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Which of the following techniques best maintains the microscopic morphology of the fungus
A. Tease mount
B. Cellophane preparation
C. Slide culture
D. India ink preparation
4) List at least three types of primary isolation media and state the purpose of each

1. Medical Microbiology Medically Important Microbial Pathogens of the Respiratory Medical microbiology, College of Medicine and health Sciences, Adigrat University Haftom L . (Ass. Prof. Microbiology) 2016 E.C. Pathogens of the Respiratory system 4/22/2024 1

2. Outline Learning objectives 2 . Introduction 3 . Respiratory system Bacterial infection 4 . Viral infections 5 . Fungal infections 6 . References 4/22/2024 2

3. Learning objectives Upon completion of this topic, you will be able to: List the important microbial agents involved in respiratory infections Describe the classification and characteristics of important microbes involved in causation of respiratory illnesses Introduction 4/22/2024 3

4. Introduction Respiratory system normal flora A variety of microorganisms live in upper respiratory tract However , the lower respiratory tract (lower bronchi & alveoli) is sterile 4/22/2024 4

5. Immune system The lung is the largest interface b/n the body & environment Therefore, the lungs have an important set of mechanisms to defend the body from foreign matter. An excess of immune & nonimmune defense mechanisms exists in the respiratory system. 4/22/2024 5

6. 1. Upper airways defence Saliva The flushing action of saliva is important in the oropharynx Nasal hair Trap foreign particles Turbinates Complex air passages in the nose, Help trap large inhaled particles Mucociliary apparatus Particles deposited in mucus are moved toward the mouth by the continuous beating of cilia on epithelial cells The cilia facilitate the flow of mucus from the upper airways & clear the main nasal passages 4/22/2024 6

7. Respiratory system infections The respiratory system is the most commonly infected system The most accessible system in the body - Breathing brings in clouds of potentially infectious pathogens Health care providers will see more respiratory infections than any other type 4/22/2024 7

8. Immune defects predisposes for different infections Defects in innate immunity & humoral immunodeficiency typically lead to increased incidence of infections with pyogenic bacteria Cell-mediated immune defects lead to increased infections with intracellular microbes such as mycobacteria & herpesviruses as well as Pneumocystis carinii Several lifestyle factors interfere with immune defense mechanisms & facilitate infections. For eg ., Cigarette smoke compromises mucociliary clearance & pulmonary macrophage activity, Alcohol impairs cough & epiglottic reflexes (thereby increasing the risk of aspiration), in addition, interferes with neutrophil mobilization & chemotaxis 4/22/2024 8

9. The upper respiratory tract: Infections are fairly common Usually an irritation URT infections: Rhinitis Sinusitis Pharyngitis/tonsillitis Epiglottitis Laryngotracheobronchitis (croup) The lower respiratory tract: Infections are more dangerous Can be very difficult to treat LRT infections: Bronchiolitis Pneumonia Pulmonary abscess Empyema 4/22/2024 9

10. Medically important Bacteria Causing Respiratory System Infection Distinctive features Are obligate aerobic Cell wall is unique 1. M. tuberculosis The high lipid content (~ 60%) of their cell wall Acid-fast bacilli Grows slowly 4/22/2024 10

11. Virulence Mechanisms & Virulence Factors  No exotoxins & endotoxin  However, a number of structural & physiological properties contribute its virulence ─ Sulfatides ( sulfolipids in cell envelope ) Inhibit phagosome-lysosomal fusion ─ Waxy nature of the cell envelope If phagosome-lysosomal fusion occurs, this reduces killing effect ─ Cord factor ( trehalose dimycolate ) Inhibits leukocyte migration Disrupts mitochondrial respiration & oxidative phosphorylation damage done by immune system 4/22/2024 11

12. Pathogenesis & Clinical significance Transmission: – Although other routes may be involved, most infections are acquired by direct person-to-person transmission of airborne droplets that contain M. tuberculosis bacilli from an active case to a susceptible host  It causes Pulmonary tuberculosis ─ Primary tuberculosis ─ Reactivational tuberculosis (Post primary tuberculosis) 4/22/2024 12

13. Infection leads to the development of delayed hypersensitivity . – Inhalation of the infectious agent – Immunity to infection is mediated by T cells and is characterized by delayed hypersensitivity . – Caseating granulomas & cavitation, are the result of the destructive tissue hypersensitivity. 4/22/2024 13

14. Lab identification 1. Microscopy a. Auramine-rhodamine (AR ) stain It is a fluorochrome stain used to detect acid-fast bacteria 4/22/2024 14

15. b. Ziehl Neelson /AFS staining M . tuberculosis is acid fast & stains red 4/22/2024 15

16. 4/22/2024 16

17. 2. Culture General selective medias: a . Broth media (e.g., Middlebrook 7 H9 & 7H12) b. Semi-solid media (e.g., Middle brook 7H10 & 7H11 media) c. Solid media (Lowenstein-Jensen egg based media , LJ media) 4/22/2024 17

18. 3. Biochemical tests – Organisms produce niacin – Catalase - ve 4. Molecular Techniques – Routine PCR: Gene amplification – Real Time PCR: Gene amplification plus quantification 4/22/2024 18

19. 5. Tuberculin ( Mantoux ) skin test – Infection can be detected by the tuberculin test – About 2-4 weeks after the infection has begun, intracutaneous injection of 0.1 mL of purified protein derivative (PPD) induces a visible & palpable induration (at least 5 mm in diameter) that peaks in 48-72 hours – A positive tuberculin test result signifies cell mediated hypersensitivity to tubercular antigens – It does not differentiate between infection & disease – False-negative reactions (or skin test anergy ) may be produced by certain viral infections, sarcoidosis , malnutrition, Hodgkin disease, immunosuppression , and overwhelming active tuberculous disease – False-positive reactions may also result from infection by atypical mycobacteria 4/22/2024 19

20. Treatment Long duration of treatment (6- 9 month) Treatment of latent (asymptomatic) infections consists of Isoniazid ( INH) Multidrug therapy is used to prevent drug-resistant mutants Treatment for most patients with pulmonary TB is with 3 drugs: INH, rifampin, & pyrazinamide Treatment of MDR strains usually involves the use of 4/5 drugs, including ciprofloxacin, amikacin , ethionamide , & cycloserine Directly observed therapy (DOT): patients take their medication while being supervised & observed, which is a successful strategy for achieving better treatment completion 4/22/2024 20

21. Prevention & control Early case detection & treatment Decreasing of over crowding Pasteurization of milk --- ↓ M. bovis infection Health education Immunization (BCG) It is produced from Bacille Calmette-Gurin (BCG), an attenuated strain of M. bovis 4/22/2024 21

22. 2. Streptococcus pneumoniae (Pneumococcus) Distinctive characteristics Gram positive cocci , in pairs Commensals of the nasopharyngeal areas Virulence factors Its capsule is the most important virulence factor Pneumolysin & autolysin contribute to its pathogenicity 4/22/2024 22

23. Clinical significance It is the most common cause of acute bacterial pneumonia. ─ Streptococcus pneumoniae is the most common cause of community-acquired acute pneumonia (30%) ─ Either pattern of pneumonia, lobar or bronchopneumonia, may occur • Bronchopneumonia is much more prevalent at the extremes of age. • It is responsible for more than 90% of lobar pneumonias • Pneumococcal lung infections usually originate by aspiration of pharyngeal flora (20% of adults harbor S. pneumoniae in their throats) Sinusitis Epiglottitis (rarely) 4/22/2024 23

24. Pneumococcal infections occur with increased frequency in 3 groups of individuals: 1) Those with underlying chronic diseases such as congestive heart failure, COPD, or diabetes; 2 ) Those with either congenital or acquired immunoglobulin defects (e.g., AIDS); & 3) Those with decreased or absent splenic function ( e.g., sickle cell disease or post splenectomy ); because the spleen is the major organ responsible for removing pneumococcus from the blood. 4/22/2024 24

25. Laboratory identification 1. Microscopic morphology ─ Examination of Gram-stained sputum is an important step in the diagnosis of acute pneumonia ─ The presence of numerous neutrophils containing the typical gram-positive, lancet-shaped diplococci is good evidence of pneumococcal pneumonia ─ But, it must be remembered that S. pneumoniae is a part of the endogenous flora & therefore false-positive results may be obtained by this method 4/22/2024 25

26. 2. Colony morphology ─ Isolation of pneumococci from blood cultures is more specific ─ During early phases of illness , blood cultures may be positive in 20-30% of patients ─ Smooth, glistening, wet-looking , mucoid ─ α- Hemolytic 4/22/2024 26

27. 3. Biochemical tests – Catalase test: negative – Optochin -susceptibility-test : susceptible – Bile-solubility-test: positive (the bacterial cells are lysed by bile acids ) 4. The Quellung reaction – Capsular swelling is observed when the pneumococci are treated with type-specific antisera 4/22/2024 27

28. Treatment & Prevention Pneumococcal pneumonias respond readily to penicillin treatment, but there are increasing numbers of penicillin-resistant strains of pneumococci, so whenever possible antibiotic sensitivity should be determined – In penicillin-allergic patients, erythromycin or one of its long-acting derivatives, e.g., azithromycin, can be used Commercial pneumococcal vaccines containing capsular polysaccharides from the common serotypes of pneumococcus are available 4/22/2024 28

29. 3. Haemophilus influenzae Distinctive characteristics Gram-negative, Pleomorphic (ranging from coccobacilli to long) 3. Haemophilus influenzae Non-motile Obligate parasites, requiring hemin (factor X) & NAD+ ( factor V) for growth/respiration – Culture on chocolate agar, containing these 2 growth factors – Require high CO2 levels to grow in culture Often found in oral cavity – Present in the nasopharynx of ~75% of healthy children & adults 4/22/2024 29

30. Have thick capsule ─ Based on the capsule, capsulted H. influenzae can be classified in to 6 serotype: a, b, c, d, e, & f [capsular type b ( Hib ), is highly virulent] ─ Nontypeable ( unencapsulated ) strains of H. influenza are less pathogenic Usually the non-encapsulated strains are harbored as normal flora 4/22/2024 30

31. Virulence factors ─ Capsule: An important virulence factor & Has antiphagocytic activity . ─ Fimbiae (for adherence) ─ Membrane lipooligosaccharide : May be responsible in bacterial attachment, invasiveness, and paralysis of the ciliated respiratory epithelium However , no exotoxin is produced ─ Outer membrane protein : Contribute in adhesion & invasion of host tissue ─ IgA protease: It degrades secretory IgA, facilitating colonization of the upper respiratory tract mucosa 4/22/2024 31

32. Pathogenesis & clinical manifestations H. influenzae is transmitted by respiratory droplets The organism produces IgA protease that degrades sIgA , thus facilitating attachment to the respiratory mucosa Then becoming established in the upper respiratory tract Then , inflammation 4/22/2024 32

33. H. influenzae type b ( Hib ) causes: – Pneumonia & emypyema : Commonly implicated in Community-acquired pneumonia (5%) Hib causes pneumonia mainly in infants The infection is clinically indistinguishable from other types of bacterial pneumonia (e.g., pneumococcal pneumonia ) except that Hib is more likely to involve the pleura – Acute epiglottitis in children (also in adults) • A life-threatening Hib infection involving cellulitis of the epiglottis & supraglottic tissues • It can lead to fatal acute upper airway obstruction • Sore throat & fever rapidly progress to dysphagia, drooling, & airway obstruction 4/22/2024 33

34. Non- typeable H. influenzae strains are mainly responsible for : – Chronic bronchitis • Usually in adults – Paranasal sinusitis • Among cases of community-acquired acute sinusitis, nontypable Haemophilus influenzae , & Str. Pneumoniae are the most common pathogens, accounting for 50–60% of cases 4/22/2024 34

35. Lab diagnosis Rapid diagnosis is crucial because of the potentially fulminant course of type b infections 1. Microscopy ─ Gram staining of sputum/nasopharyngeal specimen commonly reveals pleomorphic, gram-negative coccobacilli ─ Capsular swelling ( quellung ) reaction 4/22/2024 35

36. 2. Culture: – Chocolate agar: capsulated H. influenzae strains produce mucoid colonies – Cultures have a distinctive smell 3. Biochemical tests 4. Serology ─ Capsular antigen may be detected using immunologic tests • Such as latex agglutination, countercurrent immunoelectrophoresis , and radioimmune assay 5. Other tests: Biotyping , PCR, Nucleic acid analysis & sequencing 4/22/2024 36

37. Treatment Treatment is begun immediately with antibiotics effective against H. influenzae ( cefotaxime , chloramphenicol) Antibiotic sensitivity testing is necessary because of resistance to common antibiotics used to treat H. influenzae ( eg ., strains with β -lactamase-mediated ampicillin resistance 4/22/2024 37

38. Prevention & control Active immunization against Hib is effective in preventing invasive disease, and also reduces respiratory carriage of Hib ─ The current vaccine, generally given to children younger than 2, consists of Hib polyribose phosphate ( PRP) capsular carbohydrate conjugated to diphtheria toxoid or other carrier protein ─ Pentavalent vaccine Rifampin is given prophylactically to individuals in close contact with a patient infected with H. influenzae 4/22/2024 38

39. 4. Bordetella pertussis Distinctive characteristics Gram-negative Coccobacilli that grow singly or in pairs Encapsulated Non-motile Aerobic 4/22/2024 39

40. Virulence factors: ─ Toxin • Pertussis toxin The toxin can be inactivated and converted to toxoid for use in component vaccines ‹Damage cells, lymphocytosis, sensitization to histamin , activation of insulin production resulting in hypoglycemia 4/22/2024 40

41. Adenylate cyclase toxin (ACT) ‹ When taken up by phagocytic cells (e.g., neutrophils) can inhibit their bactericidal activity ‹ In the cytoplasm it acts as a calmodulin -dependent adenylyl cyclase that raises cAMP levels in the cell ‹ Generally disrupting intracellular signaling pathways ‹ Bacterial mutants that lack cyclase activity are avirulent • Tracheal cytotoxin ‹ It is a fragment of the peptidoglycan that damages ciliated cells of the respiratory tract ‹ It appears to induce nitric oxide, which kills the ciliated epithelial cells • Dermonecrotic toxin: causes vasoconstriction and ischemic necrosis • Endotoxin (LPS) 4/22/2024 41

42. Adhesins Most important adhesin is filamentous haemagglutinin – Which mediate adhesion to ciliated epithelial Cells • Agglutinogens : for attachment 4/22/2024 42

43. Pathogenesis & Clinical manifestation Spread by coughing The bacteria colonize only ciliated cells of the respiratory mucosa (trachea, bronchi) Then , they multiply rapidly & produce toxins, such as: pertussis toxin 4/22/2024 43

44. Pertussis toxin is a 2 component, A+B exotoxin – A subunit (S1) is an ADP ribosyl transferase – B component, Composed of 5 polypeptide subunits (S2 through S5), Binds to specific carbohydrates on cell surfaces Following binding of B component to host cells, A subunit is inserted through the membrane & released into cytoplasm in a mechanism of direct entry Then, the A subunit catalyzes the addition of ADP ribosyl moiety of NAD to Gi protein – (i.e., inhibitory subunit of the membrane bound G protein complex; Gi protein normally inhibits the eukaryotic adenylate cyclase ) As a result, Gi protein is inactivated & cannot perform its normal function to inhibit adenylate cyclase 4/22/2024 44

45. This results in prolonged stimulation of adenylate cyclase – Adenylate cyclase catalyzes ATP to cAMP Thus, the conversion of ATP to cAMP cannot be stopped & intracellular levels of cAMP increase. This has the effect: – To disrupt cellular function, & – In the case of phagocytes, to decrease their phagocytic activities (such as chemotaxis , engulfment, oxidative burst, & bacteriocidal killing) Since PT targets respiratory epithelium , it results in tissue damage & many of the symptoms associated with whooping cough 4/22/2024 45

46. Incubation period 1 -2 weeks It causes whooping cough (a.k.a., pertussis ) ─ An infection of the mucosa of the upper respiratory tract ─ The toxin causes secretion of mucus which leads to irritation & the spasms of coughing associated with the disease 4/22/2024 46

47. Lab Diagnosis Specimens : nasopharyngeal secretions; cough droplets Direct immunofluorescence in smear Culture : a selective & enrichment medium is recommended for the primary isolation of B. pertussis ─ Charcoal cephalexin blood agar Produces small pearly-grey, shiny (mercury-like), usually mucoid colonies ─ Bordet- Gengou media (Potato-blood-glycerol agar) Selective media containing a high percentage of blood (20-30%) Biochemical tests: Urease - ve ; oxidase - ve 4/22/2024 47

48. Treatment & prevention Erythromycin is the drug of choice, both as chemotherapy, & as chemoprophylaxis for household contacts Alternative choice: Trimethoprim- sulfamethoxazole Pertussis vaccine is available, it contains proteins purified from B. pertussis, and is formulated in combination with diphtheria & tetanus toxoids ( DTaP ) ─ Immunization is generally initiated when the infant is 2 months old ─ Currently, as pentavalent vaccine 4/22/2024 48