Actinomycetes

            Like fungi they form a mycelial network of branching filaments, like bacteria they are thin, possess cell walls containing muramic acid, prokaryotic nuclei. So actinomycetes are considered as transitional forms between bacteria and fungi. They are Gram positive, nonmotile, non sporing, non capsulated filaments that break up into bacillary and coccoid elements. Most are free living, particularly in soil. Human pathogenic actinomycetes belong to four genera : Actinomyces, Nocardia, Streptomyces & Actinomadura. Actinomyces is non acid fast and anaerobic or microaerophilic,  Nocardiais aerobe and acid fast,  Streptomyces& Actinomadura are aerobes and non acid fast. Some species of Streptomyces may cause disease rarely, but their importance is as the major source of antibiotics.

Actinomyces – are gram positive, nonmotile, non sporing, nonacid fast, 0.5-1µm in diameter, often grow in mycelial forms and  break up into bacillary and coccoid elements. they are facultatively anaerobes, best growth in the presence of 5-10% CO₂. The optimum temperature for growth is 35-37⁰C. They can be grown on brain heart infusion agar, heart infusion agar supplemented with 5% defibrinated horse, rabbit or sheep blood.

Pathogenicity – causes the disease actinomycosis. In man it is usually caused by A. israelli. They are commensals of mouth, therefore endogenous cause of disease. The disease is a chronic granulomatous infection occuring in human beings and animals. It is characterized by the development of indurated swellings, in the connective tissue, suppuration and the discharge of sulfur granules. Actinomycosis in human beings is an endogenous infection. The actinomyces species are normally present in the mouth, intestine, vagina as commensals. Actinomycosis in human beings occurs in 4 forms : (a) Cervicofacial – indurated lesions on the cheek and submaxillary regions. (b) Thoracic – lesions in the lung involving pleura and pericardium and spread outwards through the chest wall. (c) Abdominal – lesion around the cecum, neighbouring tissues and the abdominal wall, sometimes spread to the liver. (d) Pelvic – lesion on the pelvic region, associated with the use of intrauterine devices.

Laboratory Diagnosis- The specimens, pus, sinus discharge, bronchial secretions, sputum or infected tissues may contain innumerable sulfur granules.

●    Microscopy: Pus from suspected cases is shaken with sterile water in a tube. Sulfur granules settle to the bottom, and are removed with Pasteur pipette. Granules are crushed between two slides and stained with Gram and Ziehl-Neelson staining using 1% sulfuic acid for decolourization. The granules are seen to consist of gram positive hyphal fragments, surrounded by peripheral zone of swollen radiating club-shaped structures presenting a sun ray appearance.

●    Culture : Sulfur granules are thoroughly washed in sterile normal saline in  a petri dish or tube and crushed in a drop of saline with a glass rod. It is then inoculated on brain heart infusion agar, blood agar and in thioglycollate broth and incubated anaerobically and aerobically with 5-10% CO₂ at 35-37⁰C  for upto 14 days. The colonies of A. israelii are 0.5-2mm diameter, white to grey white, smooth, entire or lobulated resembling molar teeth. The identity of the isolate can be confirmed by direct flurescence microscopy and biochemical tests.

●    Biopsy: In haemotoxylin and eosin stained sections, the sulfur granules are deeply stained with haematoxylin except in the periphery which is stained by eosin, which shows short, radiate, club-like structures. On gram staining the filament are gram positive and periphery gram negative.

Treatment : Surgical removal of affected tissues with large doses of penicillin for 12-18 months are effective. Tetracycline, chloramphenicol, erythromycin, streptomycin may also be used.

Nocardia

         Resemble actinomyces morphologically but are aerobic, filamentous and acid fast. They are Gram positive, frequently found in soil and infections are exogenous. Infection with Nocardia cause cutaneous, subcutaneous or systemic lesions in humans. They readily grow on nutrient agar, Sabouraud Dextrose agar, brain heart infusion agar. The inoculated plates should be incubated at 36⁰C for upto 3 weeks. They form white, yellow, pink or brown colonies.

Pathogenicity

·         Nocardiosis : is characterized by tissue lesions and abscesses. Causative organism is N. asteroides, found in soil and water. In humans the primary infection site is lungs, also originate in skin and other organs. The disease usually occurs in immunosuppressed patients. Human-human transmission is rare, 85% mortality rate. Early diagnosis and treatment with sulfonamides in combination with triomethoprim reduces the mortality rate below 50%. Diagnosis based on finding the organism in sputum and microscopical examination.

·         Maduramycosis : Madura foot (Maduramycosis) : occurs mainly in the tropics, caused by a variety of soil organisms. The causative organism is N. asteroides. Organisms enter the body through breaks in the skin, especially in people who do not wear shoes. Initial pus filled lesions spread and form connected lesions that eventually become chronic and granulated. Uyntreated, the organisms invade muscle and bone, and the foot becomes massively enlarged. Madura foot is diagnosed by finding white, yellow, red or black granules of interwined hyphae in pus. Unless antibiotic therapy begins early in the infection and is sufficiently prolonged, amputation may be necessary. Keeping soil particles out of wounds prevents the disease.

Laboratory diagnosis - Diagnosis is by demonstration of branching filaments microscopically and by isolation in culture. Nocardia grow readily on ordinary media forming dry, granular, wrinkled colonies which produce pigments ranging from yellow to red. 

Treatment – Sulfonamides with or with out trimethoprim for 3 months or more are effective. Cortimoxazole, amikacin, cefotaxime also can be used.

This aerobic bacteria produces a substrate mycelium, ie., a mat of branching hyphae formed under the surface of agar medium. This mycelium may range from rudimentary to extensively developed. The substrate mycelium usually fragments into rod shape or coccoid cells. Some genera form an aerial mycelium that may give rise to conidiospores. Several genera possess mycolic acids and are acid fast species.

In the genus nocardia :

·         The peptidoglycan contains meso- diaminopimelic acid.

·         No glycine interpeptide bridges occur between the peptidoglycan chains.

·         The walls contain sugars arabinose and galactose.

They are saprophytes, opportunistic pathogens, causing nocardiosis and actinomycetoma in humans and animals.