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Microbiology 2011 5/6/2011 Yu Chun-Keung DVM, Ph. D Miscellaneous Small Gram-Negative Bacilli Chapter 34 Microbiology 2011 5/6/2011 Yu Chun-Keung DVM, Ph. D Miscellaneous Small Gram-Negative Bacilli Chapter 34 Haemophilus Chapter 35 Bordetella Chapter 36 Francisella Brucella

Chapter 34 Haemophilus Family Pasteurellaceae Genera Haemophilus Actinobacillus Aggregatibacter Pasteurella • Small, G(-), non-spore-forming Chapter 34 Haemophilus Family Pasteurellaceae Genera Haemophilus Actinobacillus Aggregatibacter Pasteurella • Small, G(-), non-spore-forming bacilli • Fastidious growth needs

Genus Haemophilus 嗜血桿菌 “Blood-lover” Growth require hemin (x factor) and nicrotinamide adenine dinucleotide, NAD Genus Haemophilus 嗜血桿菌 “Blood-lover” Growth require hemin (x factor) and nicrotinamide adenine dinucleotide, NAD (v factor) Heated blood (chocolate) agar for isolation Important Haemophilius species n H. influenzae (an important pathogen) n H. ducreyi (soft chancre) n H. aegyptius (acute, purulent conjunctivitis) n H. parainfluenzae (rarely pathogenic)

Classification H. influenzae (Hi) Many but not all strains is covered with polysaccharide capsule Classification H. influenzae (Hi) Many but not all strains is covered with polysaccharide capsule Serological differentiation - capsular antigens: serotype a to f Biochemical properties - indole production, urease activity, ornithine decarboxylase activity : biotype I to VIII

Pathogenesis Non-encapsulated Hi & H. parainfluenzae (non-typeable) Colonize URT in all people 10% of Pathogenesis Non-encapsulated Hi & H. parainfluenzae (non-typeable) Colonize URT in all people 10% of the flora of saliva: H. parainfluenzae Opportunistic pathogens: spread locally and cause acute and chronic otitis, sinusitis, bronchitis, and pneumonia.

Pathogenesis - encapsulated Hi type b Uncommon in the URT Common cause of disease Pathogenesis - encapsulated Hi type b Uncommon in the URT Common cause of disease in unvaccinated children Invasion: adhesins colonization of oropharynx release cell wall components damage and impair ciliary function across epithelial and endothelial cells blood Produce Ig. A 1 proteases, facilitate colonization LPS lipid A induces meningeal inflammation

Major virulence factor: antiphagocytic polysaccharide capsule – polyribitol phosphate (PRP) : ribose, ribitol, phosphate Major virulence factor: antiphagocytic polysaccharide capsule – polyribitol phosphate (PRP) : ribose, ribitol, phosphate Phagocytic engulfment of H. influenzae bacterium opsonized by antibodies specific for the capsule and somatic (cell wall) antigen. 2004 Kenneth Todar University of Wisconsin-Madison Department of Bacteriology Natural infection, vaccination, passive transfer of material antibody - anti. PRP antibody is protective (enhance phagocytosis and complementmediated bacteriocidal activity) Absence of anti-PRP antibody (complement depletion, splenectomy ) leads to invasion, bacteremia and dissemination

Clinical diseases (Hib infection) Meningitis: Hi type b was the most common cause of Clinical diseases (Hib infection) Meningitis: Hi type b was the most common cause of pediatric meningitis (3 -18 m). Age-specific incidence of bacterial meningitis caused by Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae prior to 1985 2004 Kenneth Todar University of Wisconsin. Madison Department of Bacteriology

Clinical diseases (Hib infection) Epiglotitis 會厭炎: 2 -4 yrs; swelling of the supraglottic tissue, Clinical diseases (Hib infection) Epiglotitis 會厭炎: 2 -4 yrs; swelling of the supraglottic tissue, pharyngitis, fever, rapidly progress to complete obstruction of the airways, life-threatening emergency. Cellulitis 蜂窩織炎: fever, reddish-blue patches on the cheeks or periorbital area. Arthritis: the most common form of arthritis (single large joint) in children <2 yrs.

Transmission Person-to-person transmission in -immune population non Increased disease frequency in households where there Transmission Person-to-person transmission in -immune population non Increased disease frequency in households where there is a primary case or an asymptomatic carrier. Primary risk factor for invasive disease = absence of anti-PRP antibody. Close contacts should be given chemoprophylaxis.

Diagnosis Clinical diagnosis n n n History, physical examination Sign: are those findings that Diagnosis Clinical diagnosis n n n History, physical examination Sign: are those findings that a physician can objectively detect or measure. Symptom: are those problems that a patient notices or feels. Laboratory diagnosis n n detection / identification of pathogens (i. e. , whole cell, protein, nucleic acid) detection of immune responses (i. e. , antibodies)

Diagnosis Specimens: n Oral swab: avoid contamination with oral secretions n Sputum from LRT Diagnosis Specimens: n Oral swab: avoid contamination with oral secretions n Sputum from LRT n Direct needle aspiration n Cerebrospinal fluid (CSF) and blood (>107 bacteria/ml) Microscopy: both sensitive & specific; -) bacilli in CSF in >80% cases before antibiotics treatment G(

Diagnosis (for Hib only) Particle agglutination test : n n Detect PRP antigen, rapid Diagnosis (for Hib only) Particle agglutination test : n n Detect PRP antigen, rapid and sensitive (1 ng/ml) Anti-PRP Ab-coated latex particles + specimen, if PRP present, “positive” agglutination Culture: n n Chocolate agar, colony 1 -2 mm. Blood agar: Hib grows around colonies of Staph. aureus on blood agar - Satellite phenomenon

Treatment Prompt antimicrobial therapy for systemic Hib infections, otherwise mortality 100% Serious infections: cephalosporins Treatment Prompt antimicrobial therapy for systemic Hib infections, otherwise mortality 100% Serious infections: cephalosporins Less severe infections (otitis, sinusitis): ampicillin Antibiotic chemoprophylaxis (rifampin) for high risk group (children < 2 ys with patients around)

Prevention Polysaccharide vaccine for Hib were not effective for children < 18 m (the Prevention Polysaccharide vaccine for Hib were not effective for children < 18 m (the high risk population). Hi type b conjugate vaccine n Purified capsular PRP + Carrier proteins: Meningococcal outer membrane protein Diphtheria toxoid Tetanus toxoid

 • Before the introduction of conjugated vaccine, Hib was a common pediatric disease • Before the introduction of conjugated vaccine, Hib was a common pediatric disease in children < 5 yrs. • Hib conjugate vaccine was introduced in 1987 which greatly reduced the incidence of disease (> 90%).

Epidemiology Now Hib infections only occur in nonimmune children or adults with waning immunity. Epidemiology Now Hib infections only occur in nonimmune children or adults with waning immunity. Other serotypes of encapsulated strains and non-encapsulated strains become more common. Hib remains a significant pediatric pathogen worldwide, especially in developing countries.

Haemophilus ducreyi A sexually transmitted disease; most common in Africa and Asia Painful ulcer Haemophilus ducreyi A sexually transmitted disease; most common in Africa and Asia Painful ulcer on genitalia (soft chancre, 軟 性下疳 ) with regional lymphadenopathy Differential: n Syphilis; Herpes simplex; Lymphogranuloma venereum (caused by Chlamydia trachomatis)

Genus Aggregatibacter A. actinomycetemcomitans and A. aphrophilus Mouth flora blood damaged heart valve / Genus Aggregatibacter A. actinomycetemcomitans and A. aphrophilus Mouth flora blood damaged heart valve / artificial valve subacute endocarditis Difficult to diagnosis : develop slowly and the bacteria grow slowly in blood agar (> 5 days)

Genus Pasteurella P. multocida and P. canis Primarily animal pathogen. Commensals in oropharynx of Genus Pasteurella P. multocida and P. canis Primarily animal pathogen. Commensals in oropharynx of health animals. Human infections result from animal contact (bites, scratches, shared food). The most common organism in human wounds inflicted by bites from cats and dogs.

Three general forms of disease Localized cellulitis and regional lymphadenopathy after animal bite or Three general forms of disease Localized cellulitis and regional lymphadenopathy after animal bite or scratch. Exacerbation of chronic respiratory tract disease in patients with underlying pulmonary dysfunction. Systemic infection in immunocompromised patients.

Lab diagnosis Grows well on blood and chocolate agar Large, buttery colonies with a Lab diagnosis Grows well on blood and chocolate agar Large, buttery colonies with a musty odor Treatment Susceptible to a variety of antibiotics Penicillin, macrolides, tetracycline …

Chapter 35 Bordetella Extremely small (0. 2 x 1 μm ), G(-), coccobacilli Have Chapter 35 Bordetella Extremely small (0. 2 x 1 μm ), G(-), coccobacilli Have simple nutritional requirement Some species (i. e. , B. pertussis) are highly susceptible to toxic substances and metabolites in media (need charcoal, starch, blood, or albumin to absorb toxic substances)

Important Bordetella species B. pertussis: whooping cough / pertussis (severe cough) B. parapertussis: mild Important Bordetella species B. pertussis: whooping cough / pertussis (severe cough) B. parapertussis: mild form of pertussis B. bronchiseptica: respiratory disease of animals (pigs and dogs) B. holmesii: uncommon cause of sepsis The four species are closely related, differing only in the expression of virulence genes

Pathogenesis • Exposure (aerosol) • Attachment to ciliated epithelial cells of the respiratory tract Pathogenesis • Exposure (aerosol) • Attachment to ciliated epithelial cells of the respiratory tract by means of adhesins ( 黏附因子) • Proliferation • Production of toxins • Localized tissue damage and systemic toxicity Colonization of tracheal epithelial cells by Bordetella pertussis 2004 Kenneth Todar University of Wisconsin. Madison Department of Bacteriology

Bacterial adhesins Filamentous hemagglutinin: contain RGD motif: bind (1) sulfated glycoprotein integrins on ciliated Bacterial adhesins Filamentous hemagglutinin: contain RGD motif: bind (1) sulfated glycoprotein integrins on ciliated respiratory cells; (2) CR 3 on macrophages, and trigger phagocytosis without initiating oxidative burst (intracellular survival, escape from Ab) Pertactin : contain RGD motif Fimbria : mediate binding in vitro; in vivo function unknown Pertussis toxin:A classic A-B toxin with a toxic subunit (S 1) and binding subunits (S 2 to S 5); S 2 binds lactosylceramide on ciliated respiratory cells, S 3 binds phagocytic cells

Toxins S 1 subunit of pertussis toxin Adenylate cyclase toxin / hemolysin Dermonecrotic toxin Toxins S 1 subunit of pertussis toxin Adenylate cyclase toxin / hemolysin Dermonecrotic toxin Tracheal cytotoxin LPS

S 1 subunit of pertussis toxin S 2 -S 5 binding subunit S 1 S 1 subunit of pertussis toxin S 2 -S 5 binding subunit S 1 toxic subunit Adenosine diphosphateribosylating activity for G protein, which regulates adenylate cyclase activity (convert ATP to c. AMP). Increase respiratory secretion and mucus production.

Tracheal cytotoxin: (1) target ciliated epithelial cell; ciliostasis, extrusion of ciliated cells, impair regeneration Tracheal cytotoxin: (1) target ciliated epithelial cell; ciliostasis, extrusion of ciliated cells, impair regeneration of damaged cells by interfering DNA synthesis (disrupt clearance mechanism, lead to cough); (2) IL-1 production (lead to fever) A tracheal organ culture 72 h after infection with B. pertussis. Large arrow: Bordetella Small arrow: cilia Extruded epithelial cell with attached bacteria Denuded epithelium Normal ciliated epithelial cell

Toxins Adenylate cyclase toxin / hemolysin: (1) activated by calmodulin and converse ATP to Toxins Adenylate cyclase toxin / hemolysin: (1) activated by calmodulin and converse ATP to c. AMP, increase respiratory secretion; (2) inhibit leukocyte functions Dermonecrotic toxin: vasoconstriction and tissue destruction LPS: unknown (activate complement and stimulate cytokine release)

Clinical disease Infect ciliated epithelial cells of the airways, produce disease locally, no invasion. Clinical disease Infect ciliated epithelial cells of the airways, produce disease locally, no invasion. Catarrhal phase : resemble common cold, sneezing, serous rhinorrhea, malaise, low-grade fever, 1 -2 wk, infectious (disease not recognized with high number of bacteria produced) Paroxysmal phase : a series of repetitive coughs followed by inspiratory whoop, vomiting, and exhaustion, 40 -50 paroxysms daily, lymphoctyosis, 2 -4 wk. Convalescent phase : paroxysms diminish with secondary complications, lasts for above 3 wk.

Clinical disease Classic presentation may not be seen in patients with partial immunity. n Clinical disease Classic presentation may not be seen in patients with partial immunity. n Only chronic persistent cough without whooping or vomiting Differential diagnosis n Mycoplasma pneumoniae n Chlamydophilia pneumoniae n Legionella pneumophila

Epidemiology Pertussis was considered a pediatric disease (< 1 year) Incidence (morbidity and mortality) Epidemiology Pertussis was considered a pediatric disease (< 1 year) Incidence (morbidity and mortality) has been reduced considerably after the introduction of vaccine in 1949. Still endemic worldwide with a dramatic increase in recent year Majority of infections are found in adolescents and adults (reason not known). 2018/3/1 9

Lab diagnosis – specimen collection and transport Extremely sensitive to drying, do not survive Lab diagnosis – specimen collection and transport Extremely sensitive to drying, do not survive outside the host or traditional transport medium. Inoculate (nasopharyngeal aspirate) to freshly prepared medium or transport medium at bedside. Use synthetic fiber swabs not cotton swabs (fatty acid are toxic to Bp).

Lab diagnosis - microscopy Fluoresceinlabeled rabbit anti-Bp Ab Aspirated specimen Direct Fluorescein-labeled anti-rabbit Ig Lab diagnosis - microscopy Fluoresceinlabeled rabbit anti-Bp Ab Aspirated specimen Direct Fluorescein-labeled anti-rabbit Ig Ab Rabbit anti-Bp Ab Indirect Direct or indirect fluorescent antibody tests for antigen detection Aspirated specimen microscopic slide airdried heat fixed fluorescent Ab Sensitivity 50%

Lab diagnosis - culture Regan-Lowe charcoal medium (horse blood, glycerol, peptones). 35°C, humidified, 7 Lab diagnosis - culture Regan-Lowe charcoal medium (horse blood, glycerol, peptones). 35°C, humidified, 7 days, 50% sensitivity, affected by n Patient factors (stage of illness, use of antibiotics) n Quality of specimen n Transport conditions n Culture methods

. Nucleic acid amplification Polymerase chain reaction sensitivity 80 -100% No FDA approved test, . Nucleic acid amplification Polymerase chain reaction sensitivity 80 -100% No FDA approved test, inhouse assay Serology ELISA for antibodies against filamentous hemagglutinin or pertussis toxin No FDA approved test

Treatment Primarily supportive. Recovery depends on regeneration of ciliated epithelial cells. Antibiotics (erythromycin) are Treatment Primarily supportive. Recovery depends on regeneration of ciliated epithelial cells. Antibiotics (erythromycin) are effective and can reduce duration of clinical course. However, the illness is usually unrecognized during catarrhal phase (the peak of contagiousness) Pertussis is highly contagious; prophylaxis for family members of a symptomatic patient.

Vaccination DTP vaccine (diphtheria toxoid + tetanus toxoid + inactivated whole cell of Bp), Vaccination DTP vaccine (diphtheria toxoid + tetanus toxoid + inactivated whole cell of Bp), 80 -85% effective. n DTP vaccine has not been widely accepted because of vaccine-related complications. DTa. P (acellular vaccine) : subunit vaccine D + T + inactivated pertussis toxin, filamentous hemagglutinin, and pertactin or fimbriae.

Pentavalent vaccine – PEDIACEL (Sanofi Pasteur) 1. purified polyribose ribitol phosphate (PRP) of Hib Pentavalent vaccine – PEDIACEL (Sanofi Pasteur) 1. purified polyribose ribitol phosphate (PRP) of Hib covalently bound to 20 µg of tetanus protein 10 µg 2. pertussis toxoid 20 µg filamentous haemagglutinin 20 µg fimbrial agglutinogens 2 + 3, 5 µg; pertactin 3 µg 3. diphtheria toxoid 15 Lf 4. tetanus toxoid 5 Lf 5. poliovirus type 1 (Mahoney) 40 D-antigen units poliovirus type 2 (MEF 1) 8 D-antigen units poliovirus type 3 (Saukett) 32 D-antigen units 41

Chapter 36 Francisella and Brucella Zoonotic pathogens and potential agents of bioterrorism Very small Chapter 36 Francisella and Brucella Zoonotic pathogens and potential agents of bioterrorism Very small G(-) coccobacilli, 0. 5 1. 5 m, Fastidious, slow growth on culture (>1 week)

Taxonomically unrelated α-Proteobacteria n Brucella n Rickettsia n Ehrlichia γ-Proteobacteria n Francisella n Legionella Taxonomically unrelated α-Proteobacteria n Brucella n Rickettsia n Ehrlichia γ-Proteobacteria n Francisella n Legionella n Pasteruella n Pseudomonas

Genus Francisella tularensis (Tularemia) n F. tularensis subsp. tularensis (type A) n F. tularensis Genus Francisella tularensis (Tularemia) n F. tularensis subsp. tularensis (type A) n F. tularensis subsp. holarctica (type B) n F. tularensis subsp. Mediasiatica (rare as pathogen) n F. tularensis subsp. Novicida (rare as pathogen) Francisella philomiragia (uncommon opportunistic pathogen)

Epidemiology F. tularensis subsp. tularensis Natural reservoirs and vectors: >200 species of mammals, birds, Epidemiology F. tularensis subsp. tularensis Natural reservoirs and vectors: >200 species of mammals, birds, blood-sucking arthropods Type A: n North America: lagomorphs (rabbits, hares), cats, biting arthropod Type B: n Northern hemisphere: rodents, cats Those exposed to ticks are high risk for infection in endemic areas i. e. , hunters, and lab personnel n n Infections occur in summer and winter; warm winter + wet summer (increase in tick population) Actual numbers of infections > reported cases

Pathogenesis Pathogenic strains possess antiphagocytic capsule; protect bacteria from complement-mediated killing Intracellular parasite: can Pathogenesis Pathogenic strains possess antiphagocytic capsule; protect bacteria from complement-mediated killing Intracellular parasite: can survive for prolonged periods in macrophages; inhibit phagosomelysosome fusion. IFN-γ- and TNF-α-mediated activation of macrophage is essential for controlling bacterial replication and killing in early stage. Specific T cell-mediated activation of macrophage is essential for controlling bacterial replication and killing in late stage.

Clinical disease - Tularemia (Rabbit fever / Tick fever) Clinical symptoms and prognosis determined Clinical disease - Tularemia (Rabbit fever / Tick fever) Clinical symptoms and prognosis determined by route of infection n n Ulcer Cutaneous tularemia infection microbes. historique. net Ulceroglandular form: cutaneous ulcer + swollen LN, most common Oculoglandular form: painful conjunctivitis + swollen cervical LN. n Typhoidal form: sepsis n Pneumonic form: pulmonary symptoms n Gastrointestinal form:

Lab diagnosis Specimen collection: highly contagious: able to penetrate through unbroken skin and mucous Lab diagnosis Specimen collection: highly contagious: able to penetrate through unbroken skin and mucous membrane + aerosols. extremely hazardous for physician and lab workers; wear gloves and perform work in biohazard hood Microscopy: Grain stain – not practical; direct staining with fluorescent antibody, more sensitive and specific Culture: not grow in common medium without cysteine (eg. blood agar); use chocolate agar or buffered charcoal yeast extract (BCYE) agar, take a week or longer Serology: a 4 -fold increase in Ab titer during illness or a single titer of 1: 160; antibody persist for many years (past or current infection? ); cross-reactivity between Brucella and Francisella

T/P/C Penicillin and cephalosporin are ineffective (produce β-lactamase) Streptomycin and gentamicin are effective (high T/P/C Penicillin and cephalosporin are ineffective (produce β-lactamase) Streptomycin and gentamicin are effective (high toxicity) Prompt treatment < 1% mortality rate Wear protective clothes and use insect repellents, avoid reservoirs and vectors Prophylactic antibiotics for high risk groups Live-attenuated vaccine : partly protective

Genus Brucella Six species with four species associated with human diseases B. melitensis : Genus Brucella Six species with four species associated with human diseases B. melitensis : goat and sheep (natural host) B. suis : swine, reindeer, caribou B. abortus : cattle, bison B. canis : dog, fox, coyotes

Pathogenesis No exotoxin, endotoxin low toxicity Obligate intracellular parasites Granuloma Accumulation of activated macrophages Pathogenesis No exotoxin, endotoxin low toxicity Obligate intracellular parasites Granuloma Accumulation of activated macrophages Infect monocytes/macrophages, inhibit phagolysosome fusion. Spread to spleen, liver, lymph node, bone marrow, kidneys (bacteria secrete proteins that induce granuloma)

Epidemiology Worldwide distribution Animal reservoirs; natural hosts develop mild or asymptomatic disease. Sterility, abortion, Epidemiology Worldwide distribution Animal reservoirs; natural hosts develop mild or asymptomatic disease. Sterility, abortion, and asymptomatic carriage. Animal tissues (breast, uterus, epididymis, placenta) contain erythritol (紅鮮醇 ) which is required for the growth of the organism. Milk, urine and birth products contain high number of bacteria.

Epidemiology Human infections Direct contact: a lab or occupational exposure Ingestion: consume contaminated food Epidemiology Human infections Direct contact: a lab or occupational exposure Ingestion: consume contaminated food products Inhalation Sources of Brucella infection. G. G. Alton & J. R. L. Forsyth

Clinical disease (Brucellosis, Bang’s disease, undulant fever, Malta fever) Disease spectrum depends on the Clinical disease (Brucellosis, Bang’s disease, undulant fever, Malta fever) Disease spectrum depends on the infecting organism B. melitensis : severe disease B. suis : severe and chronic B. abortus : mild B. canis : mild

Clinical disease Acute disease: incubation period 1 -3 wks, fever rises in afternoon, fall Clinical disease Acute disease: incubation period 1 -3 wks, fever rises in afternoon, fall during night with drenching sweat (undulant fever), weakness, malaise, chill, weight loss, nonproductive cough, aches, pain. Advanced disease: involve many tissues, granulomas and abscesses. 70% GI symptoms, 2060% bone lesions, 25% respiratory tract symptoms Chronic infection occur in inadequately treated patients.

Lab diagnosis Difficult Multiple sampling (blood, bone marrow, infected tissues) Microscopy: insensitive (small size Lab diagnosis Difficult Multiple sampling (blood, bone marrow, infected tissues) Microscopy: insensitive (small size and intracellular location) Culture: blood agar, > 3 days to 2 wks,

Lab diagnosis - serology 5 -10% of population in endemic area have high Ab Lab diagnosis - serology 5 -10% of population in endemic area have high Ab titer (>1: 160). A significant increase in Ab titer = evidence of current disease. Serum agglutination test (SAT): a fourfold increase in titer or a single titer >1: 160. Serologic test: use to confirm clinical diagnosis, not as a basis of diagnosis.

T/P/C Susceptible to tetracycline (doxycycline): bacteriostatic drugs, relapse is common (due to inadequate therapy, T/P/C Susceptible to tetracycline (doxycycline): bacteriostatic drugs, relapse is common (due to inadequate therapy, not antibiotic resistance); use doxycycline + rifampin for > 6 weeks. No vaccine for humans Control of disease in livestock Identification of infection (serologic testing) Elimination of infected herds Vaccination of the rest Avoidance of unpasturized dairy products Observance of safety procedures in clinical lab