Miscellaneous Small Gram-Negative Bacilli Yu Chun-Keung DVM, PhD Department of Microbiology and...
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Transcript of Miscellaneous Small Gram-Negative Bacilli Yu Chun-Keung DVM, PhD Department of Microbiology and...
MiscellaneousSmall Gram-Negative Bacilli
Yu Chun-Keung DVM, PhDDepartment of Microbiology and Immunology College of Medicine National Cheng Kung UniversityApril 30, 2008
Chapter 35 Haemophilus Pasteruella
Chapter 36 Bordetella Chapter 37 Brucella Francisella
Most of the G(-) bacilli are commonly found in the environment or as normal members of the human microbial flora.
The isolation of Bordetella, Brucella and Francisella is always associated with disease.
Extremely small (0.2 x 1 μm ), G(-), coccobacilli
Have simple nutritional requirement
Some species (B. pertussis) are highly susceptible to toxic substances (need charcoal, starch, blood or albumin to absorb toxic substance in medium, eg. agar)
Bordetella
Important Bordetella species
B. pertussis: whooping/pertussis (severe cough)
B. parapertussis: mild form of pertussis
B. bronchiseptica: respiratory disease of animals (pigs and dogs)
The three species are closely related, differing only in the expression of virulence genes
Pathogenesis
• Exposure (aerosol)
• Bacterial attachment to ciliated epithelial cells of the RT
• 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
Virulence Factors (Table 36-1)
Adhesins ( 黏附因子 )
Toxins
Bacterial adhesins Filamentous hemagglutinin: contain RGD motif: bind (1) sulfated glycoprotein integrins on ciliated respiratory cells; (2) CR3 on macrophages, and trigger phagocytosis without initiating oxidative burst (intracellular survival)
Pertactin (P69 protein) : contain RGD motif
Pertussis toxin : A classic A-B toxin. Toxic subunit (S1) and binding subunits (S2 to S5); S2 binds ciliated respiratory cells, S3 binds phagocytic cells and increase CR3 expression
Fimbria : mediate binding in vitro; function unknown
Toxins
S1 subunit of pertussis toxin: target membrance surface G protein, increase cAMP and then increase respiratory secretion and mucus secretion
Adenylate cyclase toxin/hemolysin: converse ATP to cAMP, increase respiratory secretion, inhibit leukocyte function
Dermonecrotic toxin: vasoconstriction and tissue destruction
Tracheal cytotoxin: ciliostasis, extrusion of ciliated cells, impair regeneration of damaged cells by interfering DNA synthesis (disrupt clearance mechanism, lead to cough); IL-1 production (lead to fever)
LPS: unknown (activate complement and stimulate cytokine release)
S1 subunit of pertussis toxin
Adenosine diphosphate-ribosylating activity for G protein, which regulates adenylate cyclase activity (convert ATP to cAMP). Increase respiratory secretion and mucus production.
S1 toxic subunit
S2-S5 binding subunit
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 of non-ciliated cells
Clinical diseaseInfect ciliated epithelial cells of the airways, produce disease locally, no invasion.Catarrhal phase ( 卡他期 ): resemble common cold, sneezing, serous rhinorrhea, malaise, low-grade fever, 1-2wk, infectious (disease not recognized with high number of bacteria produced)Paroxysmal phase ( 突發期 ): repetitive coughs and inspiratory whoop, vomiting and exhaustion, 40-50 paroxysms daily, bronchopneumonia, 2-4wk.Recovery phase ( 恢復期 ): lasts for above 3 wks.Classic presentation may not be seen in patients with partial immunity.
Epidemiology
Pertussis has been considered a pediatric disease (< 1 year)Incidence (morbidity and mortality) has been reduced considerably after the introduction of vaccine in 1949. Still endemic worldwide.Immunity is not lifelong.Infection is seen in nonimmune infants, young children and adults with waning immunity.
Diagnosis
Clinical diagnosis Sign, Symptom, Syndrome
Laboratory diagnosis Detection / identification of antigens
(whole cell, protein, nucleic acid)
Detection of immune responses (i.e. antibodies)
Lab diagnosis – specimen collection and transport
Extremely sensitive to drying, cannot survive outside the host or traditional transport medium.
Inoculate (nasopharyngeal aspirate) to freshly prepared medium (charcoal-horse blood agar) or Regan-Lowe transport medium at bedside.
Use synthetic fiber swabs not cotton swabs (fatty acid are toxic to Bp).
Lab diagnosis - culture
Regan-Lowe medium (charcoal medium with horse blood, glycerol, peptones).
35°C, humidified, 7 days, sensitivity 50%, depend on stage of illness, use of antibiotics etc.
Direct or indirect fluorescent antibody tests for antigen detection
Sensitivity 50%
Lab diagnosis - microscopy
Aspirated specimen
檢體玻片風乾熱固定螢光抗體染色
Fluorescein-labeled rabbit anti-Bp Ab
Fluorescein-labeled anti-rabbit Ig Ab
Rabbit anti-Bp Ab
Serology
No FDA approved test
ELISA for antibodies against filamentous hemagglutinin and pertussis toxin.
Nucleic acid amplificationPolymerase chain reaction sensitivity 80-100%
TreatmentPrimarily 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)
Prophylaxis for unimmunized infants (Pertussis is highly contagious, family members will become carrier).
Vaccination
DTP vaccine (diphtheria, toxoid of tetanus, and inactivated whole cell of Bp,), 80-85% effective.
Vaccine has not been widely accepted because of vaccine-related complications.
DTaP (acellular vaccine) with inactivated pertussis toxin and one or more bacterial components (eg. filamentous hemagglutinin, pertactin or fimbriae.
DTaP vaccination: 2, 4, 6, 15-8 m, and 4-6 y, high level of protection
Chapter 37
Brucella 布魯氏桿菌屬
Francisella 法蘭西氏菌屬
Brucella and Francisella
Zoonotic pathogens and potential agents of bioterrorists
Very small coccobacilli, 0.5 1.5 M, G(-)
Fastidious, slow growth on culture (>1 week)
Taxonomically unrelatedα-Proteobacteria
Brucella Rickettsia Ehrlichia
γ-Proteobacteria Francisella Legionella Pasteruella Pseudomonas
Brucella
Six species with four species associated with human diseases
B. melitensis : goat and sheep B. suis : swineB. abortus : cattleB. canis : dog, fox
Sources of Brucella infection. G.G. Alton & J.R.L. Forsyth
Pathogenesis
No exotoxin, endotoxin low toxicity
Obligate intracellular parasites of animals and humans
Infect monocytes/macrophages, replicate in phagolysome under the control of virulence genes in virB operon
Spread to spleen, liver, lymph node, bone marrow, kidneys (bacteria secrete proteins that induce granuloma)
Activated macrophages
Clinical disease (Brucellosis, Bang’s disease, undulant fever, Malta fever)
Disease spectrum depends on the infecting organismB. melitensis : severe disease in humansB. suis : severe and chronicB. abortus : mildB. canis : mild
Clinical disease
Acute stage: incubation period >2 months, fever rises in afternoon, fall during night with drenching sweat (undulant fever), weakness, malaise, chill, weight loss, nonproductive cough, aches, pain.
Chronic stage: involve many tissues for many years, granulomas and abscesses. 70% GI symptoms, 20-60% bone lesions, 25% respiratory tract symptoms
Epidemiology
Worldwide distribution
Animal reservoirs; natural hosts develop mild or asymptomatic disease.
Animal tissues (breast, uterus, epididymis, placenta) contain erythritol ( 紅鮮醇 )which is required for the growth of the organism.
Epidemiology
In animals: sterility, abortion, and asymptomatic carriage. Milk, urine and birth products contain high number of bacteria.Human infections:
Direct contact (a lab or occupational exposure) Ingestion: consume unpasteurized milk, milk p
roducts or cheese Inhalation
Lab diagnosis
Multiple sampling (blood, bone marrow, infected tissues)
Microscopy: insensitive (small size and intracellular location)
Culture: blood agar, > 3 days
Lab diagnosis - serology
Antibodies detected in all patients (IgM, then IgA and IgG) and persist for months and years.
A significant increase in Ab titer = evidence of current disease
Serum agglutination test: use for confirming clinical diagnosis; a fourfold increase in titer or a single titer >1:160
B. abortus Ag cross reacts with those of B. melitensis and B. suis but not B. canis
T/P/CTetracycline (doxycycline): bacteriostatic drugs, relapse is common; use doxycycline + rifampin for > 6 weeks.Control of disease in livestock Identification (serologic testing) Elimination of infected herds VaccinationAvoidance of unpasturized dairy productsObservance of safety procedures in clinical lab
Genus Francisella
F. tularensis spp. tularensis (type A)
F. tularensis spp. holarctica (type B)
F. tularensis spp. mediaasiaticaF. tularensis spp. philomiragia
Francisella tularensis
G(-), very small size (0.2 0.7 m) bacilli, able to penetrate through skin and mucous membrane + aerosols
Highly contagious: extremely hazardous for physician and lab workers.
Fastidious growth requirement (iron and cysteine)
EpidemiologyNorthern hemisphere (20°N to Arctic circle, not in southern regions
Animal reservoirs: wild mammals (rabbits, hares, voles), domestic animals, bird, fish, blood-sucking arthropods (ticks)
Human infections result from: Arthropod biting (10 organisms) Direct contact with infected animals or pets (10) Inhalation of infectious aerosol (mostly in lab ) (50) Ingestion of contaminated meat or water (108)
Hunters, lab personnel and those exposed to ticks are high risk for infection in endemic areas
Pathogenesis
Intracellular parasite
Can survive for prolonged periods in macrophages; inhibit phagosome-lysosome fusion.
Pathogenic strains possess antiphagocytic capsule; protect bacteria from complement-mediated killing
Tularemia (Rabbit fever / Tick fever)
Fever, chills, malaise, fatigueClinical symptoms and prognosis determined by route of infection
Ulceroglandular form: skin, most common Oculoglandular form: eyes, painful conju
nctivitis. Typhoidal form: blood, sepsis with multi-org
an involvement Pneumonic form: respiratory tract Gastrointestinal form: oral
UlcerCutaneous tularemiainfection microbes.historique.net
Lab diagnosisSpecimen collection: great hazardous for physicians and lab workers; wear gloves and perform work in biohazard hoodMicroscopy: Grain stain – not practical; direct staining with fluorescein antibodyCulture: not grow in common medium without cysteine (eg. blood agar); chocolate agar or buffered charcoal yeast extract (BCYE) agar, take a week or longerSerology: a 4-fold increase in Ab titer during illness or a single titer of 1:160; cross-reactivity between Brucella and Francisella
T/P/C
Streptomycin (high toxicity) and gentamicin are effectivePenicillin and cephalosporin are ineffective (β-lactamase)Wear protective clothes and use insect repellentsProphylactic antibiotiesLive-attenuated vaccine : partly protective
Chapter 35 HaemophilusFamily Pasteurellaceae (巴斯德桿菌科 ) Genera Haemophilus ( 嗜血桿菌屬 )
Actinobacillus ( 放線桿菌屬 ) Pasteurella ( 巴斯德桿菌屬 )
Small, G(-), non-spore-forming bacilli Fastidious growth needs
Haemophilus
“Blood-loving”, obligate parasite of mucus membrane.
Growth require x factor (hemin) and v factor (nicrotinamide adenine dinucleotide, NAD)
Heated-blood (chocolate) agar for isolation
Haemophilus
H. influenzae (an important pathogen)
H. parainfluenzae (rarely pathogenic)
H. ducreyi (STD – soft chancre)
H. aegyptius (acute, purulent conjunctivitis)
H. influenzae biogroup aegyptius (Brazilian purpuric fever)
Classification
Serological differentiation : polysaccharide capsular antigens: type a to f
Biochemical properties : biotypes I to VIII; indole production, urease activity, ornithine decarboxylase activity
Clinical presentation : two biogroups; biogroup aegypticus causes Brazilian purpuric fever
Pathogenesis Nonencapsulated Hi / H. parainfluenzae
Colonize URT in all people
10% of the flora of saliva: H. parainfluenzae
Opportunistic pathogens: spread locally and cause acute and chronic otitis and sinusitis, exacerbation of chronic bronchitis
Pathogenesis - Encapsulated Hi type b
Uncommon in the URT
Common cause of disease in unvaccinated children
Adhesins colonization of oropharynx release cell wall components damage and impair ciliary function
Produce IgA1 proteases, facilitate colonization
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
Major virulence factor: antiphagocytic polysaccharide capsule – (PRP: polyribitol phosphate)
Anti-PRP antibody is protective (enhance phagocytosis and complement-mediated bacteriocidal activity)
Absence of anti-PRP antibody (complement depletion, splenectomy ) leads to invasion, bacteremia and dissemination
Clinical diseasesMeningitis: Hi type b was the most common cause of pediatric meningitis results from bacterimic spread from nasopharynx; cannot be differentiated from other causes of bacterial meningitis (S. pneumoniae, N. meningitidis, E. coli).
Epiglotitis: swelling of the supraglottic tissue, rapidly progress to complete obstruction of the airways, life-threatening emergency.
Cellulitis: reddish-blue patches on the cheeks or periorbital area.
Arthritis: the most common form of arthritis (single large joint) in children <2 years old.
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
Epidemiology
Before the introduction of vaccine, Hib was responsible for >95% invasive diseases, epiglottitis, orbital cellulitis, meningitis in children 5 m to 5 y (<3 m protected by maternal antibody).
Hi type b conjugated vaccine was introduced in 1987 which greatly reduced the incidence of disease (>90%). Now infections occur in nonimmune children or adults with waning immunity, especially in many developing counties.
Hi type c and f and nonencapsulated strains become more common.
The decline of Hib meningitis associated with the introduction of new vaccines
• Polysaccharide vaccine for Hib were not protective for children < 18m.
• Hib conjugated vaccine, which can work for infant >2m, was introduced in 1987 which greatly reduced the incidence of disease (>90%).
Transmission
Person-to-person transmission
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
Samples: Cerebrospinal fluid and blood (>107 bacteria/ml)Microscopy: both sensitive & specific; G(-) bacilli in CSF in >80% cases before antibiotics treatmentCulture: chocolate agar
Satellite phenomenon: grow around colonies of Staph. aureus on unheated blood agar.Particle agglutination: detect PRP antigen, rapid and sensitive (1 ng/ml)Anti-PRP Ab-coated latex particles + specimen, if PRP present, “positive” agglutination
Treatment
Prompt antimicrobial therapy for systemic Hi infections, otherwise mortality 100%Serious infections: cephalosporins Less severe infections: ampicillinAntibiotic chemoprophylaxis (rifampin) for high risk group (children < 2ys with patients around)
Prevetion - Hi type b conjugate vaccines
Hib polysaccharide vaccine: not effective for children < 18 months
Hib conjugate vaccine: purified capsular PRP with different carrier proteins: Neisseria meningitidis outer membrane protein Diphtheria toxoid
Three doses of vaccine (the same type) before age of 6 months followed by booster doses.
Haemophilus ducreyi
Cause chancroid (soft chancre, 軟性下疳 ), a sexually transmitted disease; most common in Africa and Asia
Indurated ulcer on genitalia with regional lymphadenopathy
Differential: syphilis, herpes simplex, lymphogranuloma venereum (Chlamydia trachomatis)
H. aegyptiusAcute, purulent conjunctivitis
H. influenzae biogroup aegyptiusBrazilian purpuric fever (conjun
ctivitis fever, vomiting and abdominal pain petechia, purpura and shock)
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 scratch.
Exacerbation of chronic respiratory tract disease in patients with underlying pulmonary dysfunction.
Systemic infection in immunocompromised patients.
Lab diagnosisGrows well on blood and chocolate agarLarge, buttery colonies with a musty odor
TreatmentSusceptible to a variety of antibioticsPenicillin, macrolides, tetracycline …