Mycoplasmas and Fastidious Gram-negative Bacteria

Haemophilus * Bordetella

Legionella *

Moraxella * Mycoplasma

Ureaplasma* Brucella * Francisella

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

Objectives• To review and discuss

• microbiological characteristics• epidemiology• virulence factors• associated diseases • laboratory detection

HaemophilusBordetellaMycoplasmaUreaplasmaLegionella

Brucella Franciscella

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

Detection: H. influenzae

• Growth on chocolate but not blood or MacConkey agar

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

– varies among species

• 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

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 which are still common

Other Haemophilus sp

• H. ducreyi

–chancroid - genital ulcer

• H. aegyptius

– conjunctivitis

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

http://www.whoopingcough.net/sound%20of%20whooping

%20cough%20with%20some%20whoop.htm

Pertussis Epidemiology

• Outbreaks described in 16th Century

• Highly contagious respiratory droplets

• > 285,000 deaths worldwide in 2001

• > 10,000 US cases in 2007

• 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

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 the disease reservoir

Pertussis Pathogenesis• Attachment to ciliated respiratory

epithelium by various adhesins

– Filamentous hemagglutinin (FHA)

– Pertussis toxin (PTx)

• Evasion of host defense

– impaired chemotaxis

• Local tissue damage & systemic disease due to exotoxins

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

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

Legionella pneumophila • Fastidious

• Catalase-negative • Facultatively intracellular

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

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 or immunofluorescence

Legionella on BCYE agar

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)

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-

respiratory infections• Most strains produce beta

lactamase

MYCOPLASMA PNEUMONIAE

Eukaryotic Cell

Chlamydia elementary body Treponema

Streptococci

Herpesvirus

Mycoplasma

HIV

1 m

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

Pathogenesis• Cytadherence

– P1 & other proteins

– Immunogenic• Cytotoxicity (H2O2) hemolysin ciliostasis• Induction of inflammation• Cytokine cascade• Antigenic variation• Autoimmunity• Superoxide anions inhibit host catalase increasing oxidative

damage• MP CARDS toxin - ADP ribosyl transferase induces

vacuolation & ciliostasis of epithelium

Mycoplasma Detection• Enriched agar medium

- SP4 + serum (sterols)• Slow growth - 5-20

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

(children) or seroconversion

• PCR Microscopic spherical colonies < 100 m

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

Other Mycoplasmas• Mycoplasma hominis

• Mycoplasma genitalium

• Ureaplasma species

UreaplasmaM. hominis

Diseases due to Genital Mycoplasmas

• Commensals in lower urogenital tract in normal sexually active adults

Diseases due to Genital Mycoplasmas

Condition Ureaplasma M. hominis M. genitalium

Male urethritis + - +

Prostatitis + - +

Urinary calculi + - -

Pyelonephritis + + -

Bacterial vaginosis + + -

Cervicitis - - +

Pelvic inflam. disese - + +

Infertility + - -

Chorioamnionitis + + -

Spont. Abortion + + -

Low birth weight + - -

Postpartum fever + + -

Neonatal pneumonia/meningitis

+ + -

Extragenital disease adults (arthritis)

+ + +

Detection of Genital Mycoplasmas and Ureaplasmas

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

• Serology – not useful

• PCR – needed for M. genitalium

Brucella• Small gram-neg. coccobacilli

– B. abortus (cattle)

– B. melitensis (goats, sheep) • most common cause of human infection

– B. suis (pigs, deer)

– B. canis (dogs, foxes, coyotes)

• Intracellular pathogen - grows in macrophages, inhibits lysosome fusion

• Nonmotile

• Encapsulated

• Fastidious; slow-growing

• Strict aerobe

Brucellosis

Worldwide distribution Spread:

direct contact with organism consumption of contaminated food product inhalation

Clinical Acute vs. chronic infection Intermittent “undulant” fevers Multisystem involvement Manifestation & severity varies with species

Brucellosis• Lab Detection

– Blood & bone marrow culture– Prolonged incubation

• Grows on enriched blood agar after > 3 days• Positive oxidase & urease• ID by antibody agglutination

– Serology• Control of human infection:

– animal vaccination for B. abortus & B. melitensis– elimination of infected herds– avoiding unpasteurized dairy products

Bioterrorism agent

Francisella tularensis • Small gram-neg. coccobacilli • Fastidious • Strict aerobe• Grows in > 3 days• Intracellular pathogen – grows in macrophages,

inhibits lysosome fusion• Encapsulated• Common in many animals, birds, arthropods • AR, OK, MO are most common US locations ~

100 cases/yr

Tularemia• Clinical Manifestations

– Ulceroglandular– Oculoglandular– Typhoidal– Pneumonic– Oropharyngeal– Gastrointestinal

• Lab Detection– Growth on chocolate or BCYE media– Serology – four-fold rise in titer

• Vaccine available but rarely used

• Bioterrorism agent