Mycoplasmas and Fastidious Gram-negative Bacteria

1. Mycoplasmas and Fastidious Gram-negative Bacteria Haemophilus * Bordetella Legionella * Moraxella * Mycoplasma Ureaplasma* Brucella * Francisella Ken B. Waites, M.D. F(AAM)

2. Objectives • To review and discuss • microbiological characteristics • epidemiology • virulence factors • associated diseases • laboratory detection Haemophilus Bordetella Mycoplasma Ureaplasma Legionella Brucella Franciscella

3. Haemophilus • “Blood-loving” • Gram-negative coccobacillus • Facultative anaerobe • Non-hemolytic • Invasive strains • Non-encapsulated H. influenzae & other spp. common URT flora Haemophilus influenzae

4. 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

5. 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

6. Otitis media Sinusitis Bacteremia Epiglottitis Laryngotracheobronchitis Meningitis Exacerbation of chronic bronchitis in COPD Pneumonia Cellulitis Otitis media Conjunctivitis Diseases: H. influenzae

7. 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

8. Other Haemophilus sp • H. ducreyi • chancroid - genital ulcer • H. aegyptius • conjunctivitis

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

10. 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

11. 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

12. Pertussis in Alabama Reported cases82 cases in 200549 cases in 200419 cases in 200337 cases in 200237 cases in 2001

13. 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

14. 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

15. 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

16. 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

17. 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

18. Legionella pneumophila • Fastidious • Catalase-negative • Facultatively intracellular Gram-negative bacillus • Nonfermentative • Stains poorly with safranin • > 30 species • Multiple serogroups • 1st described in 1976

19. 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

20. 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

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

22. 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)

23. Prevention of Legionellosis • No vaccine • CMI more important than AB • Identify sources and eliminate them

24. 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

25. MYCOPLASMA PNEUMONIAE

26. Eukaryotic Cell Herpes virus HIV Mycoplasma Chlamydia elementary body Streptococci Treponema 1 m

27. 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

28. 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

29. 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

30. 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

31. Other Mycoplasmas • Mycoplasma hominis • Mycoplasma genitalium • Ureaplasma species Ureaplasma M. hominis

32. Diseases due to Genital Mycoplasmas • Commensals in lower urogenital tract in normal sexually active adults

33. Diseases due to Genital Mycoplasmas

34. Detection of Genital Mycoplasmas and Ureaplasmas • Culture – good for rapid growing M. hominis and Ureaplasma • Serology – not useful • PCR – needed for M. genitalium

35. 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

36. 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

37. 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

38. 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

39. 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