Miscellaneous GPB knwopjffpj'w02.09.25.pptx

Actinomycetes and Nocardia

1. Chapter 29 Skin, Soft Tissue and Musculoskeletal Infections (B) Dr Sonal Saxena Dr Arpita Saxena

2. BACTERIAL INFECTIONS OF SKIN & SOFT TISSUES ANAEROBIC BACTERIAL INFECTIONS BACTERIAL MYCETOMA OTHER BACTERIAL INFECTIONS

3. ANAEROBIC BACTERIAL INFECTIONS OF SKIN Gas gangrene Clostridium perfringens Clostridium septicum Clostridium novyi Clostridium histolyticum This Photo by Unknown Author is licensed under CC BY-SA

4. ANEROBIC GRAM-POSITIVE RODS Genus Clostridium Gram-positive anaerobic bacteria with spores Motile (with peritrichate flagella) except C. perfringens and C. tetani type VI Stately motility Non-capsulated except C. perfringens and C. butyricum Classification Molecular classification: Based on 16SrRNA sequences Phenotypic classification: based on morphology, culture an biochemicals

5. Morphological and biochemical classification of Clostridia

6. Spores of Clostridia Wider than bacillary bodies Arrangement of spores:  Central (equatorial): C. bifermentans - spindle-shaped bacillus  Subterminal: C. perfringens - club-shaped bacillus  Oval and terminal: C. tertium - tennis racket-shaped bacillus  Spherical and terminal: C. tetani - drumstick appearance Sporulating clostridia (Source: Dept. of Microbiology, PIMS)

7. VIABILITY OF CLOSTRIDIAL SPORES

8. ANAERBIC GRAM- POSITIVE COCCI Peptostreptococci Normal flora of vagina, intestines and mouth Can grow well under 10% CO2 in aerobic atmosphere Infections Puerperal sepsis ( Peptostreptococcus anaerobius ) Other genital infections Wound infections, UTI Osteomyelitis Abscesses of internal organs ( P. magnus )

9. ANAEROBIC CULTURE METHODS CULTURE METHODS McIntosh and Filde’s jar Culture plates in a jar with lid tightened Catalyst: alumina pellets coated with palladium Indicator for anaerobiosis: reduced methylene blue 2) Anoxomat Automated microprocessor-controlled system For anaerobic, microaerophilic and capnophilic bacteria

10. Fig. 2.41 (a) and (b) McIntosh and Filde’s anaerobic jar (actual jar and line diagram)

11. CULTURE METHODS Fig. 2.42 Gaspak airtight anaerobic jar (Source: Microbiology Laboratory, PIMS) 3) Gaspak Method of choice; simple and effective Disposable envelope with chemicals that generate hydrogen and carbon dioxide on addition of water. Catalyst present

12. CULTURE METHODS 4) Pre-reduced anaerobic system (PRAS) 5) Anaerobic chamber (glove box) 6) Other methods of anaerobiosis Robertson’s cooked meat (RCM) medium Thioglycolate broth Robertson’s cooked meat medium- widely used medium for transport and culture of anaerobic organisms (Source: Dept. of Microbiology, PIMS) Growth in thioglycolate broth: (a)uniform turbidity, (b)surface growth and (c)granular turbidity

13. ANAEROBIC WOUND INFECTIONS

14. Gas gangrene A 50-year-old man met with a road traffic accident in which he sustained multiple fractures with open wounds and a crush injury of the leg. He was taken to the nearest hospital two days later, at which time, he was found to be in shock. He was started on supportive therapy and antibiotics. There was edema and pain at the site of injury with increased discolouration and a serous discharge. The area around the wound exhibited crepitus on palpation. Microscopic examination of the wound discharge showed the presence of thick, brick-shaped, gram- positive bacilli along with gram-positive cocci. Based on a provisional diagnosis of gas gangrene, immediate surgical treatment with extensive excision of the local area (to prevent further spread) and intravenous penicillin with clindamycin were given. The exudate was also inoculated into Robertson’s cooked meat medium. Clostridium perfringens and peptostreptococci grew in the culture. What is gas gangrene? Describe its pathogenesis.

15. GAS GANGRENE Gas gangrene is a rapidly spreading, edematous myonecrosis, occurring characteristically in association with severe crush injuries, leading to wounds of extensive muscle masses contaminated with pathogenic clostridia, particularly Clostridium perfringens . The disease has been referred to in the past as malignant edema, anaerobic (clostridial), myositis, and clostridial myonecrosis.

16. Etiology C. perfringens (60% of cases) C. novyi (40%) C. septicum (20%) C. histolyticum (10%) C. bifermentans (10%) C. fallax (5%) C. sordellii C. sporogenes C. tertium

17. Pathogenesis of gas gangrene

18. Pathogenicity Virulence factors C. perfringens strains are classified into five types, A to E, based on the toxins they produce. Typing is based on neutralisation tests using intracutaneous injections of specific antitoxins in guinea pigs or intravenous injection in mice.

19. CLINICAL PRESENTATION Incubation period – 7 hours to 6 weeks C. perfringens – 10–48 hours C. septicum – 2–3 days C. novyi – 5–6 days Increasing pain, tenderness and edema over the affected part Accumulation of gas – crepitus Untreated – profound toxemia and prostration

20. GAS GANGRENE Commonly seen in association with other clostridia Wound contamination Anaerobic cellulitis Muscle tissues are invaded – anaerobic myositis

21. Clostridium perfringens Large rectangular, stout gram-positive, capulated , non-motile bacillus, 4–6 micro x l µm Pleomorphic, single, chains Spores: Subterminal, Not produced in artificial media Autoclaving at 120°C for 15 minutes kills spores but are resistant to commonly used antiseptics and disinfectants.

22. VIRULENCE FACTORS Strains – five types A – E Toxins – most prolific of toxin producing bacteria Four major toxins – alpha, beta, epsilon and iota Alpha toxin – most important biologically, lethal, dermonecrotic and hemolytic

23. CLOSTRIDIUM PERFRINGENS Enzymes Neuraminidase destroys myxovirus receptors Hemagglutinin – active against RBCs Fibrinolysin Bursting factor – muscle lesions in gas gangrene Circulating factor – increase in adrenal sensitivity

24. CLOSTRIDIUM SEPTICUM Pleomorphic bacillus, motile due to the presence of peritrichate flagella Oval, central/subterminal spores Anaerobic. saccharolytic, abundant gas Four distinct toxins Alpha toxin is hemolytic, demonecrotic , and lethal Colonies are initially irregular and transparent, turning opaque on continued incubation Hemolysis occurs on horse blood agar Growth is promoted by glucose. It is saccharolytic and produces abundant gas

25. Clostridium novyi Large, pleomorphic gram-positive bacillus Oval, subterminal spores Strict anaerobe Four types (A–D) are recognised, based on the production of toxins Only type A is of medical importance, as it causes gas gangrene Type A – causes gas gangrene Large amounts of edema fluid, little or no observable gas, high mortality

26. Clostridium histolyticum Oval, subterminal, bulging spores Actively proteolytic organism Gas gangrene in humans Infection – exogenous/endogenous Exogenous – implanted foreign particles Endogenous – clean surgical procedures It forms at least five distinct toxins and is infrequently associated with gas gangrene in humans

27. LABORATORY DIAGNOSIS OF GAS GANGRENE Diagnosis of gas gangrene primarily clinical & confirmation by lab. The mere presence of clostridia in wounds does not constitute a diagnosis of gas gangrene. Bacteriological examination also helps to differentiate gas gangrene from anaerobic streptococcal myositis, which may be indistinguishable from it clinically in the early stages. In the latter, gram-stained films show large numbers of streptococci and pus cells but not bacilli, contrasting with the scanty pus cells and diverse bacterial flora seen in films from gas gangrene.

28. LABORATORY DIAGNOSIS OF GAS GANGRENE SPECIMEN Films – edge of affected area, tissue from necrotic area, exudate from deeper part of wound Exudate collected from depth of wound – collected by capillary pipette or swab Necrotic tissue/muscle fragments Blood cultures in C. perfringens and C. septicum infections However, C. perfringens bacteremia may occur without gas gangrene

29. MICROSCOPY C. perfringens: Brick-shaped, gram-positive bacilli without spores C. tetani/C. tetanomorphum : Slender bacilli with round, terminal spores C. novyi : Large bacilli with oval, subterminal spores C. septicum : ‘Citron bodies’ and boat- or leaf- shaped pleomorphic bacilli with irregular staining and no spores Methylene blue-stained culture of Clostridium septicum bacteria

30. CULTURE

31. CULTURE

32. CULTURE

33. NAEGLER REACTION C. perfringens grown on media containing Fildes peptic digest of sheep blood and human serum, with antitoxin on one half of the plate. Colonies on the half without antitoxin will be surrounded by a zone of opacity. No opacity around the colonies on the half of the plate with antitoxin.

34. CULTURE Reverse CAMP test

35. TREATMENT AND PROPHYLAXIS Surgery: Most important therapeutic and prophylactic Damaged tissue removed extensively and promptly Hyperbaric oxygen Antibiotics: metranidazole I/V – before surgery and every 8 hours Mixed aerobic and anaerobic infection: combination of metranidazole with amoxicillin and gentamicin. Passive prophylaxis – anti-gas gangrene serum given I/M 10,000 IU – C. perfringens 10,000 IU – C. novyi 5000 IU – C. septicum

36. BACTERIAL MYCETOMA Actinomycosis—Actinomycetes Nocardiosis—Nocardia Botryomycosis

37. A 20-year-old woman presented to the surgery outpatient department with a soft, painless swelling on her left jaw, which was covered with exudates from a discharging sinus. She gave a history of trauma to the jaw. On macroscopic examination of the pus, a light-coloured granule was found in the exudate. Gram stain of the granule showed the presence of filamentous, branching, gram-positive bacilli. The condition was diagnosed as cervicofacial actinomycosis, and the woman was treated with intravenous penicillin G (8 million units per day) for two weeks followed by oral penicillin. What is actinomycosis? What are the bacterial agents that cause mycetoma? How is it diagnosed?

38. MYCETOMA Mycetoma is a progressive chronic granulomatous infection of the skin and subcutaneous tissue. Causative agents and types 1. Eumycetoma (e.g., maduramycosis ): It is caused by fungi, namely Scedosporium ( Pseudallescheria ), Madurella mycetomatis , M. grisea , Acremonium spp., Exophiala spp., Aspergillus nidulans , and Fusarium spp. 2. Actinomycetoma : It is caused by actinomycetes, namely Actinomadura , Streptomyces , and Nocardia. 3. Botryomycosis: It is most commonly caused by Staphylococcus aureus, followed by Pseudomonas aeruginosa, Escherichia coli, and Proteus .

39. Epidemiology Mycetoma has been referred to in the Atharva Veda as padavalmika (foot anthill) . The disease was first reported by John Gill (1842) from Madurai, South India. Vandyke Carter (1860) established its fungal origin. It came to be known as maduramycosis . Mycetomas are seen chiefly in the tropics, though occasional cases have been reported from the temperate countries. In India, it is common in Tamil Nadu but rare in Kerala and northern parts of the country. The disease is endemic in regions with long dry seasons and short rainy spells such as Central and South America, West and East Africa, India, and Sri Lanka. Actinomycetoma occurs more commonly than eumycetoma. Persons engaged in agriculture are especially at risk.

40. Actinomycosis Actinomycosis is characterised by indurated swellings— chiefly in the connective tissue—suppuration and discharge of pale granules called sulphur granules . The lesions often have multiple sinuses. Actinomycotic mycetoma is a localised, chronic, granulomatous involvement of the subcutaneous less often, the hand and other parts, and presenting as a tumour with multiple discharging sinuses with sulphur granules. Actinomyces bovis causes ‘lumpy jaw’ in cattle.

41. Clinical Features Actinomyces israelii is the most common causative agent of actinomycosis Four main clinical types are seen: Cervicofacial: Indurated lesion on the cheek and submaxillary region Thoracic: Lesions in the lung, may involve the pleura and pericardium, spreading outwards through the chest wall Abdominal: Lesion is usually around the cecum, involving neighbouring tissues and abdominal wall Pelvic: Associated with the use of intrauterine device (IUD), abscess in bone and soft tissues with chronic draining sinuses to the exterior

42. Actinomyces israelii Causes disease of gums, gingivitis, periodontitis and sublingual plaques leading to root surface caries May present as mycetoma, treated with penicillin for several weeks

43. Actinomycetes Actinomycetes are bacteria that possess a cell wall containing muramic acid They have prokaryotic nuclei and are susceptible to antibiotics Superficially resemble fungi due to branching filaments Gram-positive filaments may break into bacillary or coccoid elements, non-motile, non- sporing , non-capsulated Most are free-living in soil

44. Actinomyces Anaerobic Actinomyces: Non-acid fast, anaerobic or microaerophilic; Arachnia , Bifidobacterium and Rothia Aerobic Nocardia: Aerobic, may be acid fast; Nocardia , Actinomadura , Dermatophilus , and Streptomyces Streptomyces may cause disease but they are a major source of antibiotics

45. Actinomyces (Ray Fungus) Ray-like appearance of organisms in granules Chronic granulomatous infection in humans and animals Indurated swelling, mainly in connective tissues, with suppuration, discharge from multiple sinuses Discharge has yellowish, soft, waxy granules called s ulphur granules Trauma, foreign body and poor oral hygiene may favour tissue invasion

46. Actinomyces

47. Laboratory Diagnosis of Actinomycosis Specimens collected are pus or tissues; in pulmonary disease, sputum Sulphur granules are demonstrated in pus Direct microscopy: Dense network of thin Gram-positive filaments surrounded by peripheral zone of swollen, radiating, club-shaped structures presenting a sun ray appearance; the clubs are antigen–antibody complexes

48. Laboratory Diagnosis of Actinomycosis Fig 43.1

49. Laboratory Diagnosis of Actinomycosis Isolation in culture : Sulphur granules or pus inoculated into thioglycollate liquid medium or streaked on brain–heart infusion agar incubated anaerobically at 37°C Liquid medium, A. israelii produces fluffy ball colonies at the bottom Solid medium, Actinomyces israelii produces spidery colonies in 48–72 hours, becomes heaped up, white, irregular smooth large colonies in 10 days Treatment : Penicillin, tetracycline for months supplemented with surgery

50. Nocardiosis A 60-year-old, HIV-positive man developed a nodule on his hand. His CD4 count was <200/mm3). He gives a history of trauma to his hand after which a nodule developed which became swollen, painful, warm, and red. On examination, the nodules were warm and tender. Pus was drained from the nodule, which showed fine, branching filaments, which were acid-fast (to 1% sulphuric acid). Culture on blood agar showed the growth of dry, buff-coloured colonies identified as N. asteroides . The patient responded to treatment with cotrimoxazole and amikacin. What is nocardiosis? What are the clinical forms in which nocardiosis can exist? How does the causative agent differ from Actinomyces ?

51. Nocardia Resembles Actinomycetes morphologically but it is aerobic Nocardia are gram-positive and some species like N. asteroides and N. brasiliensis are acid fast Found in soil and infection is exogenous Causes cutaneous, subcutaneous and systemic lesions Common species are N. asteroides , N. brasiliensis and N. caviae

52. Epidemiology Nocardiosis is an opportunistic infection in immunocompromised hosts caused by Nocardia. Nocardia can affect nearly every part of the human body including skin and skin structures, the pleural, or the pulmonary system, or it can be a disseminated disease impacting other organ systems. Pulmonary nocardiosis and disseminated forms of the infection can occur in patients deficient in T cell- mediated immunity.

53. CLINICAL FEATURES Clinical forms: Cutaneous: Local abscess, cellulitis or lymphocutaneous lesions, subcutaneous actinomycotic mycetoma Lymphocutaneous nocardiosis: Cutaneous with ascending regional lymphadenopathy. In severe cases, the lymph nodes may ulcerate to have weeping necrotic or purulent drainage. Systemic : Manifests as pulmonary disease, pneumonia, lung abscess or resembles tuberculosis Metastatic manifestation : May involve the brain, kidneys and other organs Systemic nocardiosis occurs more often in immunodeficient persons

54. Nocardia Morphology: Filaments, rod-shaped bacteria that do not produce spores, non-motile, catalase positive and weakly acid fast by Kinyoun’s acid fast staining method N. asteroides is most commonly involved in human disease Transmission is through contaminated soil and not from humans or animals

55. Laboratory Diagnosis of Nocardiosis Direct microscopy: Weakly acid fast; branching, beaded filaments in smears from tissues Isolation in culture : Grows on ordinary media forming dry, granular wrinkled colonies, producing pigment ranging from yellow to red TREATMENT Resistant to penicillin; cotimoxazole and minocycline are used Surgery is performed to remove mycetomas In immunocompromised, amikacin and cefotaxime are used

56. Botryomycosis- Mycetoma from Bacterial Causes Usually caused by Actinomyces israelii , A. bovis N. asteroides, N. brasiliensis, N. caviae Actinomadura madurae, A. pelletierii Streptomyces somaliensis Botryomycosis: Mycetoma-like lesion produced by Staphylococcus aureus and other pyogenic bacteria

57. OTHER BACTERIAL INFECTIONS Melioidosis —Burkholderia pseudomallei Glanders— Burkholderia mallei Lyme disease —Borrelia burgdorferi

58. Melioidosis Melioidosis, a glanders-like disease, epizootic in rodents in Southeast Asia, India, and North Australia. The disease came into prominence after the Vietnam war and hence is called the Vietnam time-bomb disease. In India, cases are reported from the western and southern belts. The infections usually spike following the rainy season. The name melioidosis is derived from melis , a disease of asses (glanders; see next section) and eidos , meaning resemblance. It is caused by Burkholderia pseudomallei .

59. Burkholderia pseudomallei Burkholderia are small, irregularly staining, gram-negative bacilli that are abundant in nature, occupying diverse ecological niches such as soil, plants, animals, and hospital environments. Many members of the genus can cause infections in humans and animals. Three species of Burkholderia that can act as human pathogens are: Burkholderia pseudomallei causes meliodosis . B. mallei causes glanders. B. cepacia causes nosocomial infections.

60. Pathogenesis B. pseudomallei produces two thermolabile exotoxins , one lethal and the other necrotising. Human infection is usually acquired by the contamination of wounds, skin abrasions, or inhalation . Soil and water containing the organism are the sources of infection. Underlying diseases (like diabetes mellitus andchronic liver disease) may increase the risk of infection.

61. Clinical features The human disease may take one of two forms: i . Acute: It may be a generalised infection presenting as acute septicemia , a subacute typhoid-like disease, or pneumonia with hemoptysis resembling tuberculosis. It has a high case fatality rate. ii. Chronic : There may be multiple caseous or suppurative foci, with abscess formation in the skin and subcutaneous tissues, bones, and internal organs. Latency: Long latency and reactivation may occur as the bacillus can survive intracellularly in the reticuloendothelial system.

62. Laboratory diagnosis Microscopy: Small, irregularly staining, gram-negative bacilli, bipolar ‘safety- pin appearance’ with methylene blue stain. Culture Dry, wrinkled colonies on blood and MacConkey agars. Selective medium : Ashdown’s selective agar (ASA) medium (gentamicin and crysta )l violet Biochemical tests: non-fermentative, oxidase positivity, esculin hydrolysis and reduction of nitrate, intrinsic resistance to polymixin B PCR test or an automated VITEK system Serology: Not much useful

63. Treatment Carbapenems (imipenem, meropenem), and ceftazidime are the drugs of choice in the acute phase of the disease. For maintenance therapy, cotrimoxazole, tetracycline, or amoxicillin-clavulanate are required for many months to prevent recurrence

64. Glanders Zoonotic disease caused by Burkholderia mallei . Glanders ( malleus , in Latin), is primarily a disease of equine animals—horses, mules, and asses—and is capable of being transmitted to other animals and human beings. Human infection is usually occupational, found among ostlers, grooms, and veterinarians. It may be acute or chronic and is protean in character, with localisation in the respiratory tract, skin, or subcutaneous tissues. Acute glanders: fever, mucopurulent nasal discharge, and severe prostration, is usually fatal within 7–10 days of onset. Chronic glanders: death within months; while the survivors remain carriers of the disease. While human infection is acquired only rarely from infected animals, laboratory cultures are highly infectious.

65. Burkholderia mallei S lender, non-motile, gram-negative bacillus, 2–5 × 0.5 mm stains irregularly with beaded appearance Aerobe and facultative anaerobe, growing on ordinary media under a wide temperature range Colonies: Small and translucent initially, become yellowish and opaque Biochemically inert

66. Burkholderia cepacia B. cepacia is increasingly being recognised as an opportunist pathogen, particularly for those with cystic fibrosis or chronic granulomatous disease, in whom it causes fatal necrotising pneumonia. It is nutritionally very versatile. It can grow in many common disinfectants and can even use penicillin G as its sole source of carbon. Hence, it is often associated with hospital-acquired infections. It is oxidase-positive and oxidatively breaks down mannitol, sorbitol, and sucrose. C an cause urinary, respiratory, and wound infections, peritonitis, endocarditis, and septicemia . It is inherently resistant to most antibiotics.

67. Lyme disease Most common vector-borne disease in the United States. It is a zoonotic disease caused by the spirochete Borrelia burgdorferi and is transmitted to humans through the bite of infected blacklegged ticks ( Ixodid ). This Photo by Unknown Author is licensed under CC BY-NC

68. Borrelia burgdorferi It is a spirochete with an outer membrane and inner membrane with a thin layer of peptidoglycan in between. It has a flexible cell wall, and the outer membrane lacks lipopolysaccharide. It is a microaerobic, motile spirochete with seven to 11 bundled perisplasmic flagella set at each end. It circulates between Ixodes ticks and a vertebrate host in an enzootic cycle. It is known to exist in ecological niches with deer and rodents, and mice can act as reserviours in some areas.

69. PATHOGENESIS

70. Clinical features

71. Laboratory Diagnosis Isolation of the organism is very difficult. Serology: Acute (IgM) and convalescent (IgG) antibody titers 2 weeks apart are helpful. However, seroconversion may occur after 4 weeks, or may occasionally be absent, causing a delay in diagnosis. Detection IgG titers alone indicate past exposure. PCR in CSF or synovial fluid is often positive in the later stages. Treatment: Doxycycline or ceftriaxone .

72. Non-venereal treponemal diseases Occur in endemic foci in several parts of the world in three distinct forms: ( i ) Bejel / siti or endemic syphilis (ii) Yaws (iii) Pinta (These are caused by treponemes and are discussed in Chapter 27.)