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PAMB 650 Medical Microbiology PAMB 650 Medical Microbiology Lecture: 41Lecture: 41

LegionellaLegionella, , BordetellaBordetella and and HaemophilusHaemophilus

Gram Negative Rods of the Respiratory Tract Gram Negative Rods of the Respiratory Tract

Organization of LectureOrganization of Lecture

1.1. OverviewOverview

2.2. Organisms of Clinical ImportanceOrganisms of Clinical Importance

3.3. MicrobiologyMicrobiology

4.4. Public Health Public Health

5.5. Pathogenic MechanismsPathogenic Mechanisms

6.6. Clinical Presentation Clinical Presentation

LEGIONELLALEGIONELLA

LEGIONELLAELEGIONELLAEOverviewOverview

• Facultative intracellular pathogenFacultative intracellular pathogen

• Gram negative rodGram negative rod

• Requires specialized media to growRequires specialized media to grow

• Stains poorly with gram stainStains poorly with gram stain

• Transmitted via contaminated aerosolsTransmitted via contaminated aerosols

• No person to person transmissionNo person to person transmission

22 SpeciesSpecies of Clinical Importance of Clinical Importance

• LegionellaLegionella– One genusOne genus

– 50 species50 species

– ½ of species implicated in human disease½ of species implicated in human disease

• Legionella pneumophilaLegionella pneumophila– Causes ~ 90% of all cases of legionellosis Causes ~ 90% of all cases of legionellosis

– Majority of all confirmed cases are caused by serogroups 1-6Majority of all confirmed cases are caused by serogroups 1-6

• Legionella micdadeLegionella micdade– Most common after Most common after L. pneumophilaL. pneumophila

Legionella micdadeiLegionella micdadei

• Caution:Caution:– This strain can stain weakly acid fast on primary This strain can stain weakly acid fast on primary

isolation, but loses this property when grown in isolation, but loses this property when grown in vitro.vitro.

– NONO RELATIONSHIP TO MYCOBACTERIA RELATIONSHIP TO MYCOBACTERIA

Will not grow Will not grow on standard Sheep Blood Agaron standard Sheep Blood Agar

Buffered Charcoal Yeast Extract Agar (BCYE)Buffered Charcoal Yeast Extract Agar (BCYE)1. 1. CysteineCysteine is essential for growth is essential for growth2.2. IronIron is essential for growth is essential for growth

Growth conditions:Growth conditions:1.1. 35350 0 CC2.2. 3-7 days3-7 days

MicrobiologyMicrobiology

Colony Appearance:Colony Appearance:

• Ground glassGround glass

• Small 1-3 mmSmall 1-3 mm

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• CultureCulture of of LegionellaLegionella organism from organism from normally normally sterile tissuesterile tissue

• Detection of Detection of L. pneumophila L. pneumophila antigen in urineantigen in urine

• SeroconversionSeroconversion: 4 fold or greater rise in specific : 4 fold or greater rise in specific serum antibody titer serum antibody titer L. pneumophilaL. pneumophila

• Direct fluorescent antibody Direct fluorescent antibody (DFA) staining(DFA) staining

Laboratory Diagnosis of Laboratory Diagnosis of LegionellaLegionella

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Legionnaires Legionnaires disease-disease-PublicPublic Health Health

• Disease - Worldwide Disease - Worldwide • Sporadic Sporadic • Epidemic community-acquired pneumonia Epidemic community-acquired pneumonia • Nosocomial infectionsNosocomial infections

•Exposure - Water-based aerosols Exposure - Water-based aerosols • Air conditioning cooling towersAir conditioning cooling towers• Whirlpool spasWhirlpool spas• Sauna or misterSauna or mister

•Survival – EnvironmentSurvival – Environment• Amoebae Amoebae • BiofilmsBiofilms

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• Legionnaire'sLegionnaire's disease disease– Incubation period 2-10 daysIncubation period 2-10 days– pneumoniapneumonia– 15-75% mortality15-75% mortality– erythromycin erythromycin

•Pontiac feverPontiac fever–Incubation period 1-2 daysIncubation period 1-2 days– flu-likeflu-like– milder (no mortality)milder (no mortality)– self-limiting self-limiting

22 Clinical Presentations Clinical Presentations22 Clinical Presentations Clinical Presentations

PATHOGENESIS OF PATHOGENESIS OF LEGIONELLALEGIONELLA

•Phagocytosis into the monocytes Phagocytosis into the monocytes

‒binding to complement receptorsbinding to complement receptors

•Inhibition of phagolysosome fusionInhibition of phagolysosome fusion

•Replication within the phagosomeReplication within the phagosome

•Lysis of the phagosome leads to apoptosis and Lysis of the phagosome leads to apoptosis and

release of the organismrelease of the organism

•TH1 cells and IFN-TH1 cells and IFN-γγ

BordetellaBordetellaBordetellaBordetella

Bordetella pertussisBordetella pertussis

• Strict aerobeStrict aerobe

• Gram negative Gram negative

• Small Coccobacillus -singly or in pairsSmall Coccobacillus -singly or in pairs

• Transmission by aerosolized dropletsTransmission by aerosolized droplets

• Non-invasiveNon-invasive

• Strictly human pathogenStrictly human pathogen

B. pertussisB. pertussis Small, transparent hemolytic colonies on Small, transparent hemolytic colonies on

Bordet-Gengou mediumBordet-Gengou medium

DiagnosisDiagnosis

• Based on symptomsBased on symptoms

• Culture of respiratory secretions on Bordet-Culture of respiratory secretions on Bordet-

GengouGengou medium medium

• Direct fluorescent antibody testingDirect fluorescent antibody testing

• PCRPCR

• Slide agglutination Slide agglutination

Public Health Aspects of Public Health Aspects of B. B. pertussis (pertussis (Whooping CoughWhooping 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 349 illnesses reported for the same period last year. In addition, a San Diego County infant has become the seventh to die from pertussis this year.

“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.”

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 reports1.105 (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.

Morbidity and Mortality Weekly Report (MMWR)

July 9, 2010 / 59(26);817

Pertussis Among Adolescents and Pertussis Among Adolescents and AdultsAdults

• Disease often milder than in infants and childrenDisease often milder than in infants and children• Infection may be asymptomatic, or may present Infection may be asymptomatic, or may present

as classic pertussisas classic pertussis• Persons with mild disease may transmit the Persons with mild disease may transmit the

infectioninfection• Older persons often source of infection for Older persons often source of infection for

childrenchildren

Pertussis- DiseasePertussis- Disease

• Primarily a toxin-mediated diseasePrimarily a toxin-mediated disease

• Exotoxins are controlled by central locus Exotoxins are controlled by central locus – BvgASBvgAS two-component signal transduction system to sense the two-component signal transduction system to sense the

environment and regulate gene expression environment and regulate gene expression

Pertussis- DiseasePertussis- Disease

• Inflammation interferes with clearance of Inflammation interferes with clearance of pulmonary secretionspulmonary secretions– Cough progresses from mild (catarrhal stage) to sever Cough progresses from mild (catarrhal stage) to sever

(paroxysmal stage) (paroxysmal stage)

– Resolves slowlyResolves slowly

• Evasion of host defensesEvasion of host defenses– Pertussis antigens allow evasion of host defenses Pertussis antigens allow evasion of host defenses

– Lymphocytosis promoted but impaired chemotaxisLymphocytosis promoted but impaired chemotaxis

Pertussis PathogenesisPertussis Pathogenesis

• Two-stage process of disease Two-stage process of disease – Respiratory colonizationRespiratory colonization

• 7-10 days7-10 days

• NO symptomsNO symptoms

• Positive cultures toward the end of this stagePositive cultures toward the end of this stage

– Toxin-mediated diseaseToxin-mediated disease

ColonizationColonization

• Attachment requires multiple factorsAttachment requires multiple factors– Pertussis ToxinPertussis Toxin– Filamentous hemagglutininFilamentous hemagglutinin– FimbriaeFimbriae

1.1. Filamentous hemagglutininFilamentous hemagglutinina.a. Dominant adhesin Dominant adhesin b.b. Required for tracheal colonization Required for tracheal colonization c.c. Highly immunogenic Highly immunogenic d.d. Primary component of acellular pertussis vaccinesPrimary component of acellular pertussis vaccines

2.2. FimbriaeFimbriaea.a. Required for persistent tracheal colonizationRequired for persistent tracheal colonizationb.b. Component of some acellular pertussis vaccinesComponent of some acellular pertussis vaccinesc.c. Required for protective immunity to infectionRequired for protective immunity to infection

AdhesinsAdhesins

Bordetella pertussis Bordetella pertussis ToxinsToxins

SIXSIX

DIFFERENT TOXINSDIFFERENT TOXINS

Systemic effects of Pertussis ToxinSystemic effects of Pertussis Toxin

1.1. T cell Lymphocytosis with ↓ mitogenicityT cell Lymphocytosis with ↓ mitogenicity

2.2. ↑ ↑ insulin secretioninsulin secretion

3.3. Histamine sensitizationHistamine sensitization

4.4. ↑ ↑ IgE productionIgE production

5.5. Impaired phagocyte functionImpaired phagocyte function

6.6. ADP-ribosylates G proteinsADP-ribosylates G proteins

7.7. Strong adjuvant Strong adjuvant

8.8. Primary component of pertussis vaccinesPrimary component of pertussis vaccines

2. Adenylate cyclase Toxin2. Adenylate cyclase Toxin• Calmodulin-activated with Calmodulin-activated with adenylate cyclase and hemolysin adenylate cyclase and hemolysin activityactivity

• Acts as anti-inflammatory and antiphagocytic factorActs as anti-inflammatory and antiphagocytic factor

• SecretedSecreted invasiveinvasive toxintoxin

calmodulin

↑cAMP

B. pertussis

Adenylate cyclase toxin

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

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

Phenotypic Modifications seen1.Reorganization of

actin cytoskeletal focal adhesions stress fibers

2.Alterations in cell morphology

Rho

GDP

Rho

GDP

Polyaminated

DNT

Effectors

Rho

GTP

Polyaminated

Other Toxins:Other Toxins:

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

5.5. Type III secretion systemType III secretion systemA.A. Allows Allows Bordetella to translocate effector proteins directly into Bordetella to translocate effector proteins directly into host cellshost cellsB.B. Required for persistent tracheal colonizationRequired for persistent tracheal colonizationC.C. Inhibits host immune responseInhibits host immune responseD.D. Induces necrotic cell deathInduces necrotic cell death

6.6. Lipopolysaccharide (LPS)Lipopolysaccharide (LPS)A.A. PyrogenicPyrogenicB.B. MitogenicMitogenicC.C. Activate and induce tumor necrosis factor production in macrophagesActivate and induce tumor necrosis factor production in macrophagesD.D. LPS lacks a repetitive O-antigenic structureLPS lacks a repetitive O-antigenic structure

Other Toxins:Other Toxins:

TreatmentTreatment

• Erythromycin Erythromycin

• VaccineVaccine• killed bacterial cell suspension -DTP vaccinekilled bacterial cell suspension -DTP vaccine• Vaccine- induced immunity wanes after five Vaccine- induced immunity wanes after five

to ten years to ten years • acellular vaccines (DTaP)acellular vaccines (DTaP)

• Multicomponent acellular vaccinesMulticomponent acellular vaccines

HaemophilusHaemophilus

Overview- HaemophilusOverview- Haemophilus

• SmallSmall

• Non-motileNon-motile

• Gram-negative rodsGram-negative rods

• Transmitted via respiratory droplets, or Transmitted via respiratory droplets, or direct contact with contaminated secretionsdirect contact with contaminated secretions

• Normal flora of the human respiratory tract Normal flora of the human respiratory tract and oral cavity.and oral cavity.

HaemophilusHaemophilus species of species of clinical importanceclinical importance

1.1. H. influenzae H. influenzae

--type b is an important human pathogentype b is an important human pathogen

2.2. H. ducreyi H. ducreyi

--sexually transmitted pathogen (chancroid)sexually transmitted pathogen (chancroid)

3. Other3. Other Haemophilus Haemophilus are normal flora are normal flora

- - H. parainfluenzae – H. parainfluenzae – pneumonia & endocarditispneumonia & endocarditis

-- H. aphrophilus – H. aphrophilus – pneumonia & endocarditispneumonia & endocarditis

- - H. aegyptius – H. aegyptius – pink eye (purulent conjunctivitis)pink eye (purulent conjunctivitis)

Differentiation of SpeciesDifferentiation of Species

HemolysisHemolysisX YX Y

GrowthGrowthFactorFactor

Public Health Aspects-Public Health Aspects-H. influenzaeH. influenzae

• Typing based on capsule polysaccharide a Typing based on capsule polysaccharide a → f→ f

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

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

• 95% of invasive disease caused by type b95% of invasive disease caused by type b

Public Health AspectsPublic Health Aspects

• H. influenzaeH. influenzae type b incidence has fallen type b incidence has fallen 99% post-vaccine99% post-vaccine

• Pre-immunization Pre-immunization

– Serotype b was the most common invasive Serotype b was the most common invasive speciesspecies

• Post-immunizationPost-immunization

– Most cases in unvaccinated or incompletely Most cases in unvaccinated or incompletely vaccinated children.vaccinated children.

– Non-encapsulated and serotype f are the most Non-encapsulated and serotype f are the most common common

– Children - Pneumonia and meningitis less common Children - Pneumonia and meningitis less common

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

Disease caused by Disease caused by H. influenzaeH. influenzaeSerotype bSerotype b

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

Invasive Diseases post-immunizationInvasive Diseases post-immunization

• Septic arthritisSeptic arthritis

• OsteomyelitisOsteomyelitis

• CellulitisCellulitis

• PericarditisPericarditis

• Pneumonia - most frequent is serotype fPneumonia - most frequent is serotype f

• Otitis media Otitis media

– Streptococcus pneumoniaeStreptococcus pneumoniae and then non-typeable Hi and then non-typeable Hi

Pathogenic MechanismsPathogenic Mechanisms

• H. influenzaeH. influenzae

– Antiphagocytic Antiphagocytic polysaccharide capsule polysaccharide capsule is the is the major pathogenesis factormajor pathogenesis factor

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

– All virulent strains produce All virulent strains produce neuraminidaseneuraminidase and and an an IgA proteaseIgA protease

– No exotoxinsNo exotoxins

• Hib conjugate vaccine (PRP capsule) Hib conjugate vaccine (PRP capsule)

• The Hib conjugate vaccine does not protect The Hib conjugate vaccine does not protect against nontypeable strains. against nontypeable strains.

• Persons at risk for invasive Persons at risk for invasive H influenzaeH influenzae disease disease – AspleniaAsplenia

– Immunocompromised Immunocompromised

Pathogenesis – Host FactorsPathogenesis – Host Factors

Public Health Aspect of other Public Health Aspect of other HaemophilusHaemophilus strains strains

• H. ducreyiH. ducreyi– Sexually transmitted disease - chancroidSexually transmitted disease - chancroid

• H. influenzaeH. influenzae biogroup biogroup aegyptiusaegyptius– Brazilian Purpuric FeverBrazilian Purpuric Fever

• H. aegyptius H. aegyptius – ““pink eye” (purulent conjunctivitis)pink eye” (purulent conjunctivitis)

• H. aphrophilusH. aphrophilus– pneumoniapneumonia

– Infective endocarditisInfective endocarditis

Haemophilus Haemophilus influenzaeinfluenzae biogroup biogroup aegyptiusaegyptius

• Brazilian purpuric fever in childrenBrazilian purpuric fever in children• High fever High fever • Death within 48 hoursDeath within 48 hours

Case StudyCase Study

• HistoryHistory• 13 year old white male13 year old white male• fully vaccinatedfully vaccinated• cold-like symptoms and persistent cold-like symptoms and persistent

cough- 10 days durationcough- 10 days duration• 2 weeks later2 weeks later

• progressive coughing spells with progressive coughing spells with inspiratory whoopinspiratory whoop

• posttussive vomitingposttussive vomiting

Case StudyCase Study

• TestsTests• Nasopharyngeal swabs Nasopharyngeal swabs

• Bordet-GengouBordet-Gengou mediummedium

• Blood samples for serologyBlood samples for serology• positive IgM and IgA antibodiespositive IgM and IgA antibodies

• TreatmentTreatment• azithromycinazithromycin

Case StudyCase Study• HistoryHistory

• 4 month old white female4 month old white female• 1 day history 1 day history

• 1031030 0 fever, lethargy, irritability, stiff fever, lethargy, irritability, stiff neckneck

• TestsTests• Cerebral spinal fluid cultureCerebral spinal fluid culture

• IsoVitaleX-enriched IsoVitaleX-enriched chocolate agarchocolate agar