MiscellaneousSmall Gram-Negative Bacilli

Chapter 34 Haemophilus Chapter 35 Bordetella

Chapter 36 Francisella Brucella

Microbiology 20115/6/2011Yu Chun-Keung DVM, PhD

Chapter 34 Haemophilus

Family Pasteurellaceae

Genera Haemophilus Actinobacillus

Aggregatibacter

PasteurellaSmall, 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 (v factor)

Heated blood (chocolate) agar for isolation

Important Haemophilius species H. influenzae (an important pathogen) H. ducreyi (soft chancre) H. aegyptius (acute, purulent conjunctivitis) H. parainfluenzae (rarely pathogenic)

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 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 in unvaccinated children

Invasion: adhesins colonization of oropharynx release cell wall components damage and impair ciliary function across epithelial and endothelial cells blood

Produce IgA1 proteases, facilitate colonization

LPS lipid A induces meningeal inflammation

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 – polyribitol phosphate (PRP) : ribose, ribitol, phosphate

Natural infection, vaccination, passive transfer of material antibody - 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 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, 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 non-immune population

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.

DiagnosisClinical diagnosis

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 detection / identification of pathogens (i.e., whole

cell, protein, nucleic acid) detection of immune responses (i.e., antibodies)

Diagnosis

Specimens: Oral swab: avoid contamination with oral

secretions Sputum from LRT Direct needle aspiration Cerebrospinal fluid (CSF) and blood (>107

bacteria/ml)

Microscopy: both sensitive & specific; G(-) bacilli in CSF in >80% cases before antibiotics treatment

Diagnosis (for Hib only)

Particle agglutination test : Detect PRP antigen, rapid and sensitive (1

ng/ml) Anti-PRP Ab-coated latex particles +

specimen, if PRP present, “positive” agglutination

Culture: 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 Less severe infections (otitis, sinusitis): ampicillin

Antibiotic chemoprophylaxis (rifampin) for high risk group (children < 2ys with patients around)

Prevention

Polysaccharide vaccine for Hib were not effective for children < 18m (the high risk population).

Hi type b conjugate vaccine

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 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.

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 on genitalia (soft chancre, 軟性下疳 ) with regional lymphadenopathy

Differential:

Syphilis; Herpes simplex; Lymphogranuloma venereum (caused by Chlamydia trachomatis)

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 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 …

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)

Chapter 35 Bordetella

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 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 respiratory cells; (2) CR3 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 (S1) and binding subunits (S2 to S5); S2 binds lactosylceramide on ciliated respiratory cells, S3 binds phagocytic cells

Toxins

S1 subunit of pertussis toxin

Adenylate cyclase toxin / hemolysin

Dermonecrotic toxin

Tracheal cytotoxin

LPS

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

S1 subunit of pertussis toxin

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

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)

Toxins

Adenylate cyclase toxin / hemolysin: (1) activated by calmodulin and converse ATP to cAMP, increase respiratory secretion; (2) inhibit leukocyte functions

Dermonecrotic toxin: vasoconstriction and tissue destruction

LPS: unknown (activate complement and stimulate cytokine release)

Clinical diseaseInfect 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. Only chronic persistent cough without

whooping or vomiting

Differential diagnosis

Mycoplasma pneumoniae

Chlamydophilia pneumoniae

Legionella pneumophila

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).

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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).

Direct or indirect fluorescent antibody tests for antigen detection

Aspirated specimen

microscopic slide air-

dried heat fixed

fluorescent Ab

Sensitivity 50%

Lab diagnosis - microscopy

Aspirated specimen

Fluorescein-labeled rabbit anti-Bp Ab

Fluorescein-labeled anti-rabbit Ig AbRabbit anti-Bp Ab

Direct

Indirect

Lab diagnosis - culture

Regan-Lowe charcoal medium (horse blood, glycerol, peptones).

35°C, humidified, 7 days,

50% sensitivity, affected by

Patient factors (stage of illness, use of antibiotics)

Quality of specimen

Transport conditions

Culture methods

.Nucleic acid amplification

Polymerase chain reaction sensitivity 80-100%

No FDA approved test, in-house 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 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), 80-85% effective.

DTP vaccine has not been widely accepted because of vaccine-related complications.

DTaP (acellular vaccine) : subunit vaccine D + T + inactivated pertussis toxin, filamentous hemagglutinin, and pertactin or fimbriae.

1. purified polyribose ribitol phosphate (PRP) of Hib covalently bound to 20 µg of tetanus protein 10 µg2. pertussis toxoid 20 µg filamentous haemagglutinin 20 µg fimbrial agglutinogens 2 + 3, 5 µg; pertactin 3 µg3. diphtheria toxoid 15 Lf4. tetanus toxoid 5 Lf5. poliovirus type 1 (Mahoney) 40 D-antigen units poliovirus type 2 (MEF1) 8 D-antigen units poliovirus type 3 (Saukett) 32 D-antigen units

Pentavalent vaccine – PEDIACEL (Sanofi Pasteur)

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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 Brucella Rickettsia Ehrlichia

γ-Proteobacteria Francisella Legionella Pasteruella Pseudomonas

Genus Francisella

Francisella tularensis (Tularemia) F. tularensis subsp. tularensis (type A)

F. tularensis subsp. holarctica (type B) F. tularensis subsp. Mediasiatica (rare as

pathogen)

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, blood-sucking arthropods

Type A: North America: lagomorphs (rabbits, hares), cats, biting arthropod

Type B: Northern hemisphere: rodents, cats

Those exposed to ticks are high risk for infection in endemic areas i.e., hunters, and lab personnel

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 survive for prolonged periods in macrophages; inhibit phagosome-lysosome 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 by route of infection

Ulceroglandular form: cutaneous ulcer + swollen LN, most common

Oculoglandular form: painful conjunctivitis + swollen cervical LN.

Typhoidal form: sepsis

Pneumonic form: pulmonary symptoms

Gastrointestinal form:

UlcerCutaneous tularemiainfection microbes.historique.net

Lab diagnosisSpecimen 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/CPenicillin 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 : 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

Infect monocytes/macrophages, inhibit phagolysosome fusion.

Spread to spleen, liver, lymph node, bone marrow, kidneys (bacteria secrete proteins that induce granuloma)

Granuloma Accumulation of activated

macrophages

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 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 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 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, 20-60% 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 and intracellular location)

Culture: blood agar, > 3 days to 2 wks,

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, 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