Nocardia

Nocardia (microbiology)

1. Nocardia By Jagrity singh M.Sc 3 rd sem

2. Classification Kingdom: Bacteria Phylum: Actinobacteria Class: Actinobacteria Order: Actinomycetales Suborder: Corynebacterineae Family: Nocardiaceae Genus: Nocardia

3. I ntroduction Named after Edmond Nocard , in 1888 described the organism in cattle with bovine farcy . First human case of nocardiosis was reported in 1890 by Eppinger . Members of the genus  Nocardia  are associated with the group of microorganisms known as the aerobic actinomycetes and belong specifically to the family   Mycobacteriaceae . The nocardiae contain tuberculostearic acids but differ from the mycobacteria by possession of shorter-chained (40- to 60-carbon) mycolic acids . They have a type IV cell wall, characterized by a peptidoglycan made up of  meso -diaminopimelic acid, arabinose , and galactose  

4. Habitat Ubiquitous environmental saprophyte Soil , organic matter, water Tropical and subtropical regions.

5. Nocardia is present as commensal In Humans Nocardia are found worldwide in soil that is rich with organic matter. In addition, Nocardia are oral microflora found in healthy gingiva as well as periodontal pockets. Most Nocardia infections are acquired by inhalation of the bacteria or through traumatic introduction.

6. Other Species of Nocardia Gram-positive bacteria . On microscopy have branching filamentous cells . The more common human pathogen are Nocardia asteroids sensu stricto , Nocardia farcinica , Nocardia nova, Nocardia brasiliensis , Nocardia pseudobrasiliensis , Nocardia otitidiscaviarum , and Nocardia transvalensis

7. Morphology • Nocardia sp. are aerobic, gram-positive bacteria which are filamentous, relatively slow growing, and variably acid fast. Branching , beaded, filamentous bacteria and non motile • Can cause " Sulfur granules " like actinomycosis , in nocardial mycetomas . • Stains acid fast in tissue unlike the Actinomyces .

8. Culture character Plate culture of the bacteria Nocardia asteroides grown on 7H10 agar plates at 37° C . M edia: Nutrient agar, sabouraur agar, brain heart in fusion agar, yeast extract malt extract agar. S pecimens with mixed flora can over grow the Nocardia colonies Selective media may increase yield: – Thayer-Martin agar with antibiotics – paraffin agar . – Buffered charcoal-yeast extract ( BCYE ) medium

9. Culture….. Norcardia forms white or yellow or pink brown colonies. N. madurae – leathery colonies with red pigment N. asteroides – star shaped colony Colonies are formed after 3 weeks of incubation at 36oc On LJ medium after 1-2 weeks of incubation, colonies resemble saprophytic mycobacteria (some are white, some are pigmented)

10. Biochemical characters Usually not necessary to identify Nocardia spp Casein hydrolysis test can be done to differentiate N. asteroides from N. brasiliensis N. asteriodes doesnot hydrolyse casein N. brasiliensis – hydrolyse casein Both N. asteroides and N . brasiliensis are urease positive.

11. Virulence Factors Virulent strains are relatively resistant to neutrophil mediated killing . Organisms in the logarithmic growth phase are more toxic to macrophages. Inhibit Phagosome-lysosome fusion more successfully in vitro, which gives rise to L-forms, which can survive in macrophages for days L-forms have been found human and animal infections and perhaps account for treatment failure.

12. Virulence factors The inability to be killed by normal white cells takes on additional significance in the immunoincompetant who have WBC dysfunction that tips the battle between host and pathogen in favor of the Nocardia . Patients with CGD have increased risk for Nocardia infections, a double whammy where the patients cannot generate an oxidative burst and some strains have the ability to make superoxide dismutase.

13. S pecies tissue tropism's – N. asteroides complex including N. farcinica cause 80% of noncutaneous invasive disease and for most systemic and CNS disease . – N. brasiliensis : cutaneous and lymphocutaneous disease . – N. pseudobrasiliensis : systemic infections, including the CNS . – N. transvalensis and N. otitidiscavarium : Noncutaneous disease

14. Clinical presentation of Nocardia Overall, 80% of nocardiosis cases present as invasive pulmonary infection, disseminated infection, or brain abscess, 20 % present as cellulitis . Lymphocutaneous syndrome • Pulmonary :Pneumonia • CNS : Brain abscess • Disseminated disease CNS Eyes (particularly the retina Keratitis ), Skin & subcutaneous Kidneys , Joints , bone Heart

15. Clinical Syndromes: Mucocutaneous Mycetoma : a chronic progressive, destructive disease , occurring days to months after inoculation • located distally on the limbs (classically the foot ) and presents with – Suppurative granulomata – progressive fibrosis and necrosis – sinus formation and destruction of adjacent structures , – macroscopically visible infective granules – Mimics fungal mycetoma and actinomycetomata ( due to actinomycete ).

16. P ulmonary Clinical Presentations: – endobronchial inflammatory masses pneumonia – lung abscess, – cavitary disease – empyema – pneumonia (often progressive in HIV) – it can invade through surrounding tissues like actinomycosis

17. Pulmonary disease Pneumonia Subacute (more acute in immunosuppressed ) Cough ** Small amounts of thick, purulent sputum Fever , anorexia, weight loss, malaise • Endobronchial inflammatory mass • Lung abscess • Cavitary disease • Inadequate therapy =Progressive fibrotic disease • Cerebral imaging, should be performed in all cases of pulmonary and disseminated nocardiosis

18. CNS : Brain abscess Insidious presentations : mistaken for neoplasia !!! • Granulomatous , abscesses • Cerebral cortex, basal ganglia and midbrain • Less commonly: spinal cord or meninges . • However, cerebral biopsy: considered early in immunocompromised

19. LABORATORY DIAGNOSIS Specimens : Sputum , CSF, blood , Tissue, pus or exudate with granules, body sites with an abscess condition, Bronchial washings, bronchial lavage fluids Transport/Storage: Onsite collections: Transport specimens to the laboratory immediately. Offsite collections: Refrigerate specimen. Specimens must be promptly transported to the laboratory, not to exceed 24 hours from the time of collection. However, delayed transport causes a delay of test results. Special Processing : Store at 4°C until specimen can be shipped. Ship within 24 hours.

20. Diagnosis Macroscopic examination: The initial macroscopic inspection should include examination for the presence or absence of granules, and if granules are present, they should be carefully washed in sterile saline, crushed, and examined microscopically. These granules are most often seen in infections with N. brasiliensis but also can be produced by other Nocardia species and with Actinomyces species Microscopic examination: Gram sataining = gram-positive, thin, branching, filamentous organisms, usually in a background of purulence with many polymorphonuclear leukocytes. Nocardia are not so easily seen in tissue biopsies stained by the Gram or modified Ziehl - Neelsen methods, but may be seen in preparations stained by the Gram- Weigert or Gomori methenamine silver methods .

21. With the modified Kinyoun acid-fast stain using weak acid (1% sulfuric acid) for decolorization , the nocardiae are often seen as partially acid-fast filamentous bacilli (showing both acid-fast and non-acid-fast organisms). Histologically , Nocardia has delicate (< 2 microns in thickness) filaments with pronounced branching.

22. C ulture Nocardiae grow on blood agar, although growth is better on enriched media including Löwenstein -Jensen medium , brainheart infusion agar and Sabouraud's dextrose agar containing chloramphenicol as a selective agent . Growth is visible after incubation for between 2 days and 1 month; selective growth is favoured by incubation at 45°C . Colonies are cream, orange or pink coloured ; their surfaces may develop a dry, chalky appearance, and they adhere firmly to the medium On tap-water agar, Nocardia species have recursively branching hyphae with aerial hyphae .

23. Biochemical test hydrolysis of casein, tyrosine, and/or xanthine , (2) presence of urease , (3) utilization of rhamnose , and (4) positive resistance to lysozyme . Table 1: Hydrolysis Tests for differentiating  Nocardia  strain Casein hydrolysis L-tyrosine hydrolysis Xanthine hydrolysis N. asteroides complex, N. farcinica, or N. nova - - - N. brasiliensis + + - N. otitidis - + - N. caviae - - + Streptomyces or Nocardiopsis + + +

24. Chemotaxonomic methods Whole-cell hydrolysates examined for the presence of  meso -diaminopimelic acid and specific carbohydrates are useful tools in the identification of the nocardiae in laboratories that can perform these tests. Evaluation of the chromatographic patterns of  p - bromophenacyl esters of mycolic acids by HPLC may be helpful in identifying isolates resembling  Nocardia  to the genus level. However, most species of the nocardiae show similar mycolic acid patterns by HPLC and are difficult, if not impossible, to characterize to species. Gas-liquid chromatography of the short-chain fatty acids has also been used for identification of  Nocardia

25. Serology   I mmunodiffusion techniques to detect circulating antibodies  Complement fixation test were also shown to be nonspecific but were able to detect antibodies in most of the serum samples tested from immunocompromised patients  Immunoblot and enzyme-linked immunosorbent assays which detect antibodies to specific high-molecular-weight proteins that appear to be common in various  Nocardia  and  Actinomadura  species have been developed. These antigens do not react with antibodies produced in response to  Mycobacterium tuberculosis  infections

26. Molecular technology PCR83,84 and 16S rRNA sequencing are the most reliable for giving a precise spp. DNA probe  DNA sequencing Pyrosequencing Ribotyping Direct detection in paraffin-embedded tissue and clinical samples

27. Antibiotic Resistance of  N.asteroides  group members   Tobramycin Cefamandole or Cefotaxime Erythromycin N. asteroides  complex Variable Resistance Sensitive Resistant N. farcinica Resistant Resistant Resistant N. nova Resistant Sensitive Sensitive

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