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Mycoplasmas and FastidiousGram-negative Bacteria

Haemophilus * BordetellaLegionella *

Moraxella * MycoplasmaUreaplasma

Ken B. Waites, M.D. F(AAM)2008

Objectives• To review and discuss

• microbiological characteristics• epidemiology• virulence factors• associated diseases• laboratory detectionOf:

HaemophilusBordetellaMycoplasmaUreaplasmaLegionella

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Haemophilus• “Blood-loving”• Gram-negative

coccobacillus• Facultative anaerobe• Non-hemolytic• Invasive strains• Non-encapsulated H.

influenzae & other spp.common URT flora

Haemophilus influenzae

Haemophilus: Pathogenesis• Respiratory aerosol dissemination• Endogenous infection• Antiphagocytic capsule (type b)

– major virulence factor– 6 capsular serotypes (a-f) H. influenzae

• Endotoxin– damages respiratory epithelium leading to

bacteremic spread• No exotoxins• IgA protease• Beta lactamase in 30% of strains

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Detection:H. influenzae

• Growth on chocolatebut not blood orMacConkey agar

• 5-10% CO2 required• X (hemin) & V( NAD)

– varies amongspecies

• Satellitism with S.aureus

Diseases: H. influenzae• Otitis media• Sinusitis• Bacteremia• Epiglottitis

• Laryngotracheobronchitis• Meningitis• Exacerbation of chronic

bronchitis in COPD• Pneumonia• Cellulitis• Otitis media• Conjunctivitis

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Prevention: H. influenzae• Invasive disease rare in child > 5 yrs• Hib conjugate vaccine

– polysaccharide capsule type b– protein carrier– given in infancy (3-4 doses) since 1987– reduced invasive disease > 90%– No impact on non-typeable H. influenzae

infections

Other Haemophilus sp

• H. ducreyi–chancroid - genital ulcer

• H. aegyptius– conjunctivitis

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Bordetella pertussis

• Encapsulated• Gram-negative coccobacillus• Slow-growing• Fastidious• Strict aerobe• Non-fermentative• Oxidizes amino acids

B. pertussis: Whooping Cough• 5-21 day incubation - very contagious

– Catarrhal stage - cough & sneeze (1-2 wk)– Paroxysmal stage (1-6 wks)– Convalescent stage (months)

• Lymphocytosis• Recovery confers immunity

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Pertussis Epidemiology

• Outbreaks described in 16th Century• Highly contagious respiratory droplets• > 285,000 deaths worldwide in 2001• > 25,000 US cases in 2005 - increasing• No environmental or animal reservoir• Adolescents and adults > 50% of cases• Older persons often spread to children

Pertussis in Alabama

Reported cases82 cases in 200549 cases in 200419 cases in 200337 cases in 200237 cases in 2001

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Reasons Pertussis is Increasing

Under-vaccination in infants Under- or misdiagnosis Waning Immunity from childhood

vaccination Increased recognition among adolescents

and adults, which contributes to thedisease reservoir

Pertussis Pathogenesis• Attachment to ciliated

respiratory epithelium byvarious adhesins– Filamentous

hemagglutinin (FHA)– Pertussis toxin (PTx)

• Evasion of host defense– impaired chemotaxis

• Local tissue damage &systemic disease due toexotoxins

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Pertussis Toxins• Pertussis toxin

• Facilitates adherence• Adenyl cyclase/hemolysin

• Increases cAMP in cells• Inhibits phagocytic killing & monocyte

migration• Lethal toxin

• Inflammation & local necrosis• Tracheal cytotoxin

• Kills respiratory epithelial cells• Stimulates release of IL-1 (fever)

• Endotoxin (LPS)• Activates alternate complement pathway• Stimulates cytokine release

B. pertussis: Detection• NP swab collected at bedside• Bordet-Gengou or Regan-Lowe enriched

horse blood-charcoal medium– Incubate 3-7 days in moist environment– Identify by immunofluorescence or slide

agglutination• Measurement of serum antibody titers• PCR – in addition to culture• DFA on NP secretions – low sensitivity

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Pertussis Prevention• Acellular vaccine during infancy “DaPT” (1996)

– FHA, PTx, pertactin, type 2 fimbriae– Antibody prevents attachment

• 5 doses: 2 mo; 4 mo; 6 mo; 15-18 mo; 4-6 yr• Adolescent/ adult formulations “Tdap” (2005)

– Ages 11 through 64 yrs – single dose– Td, protects against tetanus & diphtheria, but

not pertussis - recommended every 10 yrs

Legionellapneumophila

• Fastidious• Catalase-negative• Facultatively intracellular

Gram-negative bacillus• Nonfermentative• Stains poorly with safranin• > 30 species• Multiple serogroups• 1st described in 1976

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Pathogenesis of Legionellosis• Organism inhaled from environment

– C3 deposits on bacteria– bacteria bind macrophage C3 receptor– bacteria uptaken by macrophages– prevent phagolysosome fusion– intracellular multiplication– bacteria produce enzymes– cell dies & bacteria are released

• No person to person transmission• Acute purulent pneumonia

& abscesses

Legionella Culture• Buffered charcoal yeast

extract agar + cysteine• Good for all species• 3-7 d or more required• ETA, TTA > sputum• ID species by

agglutination orimmunofluorescence

LegionellaonBCYEagar

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Legionella Detection• Silver staining of histopathology specimens• Direct fluorescent antibody (poor sensitivity)• Urine polysacharide antigen• Serology (IFA) - paired sera required• PCR

Legionnaires Disease• 5-10% of CAPs: 10-20,000 cases/yr in US

– Point source outbreaks– Cooling towers– Hospital water supplies– Hot tubs

• Purulent alveolar exudate• GI & renal manifestations• Risk factors

– Older men with COPD– Immunosuppressed (transplant recipients)

• Summer months (AC)

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Prevention of Legionellosis

• No vaccine• CMI more important than AB• Identify sources and eliminate them

Moraxella catarrhalis• Gram-negative coccus• May be carried in URT of

healthy children• Causes bronchitis, CAP,

sinusitis, otitis• Occasional cause of

non-respiratoryinfections

• Most strains producebeta lactamase

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MYCOPLASMAPNEUMONIAE

Eukaryotic Cell

Chlamydia elementary body Treponema

Streptococci

Herpesvirus

Mycoplasma

HIV

1 µm

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Mycoplasma• Smallest free-living organisms• > 150 species• Genome of 816,394 bp; 687 genes• Lack cell wall - pleomorphic• Specialized cell membrane• Evolved from Gram-positives• Extracellular on mucosal surfaces

M. pneumoniae:Pathogenesis

• Cytadherence– P1 & other proteins– Immunogenic

• Cytotoxicity (H2O2)hemolysin → ciliostasis

• Altered macromolecularsynthesis

• Induction of inflammation• Cytokine cascade• Antigenic variation• Autoimmunity• Intracellular growth?

MP

P1

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Mycoplasma Detection• Enriched agar

medium - SP4 +serum (sterols)

• Slow growth - 5-20days

• Glucose hydrolysis• ID colonies by PCR• Serology – IgM +

(children) orseroconversion

• PCR

Microscopic spherical colonies < 100 µm

M. pneumoniae Disease• Tracheobronchitis• Atypical interstitial “Walking” pneumonia• All ages affected but more common in younger

persons• 20-50% of all CAP• Clinically similar to other pneumonias• Extrapulmonary disease• Spread through households• Outbreaks in closed populations• Role in asthma?• Reinfection common – no protective immunity

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Other Mycoplasmas• Mycoplasma hominis• Mycoplasma genitalium• Ureaplasma species

UreaplasmaM. hominis

Diseases due to GenitalMycoplasmas

• Commensals in lowerurogenital tract in normalsexually active adults

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Diseases due to Genital Mycoplasmas

+++Extragenital diseaseadults (arthritis)

-++Neonatalpneumonia/meningitis

-++Postpartum fever

--+Low birth weight-++Spont. Abortion-++Chorioamnionitis--+Infertility++-Pelvic inflam. disese+--Cervicitis-++Bacterial vaginosis-++Pyelonephritis--+Urinary calculi+-+Prostatitis+-+Male urethritis

M. genitaliumM. hominisUreaplasmaCondition

Detection of Genital Mycoplasmasand Ureaplasmas

• Culture – good for rapid growing M.hominis and Ureaplasma

• Serology – not useful• PCR – needed for M. genitalium