Actinomycetes and Nocardia

Actinomycetes and Nocardia, Bacteria but similar to fungi usually because of its morphological feature of forming a branching filament network, causing Actinomycosis, Actinomycetoma, Farmer's Lung, etc. Demonstrated under microscope by Gram's stain and ZN staining. Cultured on BHI and Thioglycolate broth. Characteristically produce Supher granules. Penicillin is the drug of choice in allergic to penicillin can be replaced by Erythromycin or Tetracycline. In worst cases surgical removal of affected tissue required.

1. Actinomycetes and Nocardia Mbbs 2018 Mr. gunjal prasad n M.Sc. medical microbiology Assistant professor

2. ACTINOMYCETES A diverse group of Gram positive, non-motile, non-sporing, non-capsulated bacilli. Arranged in chains or branching filaments. True bacteria but similar to fungi – form mycelial network of branching filaments. Related to Mycobacterium and Corynebacterium . Most are soil saprophytes or normal human commensals.

3. Important genera Actinomyces: Anaerobe, non-acid fast , cause Actinomycosis. Nocardia: Aerobe, acid fast , cause Actinomycetoma and Pulmonary infections. Actinomadura: Aerobe, non-acid fast , causes Actinomycetoma. Streptomyces: Aerobe, non-acid fast , rarely causes Actinomycetoma in man. An imp. Source of antibiotic like Streptomycin . Thermophilic actinomyces such as Micropolyspora and Thermoactinomyces can cause hypersensitivity pneumonitis (Farmer’s lung and bagassosis)

4. Actinomycetes Actinomycetes are branching filamentous bacteria. Gram stain - Gram positive bacilli. They are facultative anaerobes, but often fail to grow aerobically on primary culture. They grow best under anaerobic or microaerophilic conditions with the addition of 5-10% carbon dioxide.

5. Morphology of Actinomycetes

6. Morphology

7. ACTINOMYCETES ECOLOGY Predominantly soil bacteria. Good at degrading compounds such as chitin & cellulose. Often active at higher pH (contrast to fungi who may dominate at lower pH ). Give soil the “earthy” smell.

8. Commensals in the Mouth Almost all species are commensals of the mouth and have a narrow temperature range of growth of around 35-37°C. They are responsible for the disease known as “Actinomycosis”.

9. Species of Actinomycetes Three-quarters of human cases are caused by Actinomyces israelii. Less common causes include – A . gerencseriae, A. naeslundii, A. odontolyticus, A. viscosus, A. meyeri, Arachnia propionica and members of the genus Bifidobacterium.

10. Actinomycetes Classification Order – Actinomycetales Show fungus-like characteristics such as branching in tissues or in culture (look like mycelia). The filaments are frequently segmented during growth to produce pleomorphic, diphtheroidal, or club shaped cells. The cell wall and the internal structures are typical of bacteria rather than fungi. Some are aerobic and others are anaerobic. All are slow growing.

11. Pathogenesis Actinomycosis is a chronic suppurative granulomatous infection characterised by multiple abscesses with formation of sinuses, discharge containing granules and later stage; fibrosis and tissue destruction. Name refers to ray like appearance of the organism in the granules( Actinomyces, meaning ray fungus).

12. Mode of infection Commensals of oral cavity. Infection is endogenous and may results from trauma, e.g. – Dental extraction. Bacteria bridge the mucosal or epithelial surface of mouth. Grow in an anaerobic condition. Induce mixed inflammatory response.

13. Forms painless indurated swelling with sinuses which may drain pus containing granules to the skin surface. The infection may spread to neighbouring organs including the bones and induce tissue destruction. Often hard indurated swellings are mistaken as malignant tumor. Mode of infection

14. Clinical presentation Cervicofacial infection – Most common form, usually presents as a painless, slow growing, hard mass with cutaneous fistulas, a condition commonly known as “Lumpy jaw”.

15. Abdominal Actinomycetes Occurs due to spillage of intestinal flora secondary to bowel surgery or other conditions of bowel such as appendicitis.

16. Pelvic form It occurs following intrauterine contraceptive devices (IUCDs) insertion. Brain abscesses. Bone destruction and soft tissue infections.

17. Disseminated form It may occur due to hematogenous spread. Lungs and liver are the common sites; where multiple nodules are formed. Dental caries and periodontal diseases: Mainly caused by A. odontolyticus and A. naeslundii.

18. Laboratory Diagnosis Specimens should be obtained Directly from lesions by open biopsy, needle aspiration or, In the case of pulmonary lesions, by fibreoptic bronchoscopy. Examination of sputum is of no value as it frequently contains oral actinomycetes.

19. Laboratory Diagnosis Material from suspected cases is shaken with sterile water in a tube. Sediment contains – Gritty, white or yellowish - Sulphur granules. < 5 mm in size. Granules crushed between two glass slides are stained by the Gram’s nd Ziehl-Neelsen (modified by using 1% sulphuric acid for decolourization) methods. Granules of actinomycosis are hard and not emulsifiable which differentiates them from granules produced in other conditions.

20. Gram’s staining (Brown – Brenn modification ) Shows central mass of Gram positive filamentous bacilli . Radiating peripherally with hyaline, club shaped ends. Clubs are composed of complexes formed due to interaction of bacteria derived polysaccharide and protein with host cell salts and polypeptides.

21. Laboratory Diagnosis Actinomycetes species can be directly detected in specimens by methods like: Fluorescent Antibody techniques using fluorescent tagged species specific monoclonal antibodies. Fluorescent in situ hybridization (FISH) using species specific probes.

22. Culture Suitable media are Blood or Brain-heart infusion agar. Glucose broth. Enriched Thioglycollate Broth - Inoculated with washed and crushed granules.

23. Culture Cultures are incubated aerobically and anaerobically for up to 14 days . Thioglycollate Broth: Growth of A. israelii resembles Fluffy balls ate the bottom of tube, can be differentiated from other species for e.g. A. bovis produces uniform turbidity. BHI Agar: Forms Spidery colonies at 48 hrs.

24. Species identification It is done when culture isolates are subjected to – Biochemical reactions. Gas Liquid Chromatography (GLC) for detection of the products of glucose metabolism. Molecular methods, such as PCR-RFLP are also available for speciation.

25. Antibiotics in Actinomycetes Penicillin is drug of choice. Given for 6 -12 months duration to prevent relapse. Erythromycin or Tetracycline can be given to people with penicillin allergy. Surgical removal of the affected tissues may be required for extensive lesions.

26. Nocardia

27. Nocardia Nocardia spp. (named after Edmond Nocard- 1898) Gram positive branching filamentous bacilli similar to Actinomyces . Differ by being Aerobic and Partially Acid fast. Environmental saprophytes found in soil and vegetation. More than 50 spp. are identified. Only 9 are associated with human infections. Nocardia asteroides – ( star shaped) N. brasiliensis – commonest pathogens.

28. Pathology and pathogenesis Nocardiosis – world wide - commonest amongst adult males Soil – natural habitat. Infections acquired by soil either by – Inhalation of fragmented bacterial mycelia – Leads to development of pulmonary nocardiosis that may disseminate later. Often associated with various species – N. asteroides, N. cyriacigeorgica, N. farcinica and N. pseudobrasiliensis.

29. Transcutaneous inoculation of the bacteria – Leads to various cutaneous & sub cutaneous manifestations for e.g. – Mycetoma. This is often associated with various species such as N. brasiliensis, N. asteroides. Person to person spread not known. Pathology and pathogenesis

30. Characteristic feature seen in nocardiosis is an – Abscess with extensive neutrophil infiltration and prominent necrosis, surrounded by granulation tissue. Nocardiae survive within the neutrophils by: Neutralization of oxidants. Prevention of phagosome-lysosome fusion. Prevention of phagosome acidification. Pathology and pathogenesis

31. Risk factors Cell mediated immunity plays an imp. role in controlling the disease. Hence, nocardiae acts as opportunistic pathogen, tend to occur frequently in immunocompromised conditions including AIDS, corticosteroid treatment, organ transplantation and tuberculosis.

32. Clinical manifestations Pulmonary Nocardiosis Lobar pneumonia (pneumonia affecting one or more lobes of the lung) is most commonest form. Characterized by onset of cough with thick, purulent sputum. Rarely spread to adjacent tissue, leading to – Pericarditis ( inflammation of the pericardium), Laryngitis ( inflammation of the mucous membrane of the larynx; characterized by hoarseness or loss of voice and coughing), Tracheitis (inflammation of the trachea ) and Bronchitis ( inflammation of the membranes lining the bronchial tubes).

33. Extrapulmonary nocardiosis In about half of pulmonary nocardiosis cases dissemination occurs via blood. It typically presents as subacute abscess. Brain is the most common site followed by skin, kidneys, bone and muscle. Meningitis is uncommon.

34. Actinomycetoma Mycetoma is chronic granulomatous condition affecting subcutaneous tissues of the feet and hands, characterized by; Subcutaneous nodular swelling, Multiple sinuses, Discharge containing granules, Tendency of spreading to adjacent bone (bony deformities).

35. Mycetoma Mycetoma usually affect people residing in tropical countries. The organism enters in skin due to abrasion or thorn prick. Broadly mycetoma is characterised in to two types: Eumycetoma: It is caused by fungi such as Madurella. Actinomycetoma: Caused by Nocardia, Actinomadura and Streptomyces somaliensis.

36. Laboratory diagnosis Specimen Depending on site affected, various specimens can be collected such as – Pus Sputum Granules from abscess.

37. Granules Granules present in discharge are collected in sterile gauze or loop by pressing the sinuses from the periphery to express them out (as in case of actinomycosis). Granules are washed several times in saline, crushed between two slides and observed under microscope. Granules are 0.5 -2 mm in sized microcolonies composed of branching filamentous bacilli.

38. Direct microscopy Gram staining (Brown-Brenn modification) – Revels Gram positive branching and filamentous bacilli of width 0.5-1um. They stain irregularly as their filaments are beaded. Sputum examination may show numerous lymphocytes and macrophages, some of which contain branching bacilli.

39. Modified acid fast staining Using 1% sulfuric acid as a decolourizer. Nocardiae are partially acid fast and appear as branching and filamentous red coloured acid fast bacilli.

40. culture Nocardiae are obligate aerobes that can grow on various media such as BHI SDA Incubated at 37 C for 2 wks. Colonies – creamy, wrinkled, pigmented (orange or pink coloured due to carotenoid like pigment). Adhere firmly to medium. Some colonies possess abundant aerial growth and have a cotton wool ball appearance.

41. Culture Recovery of Nocardia spp from samples containing Actinomadura and Streptomyces can be done by: Using selective media- Buffered yeast extract containing Polymyxin and Vancomycin. SDA with Chloramphenicol. Paraffin bait technique- media uses paraffin as a sole source of carbon proved to be an effective media for isolation of Nocardia from soil and clinical specimens.

42. Identification Non-motile. Partially acid fast. Biochemical Catalase positive Utilizes no of sugars oxidatively. Decomposition of Casein, Hypoxanthine, Tyrosine Growth in lysozyme Acetamid utilization. Growth at 450C for 3 days. Acid from rhamnose.

43. Treatment Sulphonamides are drug of choice. Cotrimoxazole can be used as alternative. Duration of treatment – 6-12 months for pulmonary, extrapulmonary forms and for Actinomycetoma. 2 months for cellulitis and lymphocutaneous syndrome. Aspiration or drainage of the abscesses should be carried out to limit the spread of infection.

44. Difference between actinomyces and Nocardia Features Actinomyces Nocardia Acid fastness Non acid fast Partially acid fast Oxygen requirement Anaerobe Obligate aerobe Sugar Fermenter Utilizes oxidatively Habitat Found as oral flora. Infections occur endogenously Usual habitat is soil. Infections occur exogenously Risk factors Disease occurs in immunocompromised host also Usually affects people with low immunity Clinical forms Cervicofacial, abdominal and others Pulmonary, CNS forms, Actinomycetoma Granules Sulfur granules: are hard and not emulsifiable, consist of branching filamentous bacilli surrounded by (sun-ray appearance) Granules are soft and lobulated and also show sun-ray appearance. Commonly found in mycetoma, rare in other conditions.

45. Difference between actinomyces and Nocardia Features Actinomyces Nocardia Culture Spidery molar teeth colony in solid media. Colonies are creamy, wrinkled, and pink. Isolation is done in; Selective media Paraffin bait technique Fluffy ball at bottom of liquid medium Drug of choice Penicillin Sulfonamide or Cotrimoxazole

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