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 …