Actinomycetes

ACTINOMYCETES

1. ACTINOMYCETES ( Actinomyces & Nocardia )

2. ACTINOMYCETES TOPICS for discussion - MORPHOLOGY CLASSIFICATION ACTINOMYCES NOCARDIA ACTINOMADURA

3. ACTINOMYCETES They are true bacteria , but similar to fungi, they form a mycelial network of branching filaments. They are also related to mycobacteria and corynebacteria . Cells of actinomycetes elongate to form branching, filamentous forms. Some organisms form filaments, or hyphae , on the agar surface or into the agar , whereas others produce hyphae that extend into the air. These organisms are aerobic, facultative anaerobic or obligate anaerobic.

4. ACTINOMYCETES Morphology- Gram-positive Non motile Non- sporing Non-capsulated bacilli Arrange in chains or branching filaments Most of them are soil saprophytes or normal human commensals .

5. ACTINOMYCETES lmportant genera include: Actinomyces : They are anaerobe & non-acid fast ; cause actinomycosis . Nocardia : They are aerobe & acid fast ; cause actinomycetoma & pulmonary infection . Actinomadura : They are aerobe & non-acid fast ; cause actinomycetoma . Streptomyces : They are aerobe & non- acid fast ; rarely cause actinomycetoma . important source of streptomycin (antibiotics). Thermophilic actinomycetes = Micropolyspora & Thermoactinomyces can cause hypersensitivity pneumonitis ( farmer's lung and bagassosis )

6. ACTINOMYCES Actinomyces are Gram positive Non–acid-fast (do not contain mycolic acids in their cell walls) Branching filaments Soil saprophytes & commensals of oral cavity . In humans they cause actinomycosis . Encountered infrequently in the clinical laboratory . Species - A . Israelis- most common species infecting man. A . naeslundii A . odontolyticus

7. Pathogenesis = Actinomycosis Actinomycosis Actinomycosis is a chronic suppurative and granulomatous infection characterized by multiple abscesses with formation of sinuses , discharge containing granules and on later stage- fibrosis & tissue destruction. Mode of infection : They are commensals of oral cavity, the infection is mostly endogenous and may result from trauma, e.g. dental extraction . The bacteria bridge the mucosal or epithelial surface of the mouth--- grow in an anaerobic niche --- induce a mixed inflammatory response , and form painless indurated swelling with sinuses which may drain pus containing granules to the skin surface. The infection may spread to the neighbouring organs including bones and induce tissue destruction . Often , the hard indurated swellings are mistaken as malignant tumors .

8. Clinical Manifestations Cervicofacial actinomycosis : This is the most common form , usually presents as a painless, slow-growing, hard mass with cutaneous fistulas, a condition commonly known as lumpy jaw. Other rare forms are : Abdominal form : It occurs due to spillage of intestinal flora secondary to bowel surgery or other conditions of bowel such as appendicitis. Pelvic form : It occurs following intrauterine contraceptive devices ( lUCDs ) insertion. Brain abscesses Bone destruction and soft tissue infections Disseminated form : due to hematogenous spread- lungs & liver are the common sites; where multiple nodules are formed. Dental caries and periodontal diseases : Mainly caused by A. naeslwuiii and A. odontolyticus .

9. Cervicofacial actinomycosis

10. Laboratory Diagnosis Specimen = discharge collected from the sinuses or fistula , rarely bronchoalveolar lavage, sputum or tissue sections . Direct Microscopy = Process Pus discharge is washed in saline in a test tube and the sediment is collected ( that contains gritty , white or yellowish sulfur granules , of < 5 mm in size). Granules are crushed between two slides and smears are made . Gram-staining (Brown- Brenn modification): shows a 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 cells salts and polypeptides Granules of actinomycosis are hard & not emulsifiable which differentiates them from granules produced in other conditions.

11. Gram-positive filamentous bacilli

12. Laboratory Diagnosis Actinomyces species can also be detected directly from the sample by methods such as: Fluorescent antibody techniques using fluorescent tagged species specific monoclonal antibodies. Fluorescent in situ hybridizarion (FISH) using species specific probes. Histopathological staining- Hematoxylin -eosin and Gomori's stained tissue sections – reveal granules composed of eosinophilic clubs surrounding basophilic filaments and inflamatory cells such as neutrophils and foamy macrophages ( Sun -rays appearance )

13. Laboratory Diagnosis Culture = Pus containing sulfur granules are washed and cultured anaerobically at 37 C on media such as Thioglycollate broth: Growth of A. israelii resembles fluffy balls at the bottom of the tube, this can be differentiated from other species ( A. bovis produces uniform turbidity). Brain heart infusion (BHI) agar: it forms small spidery colonies at 48 hours which become enlarged and heaped up in 10 days. Species Identification -done when the culture isolate is 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 .

14. TREATMENT Actnomyces Penicillin is the drug of choice, given for 6-12 months duration to prevent relapse . Erythromycin or tetracycline -people with penicillin allergy. Surgical removal of the affected tissues may be required for extensive lesions.

15. Nocardia Nocardia species (named after Edmond Nocard , 1898) – Gram-positive branching filamentous bacilli similar to Actinomyces They differ from Actinomyces by being aerobic and acid-fast. They are environmental saprophytes found in soil and vegetations, ( and are primarily responsible for the decomposition of plant material)

16. Nocardia Because they are ubiquitous, isolation of these organisms from clinical specimens does not always indicate infection, may indicate colonization of the skin and upper respiratory tract or laboratory contamination. As they grow form branched filaments that extend along the agar surface ( substrate hyphae ) and into the air (aerial hyphae ). More than 50 species have been identified, but only few (nine) species are associated with human disease. Most common pathogens are- N. asteroides (Star-shaped colonies) N. brasiliensis

17. Pathogenesis= Nocardiosis Occurs worldwide Common among adult males . Natural habitat of Nocardia - Soil . Mode of Infection- acquired from soil either by: lnhalation of fragmented bacterial mycelia: leads to pulmonary nocardiosis --- may disseminate later . Associated with various species such as N. asteroides , N. cyriacigeorgica , N. farcinica and N. pseudobrasiliensis . Transcutaneous inoculalion : leads to various cutaneous and subcutaneous manifestations ( e.g , mycetoma ). This is often associated with various species such as N. brasiliensis , N. asteroides and rarely by N. otitidiscaviarum and N. transvalensis . Person-to-person spread is not known.

18. Pathogenesis= Nocardiosis Hisiological feature of nocardiosis - 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

19. Risk Factors Nocardiae are opportunistic pathogen . Occur frequently in immunocompromised conditions including AIDS, corricosteroid treatment, organ transplantation and tuberculosis. Cell-mediated immunity plays an important role in controlling the disease . But in immunocompetent individuals Nocardia spp. cause three types of skin infections Mycetoma - a chronic, localized, painless, subcutaneous Infection Lymphocutaneous infections Skin abscesses or cellulitis . Nocardiosis

20. Clinical Manifestations Pulmonary Nocardiosis Lobar pneumonia is the most common form Characterized by subacute onset of cough which thick, purulent sputum . rarely spread directly to adjacent tissues, leading to pericarditis , mediastinitis , laryngitis, tracheitis and bronchitis. Extra pulmonary (Disseminated) Nocardiosis Mostly dissemination occurs via blood . it typically presents as subacute abscess . Brain is the most common site followed by skin, kidneys, bone and muscle . Brain abscesses are usually supratentorial , often multiloculated and may be single or multiple. Meningitis is uncommon. Disseminated nocardiosis has a very poor prognosis.

21. Actinomycetoma Mycetoma - is a chronic granulomatous condition affecting subcutaneous tissues of feet and hands , characterized by: Subcutaneous nodular swelling Multiple sinuses Discharge containing granules Tendency of spreading to adjacent bones (bony deformities). Mycetoma usually affects people residing in tropical countries . The organism enters through skin on exposure to contaminated soil . Mycetoma is classified into two types: Eumycetoma : It is caused by fungi such as Madurella Actinomycetoma : It is caused by filamentous bacteria such as Nocardia , Actinomadura and Streptomyces somaliensis .

22. Actinomycetoma

23. Laboratory Diagnosis Specimen Depending on the site affected, various specimens collected such as sputum, pus from abscess and 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 actinomycetoma ). Direct Microscopy Gram-staining ( Brown- Brenn modification ): Reveals gram-positive branching and filamentous bacilli of width 0.5- 1 µm. They stain irregularly as their filaments are beaded. Sputum examination may show numerous lymphocytes and macrophages, some of which contain branching bacilli. Modified acid-fast staining using 1% sulfuric acid as decolorizer ( Kinyoun method): Nocardiae are partially acid fast and appear as branching and filamentous red colored acid-fast bacilli. Granules are washed several times in saline, crushed between two slides and observed under microscope. Granules are 0.5- 2 mm sized microcolonies composed of branching filamemous bacilli. Histopathology (H & E stain ) of the granules: Shows multilobulated with sun ray appearance.

24. Laboratory Diagnosis Culture Nocardiae are obligate aerobes Culture on various media such as brain heart infusion agar and Sabouraud dextrose agar (SDA) when incubated al 37 c for 2 days to 2 weeks. Colonies are creamy, wrinkled, pigmented ( orange or pink colored due to carotenoid-like pigments ) and adhere firmly to the medium. Some colonies possess abundant aerial growth and have a cotton wool hall appearance.

25. Laboratory Diagnosis Recovery of Nocardia from the samples containing Actinomadura done by: Using selective media: Buffered yeast extract containing polymyxin and vancomycin Sabouraud dextrose agar with chloramphenicol Paraffin bait technique : Media using paraffin as the sole carbon source have been shown to be effective for isolation of nocardiae from soil and clinical samples. Lowenstein- Jensen medium : Produces moist glabrous colonies (differentiates from mycobacteria ).

26. Laboratory Diagnosis Biochemical Identification Nocardia species as they are- N on-motile , Catalase positive Utilize a number of sugars oxidatively . Other biochemical tests are done for species identification such as: Decomposition of casein, hypoxanthine, Tyrosine Growth in lysozyme Acetamide utilization Growth at 45°C for 3 days Acid from rhamnose

27. Nocardia asteroides - Modified acid-fast staining

28. TREATMENT Nocardia Sulfonamides are the drug of choice. Cotrimoxazole ( sulfamethoxazole and trim ethoprim ) can be used as alternative. Duration of treatment is about 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.

29. Difference b/n Actinomyces & Nocardia

30. Actinomadura Actinomadura is the most frequent cause of actinomycetoma , (significantly more than the cases caused by Nocardia ). Important species Actinomadura madurae A . petlettieri Granules - usually white to yellow ( except in case of A. pellettieri that produces red colored granules ). Microscopy of the specimens containing granules reveals branching filamentous bacilli . Colonies have a molar tooth appearance after 48 hours in culture with sparse aerial growth. Speciation is on the basis of biochemical tests . Most isolates are susceptible to amikacin and imipenem .

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