extracellular matrix Antibodies (anti-toxins) neutralize -vaccinationActiveBindingACell surfaceBA-B toxins11Cholera toxin and E. coli labile toxinADP-ribosylation of regulator adenylate cyclase activation cyclic AMP active

12、ion and water secretion diarrhea12Tetanus toxin inhibits glycine release inactivates inhibitory neurons muscles over-active rigid paralysis Normal conditionRigid paralysis13 Botulinum toxin inhibits acetylcholine release inhibits nerve impulses muscles inactiveflacid paralysis(Acetylcholine)1415Exot

13、oxinsAntibodies (anti-toxins) neutralizevaccination15Endotoxins Lipopolysaccharide (LPS) : a toxic lipid A a core polysaccharide O antigen polysaccharide side chains Cell wall lysis required Formaldehyde and heat resistant Poor antigen as free molecule Endotoxin effects 16 Endotoxin effects Fever-py

14、rogen 1 microgram/ kg Leukopenia and leukocytosis necrosis Endotoxemia and shock Disseminated intravascular coagulation (DIC).Non-specific inflammation. Cytokine releaseComplement activationB cell mitogensPolyclonal B cell activators Adjuvants1718Exotoxins Toxins produced by bacteria and secreted to

15、 the outside of the bacteria cell (different from the endotoxin) acting on cell surface By binding to certain receptorsPossess some degree of host cell specificitydenatured by formaldehyde treatment to generate toxoids which lack toxic activity but still induce protective immunity when used as vacci

16、nes.Many exotoxins have an A-B subunit structureA subunit- provides the toxic activityB subunit-mediates adherence of the toxin complex to a host cell Summary19Endotoxins ( LPS) derived from Gcell walls and are often liberated when the bacteria lyse. heat-stable, three main regions a toxic lipid A a

17、 core polysaccharide O antigen polysaccharide side chains The pathophysiologic effects of LPS are similar20. Pathway of Bacterial EntranceContact: direct or indirect (Sexual contact) Inhalation: transmitted by the respiratory route (Airborne droplets)Ingestion: ingested and transmitted through intes

18、tinal tract to the outside and get new infections by contaminating of food and water, which is called fecal-oral spread (Food, Water )Inoculation & blood transfusionAnimal vectors: transmitted by animal vectors (insect bitting)21Response to InfectioninfectionxdiseaseInnate immunityno diseaserecovery

19、adaptive immunityre-infectionno diseasex22Immunity of extracellular bacterial infection: antibodies (IgG, IgM, SIgA); phagocytes (neutrophils); complement; humoral immunity mainly.Immunity of intracellular bacterial infection: cell-mediated immunity (delayed-type hypersensitivity, DTH response (DTH)

20、 involving TH1and macrophages) mainly. 23No diseaseHost defenseBacterial infectionBalance between Infection and Immunity(immunity)24Disease develops only in the right host and under the right conditionsDiseaseHostPathogenEnvironment25DiseaseHost defenseBacterial infectionEnvironmental conditions can

21、 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 ions26Sources of InfectionB. Endogenous Infection: opportunistic pathogen infection

22、s Exogenous Infection PatientsCarriersAnimals27Types of Bacterial InfectionAccording to infectious state:Inapparent infection: without clear clinic symptoms.Apparent Infection: have evident clinic symptoms.According to infectious sites (Local infection and Generalized or systemic infectionBacteriemi

23、a: 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 enter blood stream, but the exotoxins enter blood and cause co

24、rresponding toxic symptoms Endotoxemia: multiply at location or in blood stream, release a lot of amount endotoxin released from bacterial cell rupture Carrier: 28Principles of Diagnosis and Prevention of Bacterial Infection29Manifestations of Infection: Signs and symptoms vary according to the site

25、 and severity of infection. Diagnosis requires a composite of information, including history, physical examination, radiographic findings, and laboratory data. Microbial Causes of Infection: Infections may be caused by bacteria, viruses, fungi, and parasites. The pathogen may be exogenous (acquired

26、from environmental or animal sources or from other persons) or endogenous (from the normal flora). 30The quantity material must be adequate.Collect from appropriate site.Prepare site to minimize contamination.Whenever possible, collect specimens prior to antibiotics.Transport system maximized for pa

27、thogen survival should be used.General guidelines for specimen collection31Body fluid for specimen collectionBlood septicemia Cerebrospinal fluid bacterial meningitisPeritoneal (abdominal)Pleural (chest)Synovial (joint)Pericardial (heart)Urine 32Sample collection devices33Microscopy and Stains: cell

28、ular 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 contain inhibitory substances that per

29、mit 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 of carbohydrates and other subs

30、trates, 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 in vitro to determine wheth

31、er they are susceptible to antimicrobial agents.Microbiologic Examination34Microscopy 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 lighting. The two most i

32、mportant methods, the Gram and acid-fast techniques, to classify as well as stain the organism. employ staining, decolorization, counterstaining 35Examples of Gram StainsGram Positive Rods and CocciGram Negative Rods and Cocci36Examples of Acid-Fast StainsFully Acid-Fast Rods (Mycobacterium species)

33、Partially Acid-Fast Rods (Nocardia species)37types 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 contai

34、ning 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.Culture38Selective mediaSelective media is one that grows only certain microorganisms while inhibiting (or preventing) others fro

35、m growing, 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.39Differential

36、mediaDifferential 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 betw

37、een 2 types of microorganisms, both should grow but they will have different reactions to the media, and thru their visible reactions you can tell them apart (differentiate them).40Growth on selective-differential media, such as Salmonella-Shigella (SS) medium, eosin-methylene-blue (EMB) and MacConk

38、ey agarThe select effect of the media in suppressing unwanted gram-positive organisms is exerted by bile salts or bacteriostatic dyes in the agar.The differential ability of these media is based on lactose fermentation: normal flora positive ( colored colonies) and pathogens negative (colorlesscolon

39、ies).41Separation of bacteria : plate streaking.Bacteriologic plate streaking.4243 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 somet

40、imes be useful in bacterial identification.Colonies are described as to such properties as size, shape, texture, elevation, pigmentation, effect on growth medium.Colony444546Growth on blood agar to test for hemolytic properties-hemolytic: incomplete lysis of red blood cells, resulting in a greenish

41、halo around the colony-hemolytic: complete lysis of red blood cells, resulting in a clear halo around the colony-hemolytic: non-hemolytic1. Cultural characteristics:unique nutritional requirements, pigment production, hemolytic properties Identification of Bacteria47a (alpha)partial hemolysis greeni

42、sh color (i.e. S. pneumoniae)b (beta) complete clearing Group A and Bg (gamma) no lysisEnterococcus (groupD)hemolysis reaction - sheep blood agar48MacConkeys 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.

43、 2. Biochemical characterizationIdentification of Bacteria the ability to attack various substrates or to produce particular metabolic products 492. Biochemical characterizationTriple Sugar Iron AgarIdentification of Bacteria5051A simple approach to rapid diagnosis (as an example of antigen detectio

44、n) is used in many doctors offices for the group A streptococcus. The patients throat 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.Serolog

45、ic identification of an antibody response (in patients serum) to the infecting agent can 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 Identi

46、fication of Bacteria52Serological methods used to detect both antigen and antibody in specimensThe fastest and most specific wayImmunofluorescence microscopy, FACSEnzyme-immunoassay ELISA, Western blotRadioimmunoassay quantitate antigen-antibody complex53FACS (fluorescence activated cell sorter)544.

47、 Genomic Methods16S DNA sequencingLabeled probes specific for the 16S rRNA of a species are added, and the amount of label on the double-stranded hybrid is measured. This technique is widely used for the rapid identification of many organisms.Fluorescence in situ hybridization (FISH)Polymerase Chain

48、 Reaction (PCR)Nucleic Acid Sequence Analysis Checker board DNA hybridization (DNA microarray)Identification of Bacteria55ELISA (Enzyme-Linked Immunosorbent Assay) 56E. coli 16S RNA The 16S rRNA of each species of bacteria has stable (conserved) portions of the sequence. Many copies are present in e

49、ach organism. 57FISH 58PCR (polymerase chain reaction)59BacteriamRNAcDNADNA microarrays6037oC cDNA labeledw/ fluorescein tag25oC cDNA labeledw/ rhodamine tagDNA array 6000 genes“Transcriptome”6162SummaryDirect microscopyFastGive some hints on the type of bacteriaLow sensitivityCultureHigh sensitivit

50、yCan make definitive IDSlowOnly works on culturable bacteriaSerological assayFastHigh specificityCan detect both antigen and antibodyEasy (can be used at chairside or bedside)Low sensitivityGenomic based assayFastHigh sensitivity and specificityWorks on both culturable and non-culturable bacteriaEsp

51、ecially useful for detecting slow growing bacteria such as T.b.Requires technical expertiseFalse positive or negative63Prevention of Bacterial Infection64Artificial active immunityVaccines are antigens prepared from pathogens that can raise a protective immune response, yet do not cause illness. The

52、se prepared antigens will stimulate both B cells and T cells and help to create memory cells that can later mount a vigorous immune response to an encounter with the real pathogen.65Toxoids: a modified form of the toxin that preserves its antigenicity but has lost its toxicity. This has been spectac

53、ularly successful with tetanus and diphtheria.Inactivated vaccines: Attenuated live vaccines :Special vaccines: polysaccharide vaccine, subunit vaccine, ( conjugate vaccine, bio-engineered vaccine, chemical vaccine, synthetic vaccine ), nucleic acid vaccine, idiotype vaccine, autovaccine, etc.Artifi

54、cial active immunity66Artificial passive immunity Antitoxin: e.g. Tetanus antitoxin and diphtheria antitoxin. It is raised in the horse. It is most important to give an intented recipient of equine serum a prior test dose to exclude hypersensitivity subjects who may have been sensitized by a previou

55、s dose of equine serum.Pooled immunoglobulin: It contains the normal repertoire of antibodies for an adult, and can protect against hepatitis A, and measles.Specific immunoglobulin: Preparations of specific immunoglobulin are available for passive immunization against tetanus, hepatitis B, rabies, v

56、aricella-zoster.Cytokine 67Sterilization and DisinfectionA. SterilizationA physical or chemical process that completely destroys or removes all microbial life, including spores.B. Disinfection treatment to destroy harmful microorganisms. C. Preservation The prevention of multiplication of microorgan

57、isms in formulated products, including pharmaceuticals and foods.68Sterilization and Disinfection BactericidalA specific term referring to the property by which a biocide is able to kill bacteria. Bactericidal action differs from bacteriostasis only in being irreversible; ie, the “killed” organism c

58、an no longer reproduce, even after being removed from contact with the agent. BacteriostaticA specific term referring to the property by which a biocide is able to inhibit bacterial multiplication; multiplication resumes upon removal of the agent. 69DisinfectionPhysical MethodsHeatRadiation Filtrati

59、onUltrasonic and SonicFreezing70Sterilization and Disinfection. HeatHeat energy can be applied in three ways, in the forms of Moist heat (either boiling or autoclaving)Dry heatPasteurization71Moist Heat: far more rapid and effective in sterilization than dry heat, A temperature of 100C will kill all

60、 but spore forms of bacteria within 23 minutes in laboratory-scale cultures; 121C for 15 minutes is utilized to kill spores. Autoclaves or pressure cookers are used for this purpose. Autoclaves are usually operated at 121C, which is achieved with a pressure of 15 lb/sq Its use ：any materials that ar