Misc gram +ve

Miscellaneous Gram Positive bacteria

1. Miscellaneous Gram Positive Bacteria Made By : Niteesh Kumar

2. Actinomycetes Actinomycetes are a family of bacteria that form long, branching filaments that resemble the hyphae of fungi. They are gram-positive, but some (such as Nocardia asteroides ) are also weakly acid-fast rods. Human pathogenic actinomycetes: Actinomyces, Streptomyces, Nocardia, Actinomadura

3. 1. Actinomyces 1 :- Soil saprophyte 2 :- Normal flora of oral cavity 3 :- A. israelii is most common pathogen amongst Actinomyces. 4 :- A. naeslundii and A. odontolyticus are rare pathogens. 5 :- In human they cause actinomycosis. 6 :- The name refers to ray-like appearance of the organism in the granules ( Actinomyces - meaning ray fungus).

4. Actinomycetes

5. Pathogenesis 1 :- Chronic suppurative and granulomatous infection characterised by multiple abscesses with formation of sinuses, discharge containing granules and on later stage; fibrosis and tissue destruction. 2 :- Mostly exogenous infection.

6. Clinical Manifestations Cervicofacial actinomycosis: 1. Most common form, 2. Painless, slow-growing, hard mass with cutaneous fistulas ( Lumpy jaw ) Rare form of actinomycosis : Pelvic form , abdominal form , disseminated form , brain abscess, bone destruction and soft tissue infections , dental caries and periodontal disease by A. naeslundii and A. odontolyticus .

7. Actinomycosis. Inflamed lesion with small sinus tract opening anterior to right ear. Yellowish “sulphur granule” can be seen at the opening

8. Laboratory Diagnosis Specimen :- Discharge from the sinuses or fistula, rarely bronchoalveolar lavage, sputum or tissue sections. Gram staining (Brown-Brenn modification): Shows a central mass of gram-positive filamentous bacilli, radiating peripherally with hyaline, club shaped end. Granules of actinomycosis are hard and not emulsifiable which differentiates them from granules produced in other conditions. Fluorescent antibody techniques using fluorescence tagged species specific monoclonal antibodies.

9. Culture :- Anaerobically at 37°C on the media Thioglycollate broth : A. israelii show fluffy ball appearance at the bottom of the test tube. A. bovis produce uniform turbidity. Brain heart infusion (BHI) agar : Small spidery molar tooth appearance of colony at 48 hrs which become enlarged and heap up in 10 days. Histopathological Slides :- Sun ray appearance using H&E Stain and Gomorie’s stain

10. Colony of Actinomyces species after 72 hours growth on brain– heart infusion agar, which usually yields colonies about 2 mm in diameter; they are often termed “molar tooth” colonies.

11. Granule of Actinomyces species in tissue with Brown and Breen stain. Original magnification ×400. Filaments of the branching bacilli are visible at the periphery of the granule. Such granules are commonly called “sulphur granules” because of their unstained gross yellow colour.

12. Actinomyces naeslundii in a brain abscess stained with methylamine silver stain. Branching bacilli are visible. Original magnification ×1000.

13. Treatment Drug of Choice :- Penicillin G Erythromycin or Tetracycline can be used

14. 2. Nocardia Gram-positive branching filamentous bacilli similar to Actinomyces; however, they differ from the later by being aerobic and partial acid-fast. Environmental saprophyte More tan 50 species are known but about 9 species are isolated in human disease.

15. Pathogenesis Nocardia survive in neutrophil by : Neutralisation of oxidants. Prevention of phagosome-lysosome fusion. Prevention of phagosome acidification. Infection is acquired from soil either by: Inhalation of fragmented bacterial mycelia - Pulmonary nocardiosis Transcutaneous inoculation of the bacteria Person-to-person spread is not known.

16. Clinical Manifestations Pulmonary nocardiosis : Lobar pneumonia Subacute onset of cough with thick, purulent sputum. Rarely spread directly to adjacent tissues, leading to pericarditis, mediastinitis, laryngitis, tracheitis and bronchitis. Extra pulmonary (Disseminated) Nocardiosis : Subacute abscess Brain is the most common site followed by skin, kidneys, bone and muscle Supratentorial, multiloculated, single/multiple brain abscess

17. Mycetoma :- chronic granulomatous condition affecting subcutaneous tissues of the feet and hand with : • Subcutaneous nodular swelling • Multiple sinuses • Discharge containing granules • Tendency of spreading to adjacent bones (bony deformities feet and hands ) Residents of tropical countries are more prone to mycetoma. Types are : 1) Eumycetoma – By fungi such as Madurella 2) Actinomycetoma – By Nocardia , Actinomadura

18. Actinomycotic mycetoma caused by Nocardia brasiliensis in right upper arm.

19. Laboratory Diagnosis Specimen :- Sputum, pus from abscess and granules. Gram staining (Brown-Brenn Modification) : - Gram-positive branching and filamentous bacilli of width 0.5- 1 μm irregularly stained due to beading. Modified acid -fast staining :- Nocardia appear as branching and filamentous red coloured acid-fast bacilli. Histopathology (H&E stain) of the granules :- Granules show multilobulated with sun ray appearance .

20. Petri dish culture of Nocardia brasiliensis , the cause of the disease, nocardiosis. After undergoing a 3-week incubation period, the culture gave rise to this wrinkled, roughly textured, beige-coloured colony.

21. From left to right you can see that the colonial growth of Nocardia asteroides, N. caviae, and N. brasiliensis

22. Culture :- Obligate aerobes that grow on various media such as brain heart infusion agar and Sabouraud dextrose agar (SDA) . At 37°C for 2-14 days. Creamy, wrinkled, pigmented (orange or pink coloured due to carotenoid-like pigments) colonies. Some colonies possess abundant aerial growth and have a cotton wool ball appearance.

23. Biochemical Tests Non motile Catalase positive Decomposition of casein, hypoxanthine, tyrosine Growth in lysozyme Acetamide utilization Growth at 45°C for 3 days Acid from rhamnose

24. Treatment Drug Of Choice : Sulfonamide Cotrimoxazole (sulfamethoxazole and trimethoprim) can be used as alternative. Aspiration or drainage of the abscesses should be carried out to limit the spread of infection.

25. 3. Actinomadura Most frequent cause of Actinomycetoma, Granules are usually while to yellow except in case of A. pellettieri that produces red coloured granules. Colonies have a molar tooth appearance after 48 hours in culture with sparse aerial growth. Most isolates are susceptible to amikacin and imipenem

26. Actinomadura meyerae colonies. A) A. meyerae cultivated from our patient's bone biopsy on blood agar; B) Gram staining of an A. meyerae colony.

27. 5. LISTERIA MONOCYTOGENES Listeria monocytogenes is a food-borne zoonotic pathogen that can cause serious human infections, particularly in neonates, pregnant women and elderly people.

28. Pathogenicity • It can grow in refrigerated food and can tolerate preserving agents. • Mode of transmission: Contaminated food (raw milk, MC) followed by vertical transmission (mother to fetus). • Intracellular survival: It is facultative intracellular organism. Survival inside the host cells is due to inhibition and lysis of phagosome by forming pores (mediated by listeriolysin O). • Direct cell-to-cell spread is due to host cell actin polymerization (mediated by listeriopods).

29. Clinical Manifestations • Infection in pregnancy: Before 20 weeks is rare, may lead to abortion, still birth. • Neonatal disease: Two types: (1) early onset and (2) late onset • Adult: ○○ Associated with steroid therapy, HIV, DM, malignancy (fludarabine treated) ○○ Bacteremia > meningitis ○○ MC cause of meningitis in kidney transplanted patient after 1 month ○○ Also causes Gastroenteritis: Following contaminated milk, meat and salad.

30. Lab Diagnosis Specimen :- CSF, blood and amniotic fluid. Gram-staining :- Gram-positive short coccobacilli, often confused with diphtheroids. Motility :- Tumbling type of motility at 25°C but non motile at 37°C Culture :- Blood agar , chocolate agar PALCAM agar (polymyxin, acriflavine, lithium chloride, ceftazidime, aesculin and mannitol)

31. Listeria

32. Listeria on blood agar

33. Growth improves if cultured in thioglycollate broth at 4°C (cold enrichment). CSF examination: It shows elevated pressure, increased protein, and increased lymphocyte count. Biochemical Tests :- Positive catalase & CAMP Test Ferments glucose, maltose, L-rhamnose and alpha methyl D- mannoside , producing acid without gas production. Can grow in presence of 10% NaCl and low pH . Anton test: Instillation to rabbit eye causes conjunctivitis

34. Treatment Ampicillin is the drug of choice, given for 2-3 weeks in combination with gentamicin for synergistic effect. Cotrimoxazole is given for patients with penicillin allergy. Cephalosporins are not effective. Prevention should be taken.

35. 6. TROPHERYMA WHIPPLEI Tropheryma whipplei is a gram-positive actinomycete not closely related to any known genus. It is the agent of Whipple’s disease affecting the small intestine. • Whipple’s disease is characterized by fever, abdominal pain, diarrhoea, weight loss and migratory polyarthralgia. • Mesenteric lymph nodes of the small intestine are primarily involved.

36. Laboratory Diagnosis • Histopathological staining of intestinal biopsy shows vacuoles within the macrophage containing PAS stain positive bacilli. • Culture of T. whipplei has been unsuccessful. • PCR targeting 16S rRNA can be done to identify the bacilli.

37. Whipple's disease. Light micrograph of a section through an intestinal villus of a patient with Whipple's disease. This disease is caused by the bacterium Tropheryma whipplei . Some of the bacteria (blue) are seen inside macrophages (purple)

38. Treatment • Penicillin, ampicillin, tetracycline, or cotrimoxazole for 1–2 years or doxycycline. • Hydroxychloroquine for 12 to 18 months.

39. 7. ERYSIPELOTHRIX RHUSIOPATHIAE • Gram-positive bacilli • Catalase negative, H2S positive • Causes erysipeloid skin lesion violaceous swelling with severe pain, but no pus. • Most common site is finger (called ‘seal finger’ and ‘whale finger’). • Treatment: DOC is penicillin G, however it is intrinsically resistant to vancomycin.

40. E. rhusiopathiae and seal finger

41. Continued….. Phenotypic and biochemical features of Erysipelothrix rhusiopathiae. (A) Gram stain of positive blood culture broth demonstrating Gram-positive bacilli. (B) Inset of image (A) highlighting Gram-positive bacilli appearing at times Gram negative (arrows). (C) Small, alpha- hemolytic colonies of E. rhusiopathiae growing on 5% sheep blood agar after 3 days of incubation. (D) Growth of E. rhusiopathiae isolate abuts 30-µg vancomycin disk demonstrating vancomycin resistance. (E) Hydrogen sulfide production is noted by formation of black precipitate when a triple sugar iron (TSI) Agar slant is inoculated with E. rhusiopathiae.

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