Human Diseases Caused Primarily by Gram-Positive and Gram-Negative Bacteria: The Airborne Diseases – Part I

1. Human Diseases Caused Primarily by Gram-Positive and Gram-Negative Bacteria:   The Airborne Diseases – Part I

2. Airborne Diseases

3. Diphtheria

4. Diphtheria - Corynebacterium diphtheriae • General features • Club shaped (coryne = “club” in Greek) – pleomorphic V-shaped rods • Gram positive, non-sporing, non-motile • Refractile granules give rise to heterogeneous staining • Accumulate in stationary phase – phosphate storage • Metachromatic granules • Volutin • Babes-Ernst bodies • Only humans serve as a reservoir for this microorganism

5. Acute, contagious disease • Affects mainly poor people living in crowded conditions – unvaccinated • Acute infection of the respiratory tract  usually invading the tonsilar area • Spread via respiratory route and contaminated milk (unpasteurized)

6. Virulence factors/pathogenicity • Exotoxin = diphtheria toxin (AB exotoxin) • Tox gene encoded by bacteriophage that integrates into bacterium’s genome  Prophage b (lysogenic) • Protein synthesis inhibition (involving EF-2 and translocase) • Can disseminate through bloodstream (toxemia) and affect nervous system, heart and kidneys • Cord factor • Glycolipid containing trehalose

7. Symptoms/Presentation • Dead tissue cells, WBC and RBC and bacterial cells form a dull gray exudate referred to as a pseudomembrane • Can block airway in trachea – suffocation • Tracheotomy may be required • Thick mucopurulant (mucous and pus) nasal discharge • Fever • Cough, sore throat • Malaise (vague feeling of physical discomfort) • Enlarged and tender cervical lymph nodes

8. Laboratory culture – swab the tonsilar area • Tellurite added to medium (chocolate or blood agar) • Enriches for C. diphtheriae and restricts growth of normal flora of the throat • Tellurite is reduced to tellurium metal precipitate (dark gray to black colonies)

9. Diagnosis • Detection of toxin • In vitro virulence test = ELEK test • Streak bacteria out on agar in lines • Lay a paper strip containing anti-toxin antitoxin antibodies • Include a positive control in parallel to patient’s sample • Incubate and look for line of identity (precipitate caused by immune complexes) • Inject guinea pig  morbid symptoms/mortality

10. Susceptibility test • SCHICK Test (rarely used anymore) • Diphtheria toxoid inject subcutaneously • Wait 48 hours and note formation of induration in the absence of antibodies = susceptible  immunity not present • If antibodies are present (immune) then toxin is neutralized  no induration • DTH response

11. Treatment • Antitoxin administered to neutralize the toxin • Immediate administration based on symptoms rather than waiting for laboratory results • Also administer penicillin or erythromycin

12. Prevention • Immunization (vaccination) • Has protected USA from this disease • Toxoid made from diphtheria toxin • Part of DPT vaccine • Diphtheria • Pertussis • Tetanus • Administered at 2, 4, 6, and 15-18 months, and again at 4-6 years with boosters every 10 years recommended (especially if traveling to areas where diphtheria is endemic – Asia, Africa, Central and South America and Russia)

13. Legionnaires’ Disease (Legionellosis)

14. General Features • Caused by Legionella pneumophila • Gram negative rods • This organism also causes Pontiac Fever (no pneumonia, self-limiting) • 1976 – American Legion convention in Philadelphia in one of the city’s largest hotels • 29 fatalities (182 participants) • 1985 – Stafford, England • Cooling system • 39 fatalities out of 163 people exposed

15. Not spread person-to-person • Bacteria normally found in soil and aquatic ecosystems • Bacteria also found in air-conditioning systems and shower stalls • Infection causes cytotoxic damage to lung alveoli

16. Virulence factors/pathogenicity • Mist inhaled from poor water cooling systems and mist machines in supermarkets • Soil  dust?? • Whirlpool filters, shower heads, nebulizers and hot water systems

17. Endotoxin in cell wall • Hemolysin generated • Cytotoxin produced • Bacteria divide within alveolar macrophages • Inhibition of fusion of phagosome with lysosome • Mediated by mip gene product (macrophage infectivity potentiator) • Important in establishing pneumonia

18. Symptoms • Chest pain, dry (non-productive) cough • Fever • Headache • Neuralgia • “Atypical pneumonia” (bronchopneumonia) • Abdominal cramping and gastrointestinal symptoms may also occur

19. Laboratory culture • Mueller-Hinton agar • 1% Hemoglobin + 1% Isovitalex (vitamins) • 5% CO2 • Cell culture • Grown in animals • Guinea pigs • Chick embryos • Can survive in free-living amoebae (another potential source)

20. Increased susceptibility • Smokers • Alcoholism • Endotracheal intubation • Chronic pulmonary diseases • Patients on immunosuppressive therapy • Transplant patients • Autoimmune patients • Anesthesia

21. Diagnosis and treatment • Isolation and serological identification needed • Direct immunofluorescence (organisms cultured or obtained from sputum) • Indirect immunofluorescence (patient’s serum – antibodies to organism) • Agglutination tests • ELISA • RIA on urine • Erythromycin and/or rifampin

22. MeningitisNeisseria meningitidis*, Streptococcus pneumoniae, Haemophilus influenzae*, Salmonella choleraesuis (invades from GI tracts), Listeria monocytogenes*

23. Meningitis - caused by a variety of organisms and conditions • Bacterial (septic) meningitis is diagnosed by the presence of bacteria in the cerebrospinal fluid • Bacterial meningitis is treated with various antibiotics, depending on the specific bacterium involved • Aseptic (nonbacterial) meningitis syndrome is more difficult to treat but the mortality is generally low

24. Neisseria meningitidis(epidemic meningitis)

25. General features • Gram negative dipolococci (meningococci) • Non-motile • Oxidase positive • Can oxidize dimethyl (and tetramethyl) para-phenylene diamine hydrochloride) • Must be grown >30oC • Require special media  sensitive to trace metals and fatty acids • Chocolate agar (blood agar treated at 80oC for 10’) • Decreases inhibitory effects of trace metals and fatty acids • Incubated with 10% CO2 • Causes 2000-3000 cases of meningitis annually in USA

26. Classification • Serotypes based on common antigens • Polysaccharide capsule • Outermembrane proteins • A-D, X-Z, L, W135 and 29E • A,B an C are primarily responsible for meningitis outbreaks

27. Virulence characteristics • Obligate parasite of humans • Often found in nasopharynx of asymptomatic carriers • Spread via respiratory route • Pili enable attachment to host cells • Capsule resists phagocytosis • Release large amount of endotoxin • IgA1 protease produced

28. Three stages • First stage = nasopharyngeal infection • Minor inflammation or asymptomatic • Lasts days to months • Protective antibodies made • Second state = memingococcemia • Microbes enter bloodstream • Can be acute and rapid (death in 6-8 hours!) • May take longer to ensue with fever, malaise and rash

29. Sometimes lesion in joints, lungs, skin, internal organs and adrenal glands develop • Endotoxin induced (TNF-alpha produced) • Disseminated intravascular coagulation (DIC) may develop (Hageman factor) • Clots disrupt circulation • Amputation may be necessary

30. Third stage = meninges infected following invasion of the blood brain barrier (BBB) • Headache, stiff neck, vomiting, delerium • TNF-alpha found in CSF • Concentration correlates with degree of BBB disruption and severity of the disease • Most severe form of meningococcemia is the life threatening Waterhouse-Friderichsen syndrome • High fever, shock, widespread purpura, disseminated intravascular coagulation and adrenal insufficiency, seeding of multiple organs including the meninges

31. Bleeding into the skin (petechiae and purpura), and the tissue in these areas may die (become necrotic or gangrenous).

32. Diagnosis of Meningococcal infections • Microscopy – smears • Culturing CSF, blood, skin lesion, nasopharyngeal secretions • Chocolate agar • Blood agar • Thayer-Martin medium • Nystatin, Polymyxin, Vancomycin – Antibioitics inhibit contaminants while favoring pathogenic Neisseria • 5-10% CO2 (candle jar or CO2 incubator)

33. Serology • Group-specific antisera – agglutination of bacteria • Sugar fermentation tests (Glucose+, Maltose+) • Counterimmunoelectrophoresis • At the pH used the anti-capsule antibodies have a positive charge and moves toward the anode while the capsular polysaccharides have a negative charge and moves toward the cathode • Electrophoresed for 30-60 minutes • A precipitate line forms if positive

34. Quellung reaction • Antibodies to capsular components – swelling of capsule occurs • Latex bead agglutination (group-specific polysaccharide adsorbed to beads – incubated with patient’s serum)

35. Treatment • Penicillin or erythromycin • Prevention • Quadravalent vaccine – A, C, Y + W135 – polysaccharide antigens • Used in epidemics and for military personnel • Prophylactic rifampin used for those exposed

36. Haemophilus influenzae - Meningitis

37. General features and growth requirements • Small, Gram negative rod/coccobacillus (pleomorphic) • Non-motile, aerobic • Associated with bacterial meningitis in children and epiglottis infection (can block airway) • Leading cause of invasive bacterial disease in children

38. Six capsular types • Type b = significant in human infections • Primary virulence factor - antiphagocytic • Composed of PRP • Polymer of ribose phosphate or ribulose phosphate • Polysaccharide capsule • Non-encapsulated forms in majority of asymptomatic carriers • Can cause secondary bacterial pneumoni after viral infections (influenzae0

39. Require chocolate agar supplemented with X and V factors • X factor = heat stable hematin (reguired for cytochromes and catalase) • V factor = heat labile, can be substituted with NAD

40. Virulence factors/pathogenicity • Respiratory route of infection/transmission • Menintigitis in children • 1000-3000 cases annually in USA • Mortality rate of 3-7% • Enters blood via nasopharynx, then meninges • CSF resembles meningococcal infection: • PMNLs and elevated sugar concentration

41. Other diseases caused by HIB • Epiglottitis in children • Airway obstruction owing to inflmmation, edema • Can lead to suffocation if not treated • Otitis media with effusion (OME) • Within first 3 years of life, 60-70% of children are affected (40% more than once) • Usually the unencapsulated form is responsible • Can also cause septic arthritis in children <2 years old (joint inflammation) • Elderly infections: pneumonia, meningitis, epiglottitis and female genital tract

42. Diagnosis, treatment and prevention • Centrifuge the CSF • Gram stain – pleomorphic Gram negative rods • Quellung reaction with type b antiserum • Fluorescent antibodies • Countercurrent electrophoresis of CSF versus antiserum • ELISA

43. Latex bead agglutination (beads coated with antibodies to type b capsule) • Culture CSF and blood  chocolate agar, 350C + CO2 or TSA + X,V or XV discs • Both X and V required for growth

44. Treatment of meningitis must be immediate! • Relatively high mortality rate (3-7%) • High level of complications (in 30% of survivors) • Convulsions • Hydrocephalus • Mental retardation • Blindness • Ampicillin or if ampicillin resistant then chloramphenicol or cephalosporins • Rifampin used as prophylactic for those exposed

45. Prevention • Vaccine for type b capsular antigens • Early vaccines were inefective in children <2 years • Improved vaccine • Conjugate vaccine • Polysaccharide of type b conjugated to large proteins which serve as carriers • Tetanus toxoid = carrier • Diphtheria toxoid = carrier • Administered at 2 months of age, 3 doses, 2 months apart, then a booster at 12-15 months • Tetramune: DPT + HbOC • Case Study

46. Listeria monocytoenes - Listeriosis

47. General features • Small, Gram positive, motile, pleomorphic rod (club shaped) • Facultative anaerobes • Beta hemolytic on blood agar plates (narrow band) • 11 serotypes • Most prevalent forms include types 1a, 1b and 4b

48. Pathogenicity • Transmitted by: • Animals • Contaminated water • Soil • Fecal-oral route • Poultry (major reservoir) • Unpasteurized milk • Coleslaw (raw vegetables not cleaned, contaminated with manure)

49. Even pastueurized milk can be a potential source: intracellular microbes in leukocytes in milk are resistant to heat • Meningitis occurs in >75% of infections (most common feature/manifestation in adults) • Also causes endocarditis, urethritis, conjunctivitis and abortions (miscarriage) • Most susceptible • Immunocompromised and immunosuppressed • Newborns • Neonatal listeriosis

50. Neonatal listeriosis • Early and late onset • Early onset: Transplacental or transvaginal acquisition • Acutely ill at birth or shortly thereafter • Pneumonia common • Believed to be a result of decreased cell-mediated IRs in pregnancy • Growth of bacteria within placental cells • Late onset • 1-4 weeks after birth • Meningitis usually • Probably acquired after birth as a result of person-to-person contact