PAMB 650 Medical Microbiology Lecture: 41

Legionella, Bordetella and Haemophilus

Gram Negative Rods of the Respiratory Tract

1

Organization of Lecture

1. Overview 2. Organisms of Clinical Importance 3. Microbiology 4. Public Health 5. Pathogenic Mechanisms 6. Clinical Presentation

LEGIONELLA

LEGIONELLAE Overview

• • • • • •

Facultative intracellular pathogen Gram negative rod Requires specialized media to grow Stains poorly with gram stain Transmitted via contaminated aerosols No person to person transmission

2 Species

of Clinical Importance

• • •

Legionella

– One genus – 50 species – ½ of species implicated in human disease

Legionella pneumophila

– Causes ~ 90% of all cases of legionellosis – Majority of all confirmed cases are caused by serogroups 1-6

Legionella micdade

– Most common after

L. pneumophila

Legionella micdadei

•

Caution:

– This strain can stain weakly acid fast on primary isolation, but loses this property when grown in vitro.

–

NO

RELATIONSHIP TO MYCOBACTERIA

Microbiology

Will not grow on standard Sheep Blood Agar Buffered Charcoal Yeast Extract Agar (BCYE) 1. Cysteine is essential for growth 2.

Iron is essential for growth Growth conditions: 1. 35 0 C 2. 3-7 days

Colony Appearance:

• •

Ground glass Small 1-3 mm

Laboratory Diagnosis of

Legionella

•

Culture of Legionella organism from normally sterile tissue

•

Detection of L. pneumophila antigen in urine

•

Seroconversion : 4 fold or greater rise in specific serum antibody titer L. pneumophila

•

Direct fluorescent antibody (DFA) staining

9

Legionnaires

disease Public

Health

• Disease - Worldwide • Sporadic • Epidemic community-acquired pneumonia • Nosocomial infections • Exposure - Water-based aerosols • Air conditioning cooling towers • Whirlpool spas • Sauna or mister • Survival – Environment • Amoebae • Biofilms 10

2 Clinical Presentations

•

Legionnaire's

– – – –

Incubation period 2-10 days pneumonia disease 15-75% mortality erythromycin

•

Pontiac fever

–

Incubation period 1-2 days

– – –

flu-like milder (no mortality) self-limiting

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PATHOGENESIS OF LEGIONELLA

•

Phagocytosis into the monocytes

‒

binding to complement receptors

•

Inhibition of phagolysosome fusion

•

Replication within the phagosome

•

Lysis of the phagosome leads to apoptosis and release of the organism

•

TH1 cells and IFN γ

Bordetella

Bordetella pertussis

•

Strict aerobe

•

Gram negative

•

Small Coccobacillus -singly or in pairs

•

Transmission by aerosolized droplets

•

Non-invasive

•

Strictly human pathogen

DIFFERENTIATION OF BORDETELLA SPECIES Growth on common lab media (SBA, MacConkey) Growth on Bordet Gengou agar Urease Oxidase Motility

B. pertussis

+ -

+

-

B. parapertussis B. bronchiseptica

+ +

+ +

-

+

-

+

B. pertussis

Small, transparent hemolytic colonies on Bordet-Gengou medium

Diagnosis

•

Based on symptoms

•

Culture of respiratory secretions on Bordet Gengou medium

•

Direct fluorescent antibody testing

•

PCR

•

Slide agglutination

Public Health Aspects of B.

pertussis ( Whooping Cough )

August 2, 2010

WHOOPING COUGH EPIDEMIC GROWS – HEALTH OFFICIALS URGE VACCINATION AND TIMELY DIAGNOSIS

As of July 27, the number of illnesses from the disease this year had climbed to 2,174, a six-fold increase from the the seventh to die from pertussis this year. 349 illnesses reported for the same period last year. In addition, a San Diego County infant has become “The pertussis epidemic is a sobering and tragic reminder that diseases long thought controlled can return with a vengeance,” Horton said. “We can protect ourselves and the most vulnerable in our community by getting vaccinated today.”

Morbidity and Mortality Weekly Report (MMWR) July 9, 2010 / 59(26);817

During January--June in California 1.

… 89% of cases were among infants aged <6 months  too young to be fully immunized 2. Children aged 7 to 9 years and 10 to 8 years  10.1 cases and 9.3 cases per 100,000, respectively.

Of 634 case reports 1. 105 (16.6%) patients were hospitalized, 2. 66 (62.9%) were <3 months. 3. Incidence among Hispanic infants (49.8 cases per 100,000) was higher than among other racial/ethnic populations. 4. 5 deaths were reported, all in previously healthy Hispanic infants aged <2 months at disease onset; none had received any pertussis-containing vaccines.

The incidence of pertussis is cyclical, with peaks occurring every 3--5 years in the United States (

2

). The last peak was in 2005, when approximately 25,000 cases were reported nationally.

Pertussis Among Adolescents and Adults • Disease often milder than in infants and children • Infection may be asymptomatic, or may present as classic pertussis • Persons with mild disease may transmit the infection • Older persons often source of infection for children

Pertussis- Disease

•

Primarily a toxin-mediated disease

•

Exotoxins are controlled by central locus

–

BvgAS two-component signal transduction system to sense the environment and regulate gene expression

Pertussis- Disease

•

Inflammation interferes with clearance of pulmonary secretions

–

Cough progresses from mild (catarrhal stage) to sever (paroxysmal stage)

–

Resolves slowly

•

Evasion of host defenses

–

Pertussis antigens allow evasion of host defenses

–

Lymphocytosis promoted but impaired chemotaxis

Pertussis Pathogenesis

•

Two-stage process of disease

–

Respiratory colonization

•

7-10 days

•

NO symptoms

•

Positive cultures toward the end of this stage

–

Toxin-mediated disease

Colonization

•

Attachment requires multiple factors

–

Pertussis Toxin

–

Filamentous hemagglutinin

–

Fimbriae

Adhesins

1.

Filamentous hemagglutinin a. Dominant adhesin b. Required for tracheal colonization c. Highly immunogenic d. Primary component of acellular pertussis vaccines 2. Fimbriae a. Required for persistent tracheal colonization b. Component of some acellular pertussis vaccines c. Required for protective immunity to infection

Bordetella pertussis

Toxins

SIX DIFFERENT TOXINS

Systemic effects of Pertussis Toxin

1. T cell Lymphocytosis with ↓ mitogenicity 2. ↑ insulin secretion 3.

Histamine sensitization 4. ↑ IgE production 5. Impaired phagocyte function 6. ADP-ribosylates G proteins 7. Strong adjuvant 8. Primary component of pertussis vaccines

2. Adenylate cyclase Toxin

• Calmodulin-activated with adenylate cyclase and hemolysin activity • Acts as anti-inflammatory and antiphagocytic factor • Secreted invasive toxin Adenylate cyclase toxin

B. pertussis

↑ cAMP calmodulin

Other Toxins:

3. Dermonecrotic toxin (DNT) A. Heat-labile secreted toxin B. Transglutaminase activity that acts on small GTPases of the Rho family C. Induces localized necrosis

GDP Rho GDP Rho DNT Polyaminated GTP Rho Polyaminated Effectors Phenotypic Modifications seen 1. Reorganization of  actin cytoskeletal  focal adhesions  stress fibers 2. Alterations in cell morphology

Fukui, A. et al. J Biochem 2004 136:415-419; doi:10.1093/jb/mvh155

Other Toxins:

4. Tracheal cytotoxin

A. Disaccharide-tetrapeptide monomeric by-product of peptidoglycan synthesis B. Causes damage to cilia, and loss of ciliated cells C. Increased IL-1 and nitric oxide production

5. Type III secretion system

A. Allows

Bordetella to translocate effector proteins directly into

host cells B. Required for persistent tracheal colonization C. Inhibits host immune response D. Induces necrotic cell death

6. Lipopolysaccharide (LPS)

A. Pyrogenic B. Mitogenic C. Activate and induce tumor necrosis factor production in macrophages D. LPS lacks a repetitive O-antigenic structure

Treatment

• •

Erythromycin Vaccine

•

killed bacterial cell suspension -DTP vaccine

•

Vaccine- induced immunity wanes after five to ten years

•

acellular vaccines (DTaP)

•

Multicomponent acellular vaccines

Haemophilus

Overview- Haemophilus

•

Small

•

Non-motile

•

Gram-negative rods

•

Transmitted via respiratory droplets, or direct contact with contaminated secretions

•

Normal flora of the human respiratory tract and oral cavity.

Haemophilus species of clinical importance

1. H. influenzae type b is an important human pathogen 2. H. ducreyi sexually transmitted pathogen (chancroid) 3. Other Haemophilus are normal flora H. parainfluenzae – pneumonia & endocarditis - H. aphrophilus – pneumonia & endocarditis - H. aegyptius – pink eye (purulent conjunctivitis)

Differentiation of Species

H. influenzae H. aegyptius H. ducreyi H. parainfluenzae H. aphrophilus

Hemolysis + Growth Factor X Y + + + + + + -

Public Health Aspects-

H. influenzae

•

Typing based on capsule polysaccharide a → f

•

Polyribose-ribitol phosphate (PRP) capsule (type b)

•

Nonencapsulated (nontypeable) organisms are part of normal flora of the respiratory tract

•

95% of invasive disease caused by type b

Public Health Aspects

• •

H. influenzae type b incidence has fallen 99% post-vaccine Pre-immunization

–

Serotype b was the most common invasive species

•

Post-immunization

–

Most cases in unvaccinated or incompletely vaccinated children.

–

Non-encapsulated and serotype f are the most common

–

Children - Pneumonia and meningitis less common

–

Most infections (~2/3) are currently attributed to nontypeable strains.

Disease caused by H. influenzae

Serotype b

Clinical Microbiology Reviews, April 2000, p. 302-317, Vol. 13, No. 2

Invasive Diseases post-immunization

• • • • • •

Septic arthritis Osteomyelitis Cellulitis Pericarditis Pneumonia - most frequent is serotype f Otitis media

–

Streptococcus pneumoniae and then non-typeable Hi

Pathogenic Mechanisms

•

H. influenzae

–

Antiphagocytic polysaccharide capsule is the major pathogenesis factor

–

Lipopolysaccharide lipid A component from the cell wall (major role in non capsule strains)

–

All virulent strains produce neuraminidase and an IgA protease

–

No exotoxins

Pathogenesis – Host Factors

• •

Hib conjugate vaccine (PRP capsule) The Hib conjugate vaccine does not protect against nontypeable strains.

•

Persons at risk for invasive H influenzae disease

– –

Asplenia Immunocompromised

Public Health Aspect of other Haemophilus strains

• • • •

H. ducreyi

–

Sexually transmitted disease - chancroid H. influenzae biogroup aegyptius

–

Brazilian Purpuric Fever

H. aegyptius

–

“pink eye” (purulent conjunctivitis)

H. aphrophilus

– –

pneumonia Infective endocarditis

Haemophilus

influenzae

biogroup aegyptius

•

Brazilian purpuric fever in children

•

High fever

•

Death within 48 hours

Case Study

• History • •

13 year old white male fully vaccinated

•

cold-like symptoms and persistent cough- 10 days duration

• 2 weeks later •

progressive coughing spells with inspiratory whoop

•

posttussive vomiting

Case Study

•

Tests

•

Nasopharyngeal swabs

•

Bordet-Gengou medium

•

Blood samples for serology

•

positive IgM and IgA antibodies

•

Treatment

•

azithromycin

Case Study

• History • •

4 month old white female 1 day history

•

103 0 fever, lethargy, irritability, stiff neck

• Tests •

Cerebral spinal fluid culture

•

IsoVitaleX-enriched chocolate agar