1. Bordetella, Legionella, Neisseria & Haemophilus Daniel Wozniak, Ph.D. Microbial Infection & Immunity, Microbiology Daniel.wozniak@osumc.edu Research interests: Bacterial pathogenesis and Microbial Biofilms

2. Learning Objectives  Describe the structure and microbial physiology of these four organisms and integrate this information with the human pathophysiologic correlates  Structure and composition  Underlying genetic mechanism of antimicrobial resistance (focus on, N. gonorrhoeae, N. meningitidis and H. influenzae)  Nature and mechanism of action of virulence factors  Identify the normal human immune response to these organisms At the end of this module, you will learn the following:

3. Learning Objectives  Recognize the epidemiology and ecology of infections due to these organisms  Define principles of infection prevention for these organisms  Describe and differentiate principles of laboratory diagnosis for these organisms  Recognize treatment options and accurately evaluate their role in the therapy of infections due to these organisms At the end of this module, you will learn the following:

4. Common Properties Shared by These Organisms Gram negative Opportunistic and overt pathogens Environmental reservoir not always common (usually only humans) Difficult to growth (fastidious) Treatment somewhat less problematic than “PABS” group Members of Neisseria and Haemophilus genera cause both respiratory and STD syndromes All cause respiratory tract infections

5. Legionella Adherence factors Replication in tissues Evasion of host immunity Mucosal pathogens Properties Shared by These Respiratory Tract Pathogens

6. Physiology, Structure & Classification

7. Small cocco-bacilli Gram negative Difficult to grow (“Bordet- Gengou” or “Regan-Lowe Charcoal” media) Diagnosis  Nasopharyngeal swab for culture and Polymerase Chain Reaction (PCR) and/or Serologies Clinically important species: - B. pertussis (whooping cough/pertussis) - B. parapertussis (severe cough) - B. bronchiseptica (kennel cough in animals) Epidemiology  Humans only known reservoir for B. pertussis Spread person to person via aerosol route Bordetella (Microbiology)

8. Only vaccine-preventable disease that is ↑ in incidence in U.S. Clinical relevance of B. pertussis

9. Age distribution is changing….. 20 – 30% of adults and teenagers with chronic cough lasting for more than a week are colonized with B. pertussis 1998 2005 Clinical relevance of B. pertussis

10. Clinical Presentation of B. pertussis

11. Cover photograph Infection and Immunity, June 2005, p. 3618-3626, Vol. 73, No. 6 Adherence is mediated by: Filamentous hemagglutinin Fimbriae Pertactin Bordetella Attach to Ciliated Epithelial Cells (Incubation Stage)

12. Pertussis toxin (Ptx) A-B toxin 5 non-identical subunits “B” subunits bind to cell surface receptors “A” has toxin activity Ptx targets phagocytic cells increasing cAMP levels Impairs chemotaxis and oxidative burst Stimulates release of T and B cells; induces lymphocytosis Net result of toxin release is extrusion of epithelial cells and impaired clearance of mucus - whooping cough Pertussis is a Toxin-mediated Disease (Catarrhal & Paroxysmal stages)

13. This video will play shortly (in approx 35 sec). A Child with “Whooping Cough”

14. Supportive in mild disease, otherwise needs antibiotics Macrolides (i.e. erythromycin, clarithromycin, azithromycin) Trimethoprim-Sulfamethoxazole (TMP/SMX) Post-exposure prophylaxis (PEP) indicated for those who come in close contact (i.e. face-to-face) with someone with pertussis Vaccine  children, adolescents and adults Contains inactivated Ptx, filamentous hemagglutinin & pertactin Formulated along with Diphtheria and Tetanus vaccine “Cellular” (DPT) and “acellular”(DTaP and Tdap) versions “Acellular” vaccines are now routinely used Bordetella (Treatment, Prevention & Control)

15. Clinical Features Name of organism Epidemiological Features Virulence & Pathogenesis Features Treatment, Prevention & Control Micro. Features Bordetella pertussis Pertussis (“Whooping cough”) Only found in humans Person to person transmission Carriage in young adults Pertussis toxin Numerous adhesins (FHA, pertactin & fimbriae Vaccine (DPT, DTap & Tdap) Antibiotics in severe cases (only effective in early stages) PEP for close contacts Gram neg. Cocco-bacillus Difficult to grow Summary and Take Home Message - B. pertussis

16. Slender rods which stain poorly Gram negative Difficult to grow Diagnosis  Lower respiratory tract sample obtained and cultured on Buffered Charcoal Yeast Extract (BCYE) or Urinary Antigen Test are commonly used Clinically important species: L. pneumophila (Legionnaires Disease and Pontiac Fever) Others of less significance clinically (Chapter 34; Fig. 34-1) Legionella (Microbiology)

17. Patients at high risk: elderly, compromised pulmonary function & cell-mediated immunity (steroid use/HIV/transplant recipients). Reservoir is believed to be amoebae in fresh water systems or in biofilms on cooling water surfaces. Causes community (sporadic & epidemic) and nosocomial pneumonias Intracellular pathogen - resides in humans within alveolar macrophages. No known person to person transmission. L. pneumophila is commonly found in natural bodies of fresh water, cooling towers, condensers and water systems. Legionella (Epidemiology & Clinical Manifestations)

18. Dot/Icm+ bacteria Dot/Icm- bacteria L. pneumophila (Pathogenesis)

19. Prevention and Control  Eliminate reservoir Hyper-chlorination of the water supply Maintaining elevated water temperatures Treatment challenge is to target the intracellular niche occupied Antibiotics must be able to penetrate macrophage and vacuoles Macrolides (i.e. Erythromycin, Clarithromycin & Azithromycin) Fluoroquinolones (i.e. Ciprofloxacin, Levofloxacin & Moxifloxacin) Tetracyclines (i.e. Tetracycline & Doxycycline) Diagnosis  Obtain lower respiratory tract sample for culture in BCYE or a Urine Legionella Antigen Test from patient with pneumonia in whom Legionella is suspected Legionella (Diagnosis, Treatment, Prevention & Control)

20. Legionella pneumophila Legionnaires disease Pontiac Fever Fresh water No person to persons transmission Amoebae/biofilms Lung Dz./elderly/ impaired CMI Dot/Icm secretion Intracellular pathogen (macrophages) Persists in specialized vacuole- phagosome Antibiotic must target intracellular bacteria Eliminate from fresh water sources Slender GNR Difficult to grow (BYCE) Clinical Features Name of organism Epidemiological Features Virulence & Pathogenesis Features Treatment, Prevention & Control Micro. Features Summary and Take Home Message - L. pneumophila

21. Nutritionally fastidious Gram negative diplococci Surface structures exhibit antigenic variation LOS structure atypical of most Gram negatives but highly pro-inflammatory (stimulates TNF-α) Clinically important species: N. meningitidis (meningitis, invasive sepsis) N. gonorrhoeae (gonorrhea, STD, conjunctivitis - infants) Capsule present on most N. meningitidis isolates Neisseria (Microbiology & Pathogenesis)

22. Gram negative diplococci Mucosal pathogens Closely related to commensal Neisseria (non pathogenic) Humans are only known natural source Infections spread via person-to-person contact Sample preparation for diagnosis critical Antibiotic resistance emerging Properties Shared by 2 Pathogenic Neisseria species

23. Acquired through contact with mucous exudates from infected people Second most common reportable bacterial STD in the U.S. Males Purulent urethral discharge (i.e. urethritis) (Chapter 26; Fig. 26-2) Pharyngeal & anal infections Acute epididymitis Acute prostatitis Females Often asymptomatic Mucopurulent Cervicitis (MPC) Pharyngeal & anal infections Pelvic inflammatory disease (PID) Abdominal pain Fallopian tube scarring Infertility Ectopic pregnancy Chronic pelvic pain Perihepatitis (Fitz-Hugh-Curtis Syndrome) Disseminated gonococcal infection (DGI) Dermatitis without purulent arthritis Septic arthritis Purulent conjuctivitis in neonates (“Ophthalmia neonatorum”) (Chapter 26; Figure 26-4) N. gonorrhoeae (Epidemiology & Clinical Manifestations)

24. Treatment with an extended-spectrum cephalosporin (i.e. ceftriaxone or cefixime) PLUS azithromycin or doxycycline Samples from exposed sites (i.e. endocervix, vagina, urethral / urine, pharynx and anus) Gram stain (male urethral samples) Culture (selective modified “Thayer-Martin” media) Nucleic Acid Based Testing Prevention  Decrease sexual contacts and use barrier protection (i.e. condoms). In children, treatment of eyes immediately following birth is used to prevent conjunctivitis. Control  Public health management important. Reporting of all cases to state and local county health department N. gonorrhoeae (Diagnosis, Treatment, Prevention & Control)

25. Clinical presentation  Acute meningitis or Sepsis High rates of asymptomatic carriage in nasopharynx Pathogenesis (simplified) Aerosolization of respiratory tract secretions (droplets) Carriage Stress or Viral URT infection (inflammation and damage) Organisms can enter bloodstream and cross blood-brain barrier (Capsule critical in protection in bloodstream) CNS (acute meningitis) Bloodstream (disseminated meningococcemia & septicemia) Growth and fulminant inflammatory response N. meningitidis (Pathogenesis)

26. Major cause of bacterial meningitis and septicemia Incidence = 0.4 cases / 100,000 population At-Risk Groups: Individuals in prolonged close contact (i.e. family members in same household, soldiers in boot camps and military barracks, college students living in dormitories, prisoners). Asplenic patients also a high risk for invasive disease Age distribution  Children, teenagers and young adults Immunodeficiency  Recurrent infections in patients with terminal (C5 – C8) complement deficiency!! Rapid diagnosis essential! High mortality if untreated. N. meningitidis (Epidemiology)

27. N. meningitidis (Clinical Manifestations & Diagnosis) Acute meningitis Severe headaches, fever, confusion, irritability and seizures Lumbar puncture for CSF evaluation  Cloudy, Gram negative diplococci on gram stain, elevated WBC (1000 – 5,000; Neutrophil predominance), elevated protein and decreased glucose Blood and CSF cultures Disseminated Meningococcemia & Septicemia Fever, shock, multi-organ failure, disseminated intravascular coagulation (DIC), petechial rash on trunk and extremities, hemorrhage and failure of adrenal glands (Waterhouse- Friderichsen Syndrome). Can present with or without acute meningitis Blood culture +/- CSF cultures

28. Strains may be sensitive to penicillin-G, but resistance is emerging so an intravenous 3 rd generation cephalosporin (ceftriaxone or cefotaxime) is primarily recommended. Prophylaxis with ceftriaxone, rifampin or ciprofloxacin for close contacts (i.e. direct exposure to respiratory secretions or ≥ 8 hours of close contact) Polyvalent vaccines available and based on most common capsular serotypes (A, C, Y and W-135), but NOT effective against B-serogroup Patients with recurrent meningococcal disease should be evaluated for terminal (C5 – C8) complement deficiencies. N. meningitidis (Treatment, Prevention & Control)

29. Neisseria (Anti-Bacterial Resistance) Multiple mechanisms have been described:  BIG public health concern with respect to N. gonorrhoeae  Concern for emergence of Cephalosporin-resistant and Multi- drug resistant N. gonorrhoeae strains!!  Enzymatic inhibition (i.e. beta-lactamase production)  Alteration of target binding sites (i.e. penicillin-binding proteins, DNA gyrase, topoisomerase, dihydropteroate synthase, etc.)  Alteration of outer membrane porin protein channels  Overexpression of drug efflux pumps

30. Clinical Features Name of organism Epidemiological Features Virulence & Pathogenesis Features Treatment, Prevention & Control Neisseria gonorrhoeae Neisseria meningitidis Gonorrhea Meningitis & Disseminated disease Only found in humans Person to person transmission Gonococci – STD Meningococci – High nasopharyngeal carriage rates Multiple phase variable surface antigens Numerous adhesins Meningococci have a capsule LOS is highly inflammatory 3 rd Generation Cephalosporins (add 2 nd drug for Gonococci) Gonococci - Safe sex practices, Reportable Meningococci - Rapid Dx & treatment, prophylaxis for close contacts, vaccine available Micro. Features Gram negative diplococci Difficult to grow LOS instead of LPS Meningococci have a capsule Summary and Take Home Message - Neisseria

31. H. influenzae  Capsulated (typeable) or Non-encapsulated (non-typeable; NTHi, common normal flora) Gram negative cocco-bacilli that has LOS Difficult to grow - requires hemin & NAD Clinically important species: H. influenzae - Capsulated strains cause meningitis, sepsis, and acute epiglottitis. Non-encapsulated strains cause opportunistic infections like otitis media, pneumonia, bronchitis and sinusitis. H. ducreyi - “Chancroid” (STD; not common in U.S., co-factor in HIV transmission) H. aegyptius - Conjunctivitis (“pink-eye”) Haemophilus (Microbiology)

32. Cause invasive, life-threatening infections in young children and patients with asplenia –Rarely found as normal flora Six capsular serotypes –Serotype “b” is (was) a major pathogen –Effective vaccine in western world Capsule is main virulence factor Non-compliance with Hib vaccination and recent travel to developing world are risk factors H. influenzae (Pathogenesis; Encapsulated)

33. H. influenzae (Pathogenesis; NTHi)

34. Caused by non-encapsulated strains of H. influenzae Infections are localized and can be acute or chronic in nature Source of infection is typically normal flora. Major cause of morbidity in children – Otitis Media (OM). Most common cause of pediatric office visits and reason for use of antibiotics in infants and children Costs the U.S. billions of dollars annually to treat Often polymicrobial: H. influenzae, S. pneumoniae, M. catarrhalis Can lead to severe sequelae: hearing problems, speech and language issues Normal Tympanic Membrane Otitis Media (OM) H. influenzae (Pathogenesis; NTHi)

35. Viral URT Infection Ascension of virus- compromised Eustachian tube Development of otitis media Resolution of Disease Chronicity Biofilm formation in the middle ear Adherence to mucosal surface Replication/ colonization Transmission by droplet or contact Commensal H. influenzae (Pathogenesis; NTHi)

36. Many strains are resistant to  -lactam antibiotics due to enzymatic inhibition (Beta-lactamase production) Diagnosis: Microscopy and culture (respiratory tract, middle ear, CSF and/or blood) Antigen detection (particle-agglutination test) for capsulated strains (Hib) Treatments may include the following: Beta-lactam / Beta-lactamase inhibitors (i.e. Amoxicillin/Clavulanate) 2 nd or 3 rd Generation Cephalosporin (i.e. Cefuroxime, Cefpodoxime) Macrolides (i.e. Erythromycin, Clarithromycin, Azithromycin) Tetracyclines (i.e. Doxycycline) Fluoroquinolones (i.e. Ciprofloxacin, Levofloxacin, Moxifloxacin) Patient with acute exacerbation of COPD may also necessitate corticosteroid therapy H. Influenzae (Diagnosis & Treatment)

37. No vaccine for NTHi, but effective one for invasive Hib Adults - Reduce smoking/smoke exposure. Children - NTHi high incidence in day care settings Influenza vaccine Prophylaxis with Rifampin to eliminate carriage of Hib in children at high risk for invasive disease (i.e. children < 2 y/o in family or day-care center in which invasive Hib has been documented) H. Influenzae (Prevention)

38. Haemophilus influenzae Typeable – epiglottitis, meningitis & sepsis NTHI – otitis media & opportunistic (COPD) Only found in humans Person to person transmission NTHi carriage in nasopharynx Children-otitis Elderly smokers - COPD LOS Phase variable surface structures NTHi - biofilms in inner ear Typeable - capsule Hib vaccine for typeable strain NTHi - antibiotics, resistance a problem NTHi - tympanostomy tubes Avoid smoking Influenza vaccine Gram negative cocco-bacillus Difficult to grow - requires hemin and NAD LOS not LPS Clinical Features Name of organism Epidemiological Features Virulence & Pathogenesis Features Treatment, Prevention & Control Micro. Features Summary and Take Home Message - H. influenzae

40. Thank you for completing this module I hope that I was able to teach the subject clearly. If you have any questions, write to me. [Daniel.wozniak@osumc.edu]

41. References  Medical Microbiology, 7 th Ed. Murray, Rosenthal & Pfaller; Chapters 26, 31, 32, 34.  Theresea Knott. Respiratory system.svg. http://upload.wikimedia.org/wikipedia/commons/8/8b/Res piratory_system.svg. January 11, 2010  CDC: Pertussis video  Cellular Microbiology, Volume 13, Issue 12, pages 1870–1880, December 2011Volume 13, Issue 12,  Infection and Immunity, June 2005, p. 3618-3626, Vol. 73, No. 6  Textbookofbacteriology.net

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