Nocardia and An aerobic bacteria

1. İ. Çağatay Acuner M.D., Clinical Microbiologist, AssistantProfessor Department of MicrobiologyFaculty of Medicine, Yeditepe University, Istanbul cagatay.acuner@yeditepe.edu.tr Nocardia and Anaerobicbacteria

2. Nocardia (Actinomycetes)

3. Nocardia (Actinomycetes) • Nocardiae are strict aerobic rods that form branched filamentous form in tissues and culture. • These filamentous forms resemble the hyphae formed by filamentous fungi (molds), and at one time Nocardia was thought to be a fungus. • However, the organisms have a gram-positive cell wall and other cellular structures that are characteristic of bacteria. • Most isolates stain poorly with the Gram stain and appear to be gram-negative with intracellular gram-positive beads • The reason for this staining property is that nocardiae have a cell-wall structure similar to that of mycobacteria; "weakly acid-fast"

4. Nocardia (Actinomycetes) • Cord factor is an important virulence factor that facilitates intracellular survival • Nocardia can grow on nonselective laboratory media ; growth is slow, requiring 3 to 5 days of incubation. (white to orange colonies withaerial hyphae) • Most infections are caused by a relatively few species • Identification at the genus level, with in vitro susceptibility testing, is sufficient for the management of most patients.

5. Nocardia (Actinomycetes) • Biology, Virulence, and Disease • Gram-positive, partially acid-fast, filamentous rods; cell wall with mycolic acid • Strict aerobe capable of growth on most nonselective bacteria, fungal, and mycobacterial media; however, prolonged incubation (7 days or more) may be required • Virulence associated with ability to avoid intracellular killing • Catalase and superoxide dismutase: inactivate toxic oxygen metabolites (e.g., hydrogen peroxide, superoxide) • Cord factor: prevents intracellular killing in phagocytes by interfering with fusion of phagosomes with lysosomes • Primary disease most commonly bronchopulmonary (e.g., cavitary disease) or primary cutaneous infections (e.g., mycetoma, lymphocutaneous infection, cellulitis, subcutaneous abscesses) • Dissemination most commonly to central nervous system (e.g., brain abscesses) or skin

6. Nocardia (Actinomycetes) • Epidemiology • Worldwide distribution in soil rich with organic matter • Exogenous infections acquired by inhalation (pulmonary) or traumatic introduction (cutaneous) • Opportunistic pathogen, causing disease most commonly in immunocompromised patients with T-cell deficiencies (transplant recipients, patients with malignancies, patients infected with the human immunodeficiency virus, patients receiving corticosteroids) • Diagnosis • Microscopy is sensitive and relatively specific when branching, partially acid-fast organisms are seen • Culture is slow, requiring incubation for up to 1 week; selective media (e.g., BCYE agar) may be required for isolating Nocardia in mixed cultures • Identification at the genus level can be made by the microscopic and macroscopic appearances (branching, weakly acid-fast rods forming colonies with aerial hyphae) • Identification at the species level requires genomic analysis for most isolates

7. Nocardia (Actinomycetes) • Treatment, Prevention, and Control • Infections are treated with antibiotics and proper wound care • Trimethoprim-sulfamethoxazole is used for localized infections; combination of antibiotics such as amikacin with a carbapenem or broad-spectrum cephalosporin is used for severe, progressive disease; treatment for 6 weeks or more • Exposure cannot be avoided, because nocardiae are ubiquitous

8. Nocardia (Actinomycetes) Acid-fast stain of Nocardia species in expectorated sputum. In contrast with the mycobacteria, members of the genus Nocardia do not uniformly retain the stain ("partially acid-fast"). Gram stain of Nocardia species in expectorated sputum. Note the delicate beaded filaments.

9. Nocardia (Actinomycetes) Aerial hyphae of Nocardia. Orange colonies of Nocardia

10. Nocardia (Actinomycetes) Nocardiosis: Clinical syndromes Bronchopulmonary disease: pulmonary disease with necrosis and abscess formation; dissemination to central nervous system or skin is common Mycetoma: chronic, destructive, progressive disease, generally of extremities, characterized by suppurative granulomas, progressive fibrosis and necrosis, and sinus tract formation Lymphocutaneous disease: primary infection or secondary spread to cutaneous site characterized by chronic granuloma formation and erythematous subcutaneous nodules, with eventual ulcer formation Cellulitis and subcutaneous abscesses: granulomatous ulcer formation with surrounding erythema but minimal or no involvement of the draining lymph nodes Brain abscess: chronic infection with fever, headache, and focal deficits related to the location of the slowly developing abscess(es)

11. Nocardia (Actinomycetes) Cutaneous lesion caused by Nocardia

12. Nocardia (Actinomycetes) Treatment, Prevention, and Control Nocardia infections are treated with the combination of antibiotics and appropriate surgical intervention. Trimethoprim-sulfamethoxazole is used most commonly to treat localized infections. In patients with severe, progressive disease, a combination of antibiotics is recommended, such as amikacin with a carbapenem (e.g., imipenem, meropenem) or broad-spectrum cephalosporin. In vitro susceptibility tests can be used to guide the selection of antibiotics. Because nocardiae can disseminate and produce significant disease, therapy should be extended for 6 weeks or more. Whereas the clinical response is favorable in patients with localized infections, the prognosis is poor for immunocompromised patients with disseminated disease. Nocardiae are ubiquitous, so it is impossible to avoid exposure to them. However, bronchopulmonary disease caused by nocardiae is uncommon in immunocompetent persons, and primary cutaneous infections can be prevented with proper wound care.

13. Anaerobic, non-spore forming Gram-positive bacteria: Gram-positive cocci and rods • The anaerobic gram-positive cocci and non-spore-forming rods are a heterogeneous group of bacteria that characteristically colonize the skin and mucosal surfaces. • These organisms are opportunistic pathogens, typically responsible for endogenous infections and usually recovered in mixtures of aerobic and anaerobic bacteria. • Additionally, most of these anaerobes have fastidious nutritional requirements and grow slowly on laboratory media. • Thus the isolation and identification of individual strains are difficult and often time consuming. • Fortunately, the appropriate management and treatment of most infections with these organisms can be based on the knowledge that a mixture of aerobic and anaerobic organisms is present in the clinical specimen and does not require the isolation and identification of the individual organisms.

14. Anaerobic, non-spore forming Gram-positive bacteria: Gram-positive cocci and rods

15. Anaerobic, non-spore forming Gram-positive bacteria: Gram-positive cocci

16. Anaerobic, non-spore forming Gram-positive bacteria: Gram-positive cocci • Characteristics: • At one time, all clinically significant anaerobic cocci were included in the genus Peptostreptococcus. • More sophisticated methods have since been used to reclassify many of these species into six genera. • Biochemical separation of the different genera is generally unnecessary, and knowledge that an anaerobic coccus is associated with an infection is typically sufficient. • The anaerobic gram-positive cocci normally colonize the oral cavity, gastrointestinal tract, genitourinary tract, and skin. • Diseases: • They produce infections when they spread from these sites to normally sterile sites. For example, bacteria colonizing the upper airways can cause sinusitis and pleuropulmonary infections; bacteria in the intestines can cause intraabdominal infections; bacteria in the genitourinary tract can cause endometritis, pelvic abscesses, and salpingitis; bacteria on the skin can cause cellulitis and soft-tissue infections; and bacteria that invade the blood can produce infections in bones and solid organs . • Laboratory: • Confirmation of infections with anaerobic cocci is complicated by the following three factors: • (1) care must be taken to prevent contamination of the clinical specimen with the anaerobic cocci that normally colonize the skin and mucosal surface; • (2) the collected specimen must be transported in an oxygen-free container to prevent loss of the organisms; and • (3) specimens should be cultured on nutritionally enriched media for a prolonged period (i.e., 5 to 7 days). • Treatment and prevention: • Anaerobic cocci are usually susceptible to penicillin and carbapenems (e.g., imipenem, meropenem). • Specific therapy is generally indicated in monomicrobic infections; however, because most infections with these organisms are polymicrobic, broad-spectrum therapy against aerobic and anaerobic bacteria is usually selected.

17. Anaerobic, non-spore forming Gram-positive bacteria: Gram-positive rods • The non-spore-forming, gram-positive rods are a diverse collection of facultatively anaerobic or strictly anaerobic bacteria that colonize the skin and mucosal surfaces. • Actinomyces, Mobiluncus, Lactobacillus, and Propionibacteriumare well recognized opportunistic pathogens, • whereas other genera such as Bifidobacterium and Eubacteriumcan be isolated in clinical specimens but rarely cause human disease.

18. Diseases associated with some gram-positive anaerobes

19. Anaerobic, non-spore forming Gram-positive bacteria: Gram-positive rods

20. Anaerobic, non-spore forming Gram-positive bacteria: Gram-positive rods; Actinomyces • The actinomycetes are true bacteria, although they have in the past been considered to resemble fungi because they form branching filaments. • They are related to the corynebacteria and mycobacteria in the chemical structure of their cell walls. • It is important to differentiate them from fungi because infections with actinomycetes should respond to antibacterial agents whereas similar clinical presentations caused by fungi are resistant to antibacterials (and extremely refractory to treatment by antifungal agents). • This genus contains many species, some of which are important to humans as producers of antimicrobial agents. • A few are pathogenic to humans and animals; A. israelii is a key cause of actinomycosis.

21. Anaerobic, non-spore forming Gram-positive bacteria: Gram-positive rods; Actinomycesisraelii • Characteristics: • Gram-positive anaerobic filamentous branching rods. Non-sporing, non-acid fast. • Laboratory identification: • Forms 'sulfur granules' composed of a mass of bacterial filaments in pus. These can be identified by washing pus, squashing granules and observing in stained microscopic preparations. Gram-positive branching rods also visible in stained pus. Forms characteristic breadcrumb or 'molar tooth' colonies on blood agar after 3-7 days anaerobic incubation at 35°C. • Diseases: • Actinomycosis follows local trauma and invasion from normal flora. Hard non-tender swellings develop which drain pus through sinus tracts. Cervicofacial lesions are most common, but abdominal lesions after surgery and infection related to intrauterine contraceptive devices also occur. • Transmission: • A. israelii is part of normal flora in mouth, gut and vagina. Infection is endogenous. There is no person-to-person spread. • Pathogenesis: • Virulence factors not described. • Treatment and prevention: • Penicillin is the drug of choice. Prolonged treatment is required, accompanied by surgical drainage .

22. Anaerobic, non-spore forming Gram-positive bacteria: Gram-positive rods; Actinomycesisraelii

23. Anaerobic, spore-forming, Gram-positive rods

24. Anaerobic, spore-forming Gram-positive rods

25. Anaerobic, spore-forming Gram-positive rods:Clostridium • This genus contains many species of Gram-positive anaerobic spore-forming rods; a few are aerotolerant. • Widely distributed in soil and in the gut of humans and animals. • The spores are resistant to environmental conditions. • The major diseases associated with species of the genus are gangrene, tetanus, botulism, food poisoning and pseudomembranous colitis. • In each of these, the production of potent protein exotoxins is an important cause of pathology, and in several species the genes encoding toxins are carried by plasmids or bacteriophages.

26. Anaerobic, spore-forming Gram-positive rods:Clostridium perfringens • Characteristics: • Anaerobic Gram-positive rods; spore-forming, but spores rarely seen in infected material. More tolerant of oxygen than other clostridia. • Laboratory identification: • Hemolytic colonies on blood agar incubated anaerobically. Germination of heat-resistant spores (with subsequent toxin production) may be responsible for food poisoning.Five types of C. perfringens (A-E) identified on the basis of toxins produced; type A strains can be further divided into several serotypes. • Diseases: • Gas gangrene resulting from infection of dirty ischemic wounds. Food poisoning following ingestion of food contaminated with enterotoxin-producing strains. • Transmission: • Spores and vegetative organisms widespread in soil and normal flora of humans and animals. Infection acquired by contact; may be endogenous (e.g. wound contaminated from patient's own fecal flora) or exogenous (e.g. contamination of a wound with soil, ingestion of contaminated food). • Pathogenesis: • In ischemic wounds, production of numerous toxins and tissue-destroying enzymes allows organism to establish itself and multiply in wound. Local action of toxins produces necrosis thereby further impairing blood supply and keeping conditions anaerobic, and aiding spread of organism into adjacent tissues. Food poisoning results from the ingestion of large numbers of vegetative cells, which sporulate in the gut and release enterotoxin. • Treatment and prevention: • Gangrene requires rapid intervention with extensive debridement of the wound. Penicillin is the antibiotic of choice (alternatively metronidazole, clindamycin or imipenem). Hyperbaric oxygen may also be helpful. Food poisoning does not usually require specific treatment.

27. Anaerobic, spore-forming Gram-positive rods:Clostridium tetani • Characteristics: • Gram-positive spore-forming rod with terminal round spore (drumstick). Strict anaerobe. • Laboratory identification: • Grows on blood agar in anaerobic conditions as a fine spreading colony; 'ground glass' appearance (hand lens inspection of cultures important). • Diseases: • Tetanus (lockjaw). Severe disease characterized by tonic muscle spasms and hyperflexia, trismus, opisthotonos and convulsions. • Transmission: • Organism widespread in soil. Acquired by humans by implantation of contaminated soil into wound. Wound may be major (e.g. in war, in road traffic accident) or minor (e.g. a rose-thorn puncture while gardening). No person-to-person spread. • Pathogenesis: • Tetanus results from neurotoxin (tetanospasmin) produced by organisms in wound. Toxin genes are plasmid-encoded. The organism is non-invasive, but the toxin spreads from site of infection via bloodstream and acts by binding to ganglioside receptors and blocking release of inhibitory neurotransmitters. Causes convulsive contractions of voluntary muscles. • Treatment and prevention: • Antitoxin is available (hyperimmune human gamma globulin; tetanus immune globulin). Metronidazole (or penicillin) and spasmolytic drugs indicated. Prevention readily available and effective in form of immunization with toxoid. Usually given in childhood, but if immunization status of injured patient is unknown, toxoid is given in addition to antitoxin.

28. Anaerobic, spore-forming Gram-positive rods:Clostridium botulinum • Characteristics: • Anaerobic Gram-positive rods. Not easily cultivated.Produces most potent toxins known to man. Seven immunologically distinct toxins (A, B, Cα, Cβ, D, E and F) produced by different strains of C. botulinum. Types A, B and E (and in some cases F) are most commonly associated with human disease: serotypes A and B linked to a variety of foods (e.g. meat), serotype E especially associated with fish. • Laboratory identification: • Requires strictly anaerobic conditions for isolation. Grows on blood agar, but very rarely isolated from human cases of disease.Detection of the toxin or organisms in the food or detection of the toxin or organisms in the serum or feces of the patient, respectively, is the way of confirming the diagnosis. • Diseases: • Major pathogen of birds and mammals, rare in humans. Botulism acquired by ingesting preformed toxin. Disease entirely due to effects of toxin. Infant botulism results from ingestion of organisms and production of toxin in infant's gut. Associated with feeding honey contaminated with spores of C. botulinum. Wound botulism: toxin produced by organisms infecting a wound. Extremely rare. • Transmission: • Soil is the normal habitat. Intoxication most often by ingestion of toxin in foods that have not been adequately sterilized (e.g. home-preserved foods) and improperly processed cans of food.Toxin is associated with germination of spores. There is no person-to-person spread. • Pathogenesis: • Toxin released from organism as inactive protein and cleaved by proteases to uncover active site. It is acid stable and survives passage through stomach. Taken up throughstomach and intestinal mucosa into bloodstream. Acts at neuromuscular junctions inhibiting acetylcholine release. Results in muscle paralysis and death from respiratory failure. • Treatment and prevention: • Supportive therapy is paramount. Trivalent antitoxin is available. In the rare cases of infant and wound botulism (i.e. when the organism is growing in vivo), penicillin is effective. Prevention relates to good manufacturing practice. The toxin is not heat stable, therefore adequate cooking of food before consumption will destroy it.

29. Anaerobic, spore-forming Gram-positive rods:Clostridium difficile • Characteristics : • Slender Gram-positive anaerobic rod; spore-former; motile. • Laboratory identification: • Difficult to isolate in ordinary culture because of overgrowth by other organisms; selective medium CCFA (cycloserine-cefoxitin-fructose agar) may be helpful; however, mere presence of the organism is not indicative of infection. Diagnosis by detection of toxin in feces (i.e. immunoassay or tissue culture cytotoxicity assay). • Diseases: • Pseudomembranous colitis (antibiotic-associated diarrhea). Can be rapidly fatal especially in the compromised host. • Transmission: • Component of normal gut flora; flourishes under selective pressure of antibiotics. May also be spread from person to person by the fecal-oral route. • Pathogenesis: • Toxin-mediated damage to gut wall. Produces both an enterotoxin (toxin A) and cytotoxin (toxin B). • Treatment and prevention: • Oral vancomycin or metronidazole. Other antibiotics should be withheld if possible. Prevention of cross-infection in hospitals depends upon scrupulous attention to hygiene.

30. Anaerobic, non-spore-forming Gram-negative bacteria

31. Anaerobic, non-spore-forming Gram-negative bacteria; associated diseases

32. Anaerobic, non-spore forming, Gram-negative rods • Historically, all short Gram-negative anaerobic rods or coccobacilli have been classified in the genus Bacteroides and longer rods with tapering ends in the genus Fusobacterium. • Recent applications of new techniques to the Bacteroides have resulted in the definition of two additional genera: Porphyromonas and Prevotella. • The genus Bacteroides is now restricted to species found among the normal gut flora. • Prevotellacontains saccharolytic oral and genitourinary species, including P. melaninogenica (formerly B. melaninogenicus), which produces a characteristic black-brown pigment. • The genus Porphyromonascontains asaccharolytic pigmented species, which form part of the normal mouth flora (P. gingivalis) and may be involved in endogenous infection within the oral cavity. • The most important non-sporing anaerobe causing infection is B. fragilis, • although others are much more common (e.g. in gingivitis and other endogenous oral infections).

33. Anaerobic, non-spore forming, Gram-negative rods: Bacteroides • Characteristics: • Small pleomorphic Gram-negative rods or coccobacilli. Capable only of anaerobic respiration. Non-spore forming, non-motile. • Laboratory identification: • Grows on blood agar incubated anaerobically and in other media designed for isolation of anaerobes. Plates may require up to 48 h incubation at 35°C for colonies to become visible. Cultures have a foul odor due to the fatty acid end-products of metabolism. These can be used as identifying characteristics by analysis of culture supernates by gas-liquid chromatography (GLC). The major products of Bacteroides are acetate and succinate. Full identification in the diagnostic laboratory is based on biochemical tests and antibiogram. Commercial kits are available. • Diseases: • Intra-abdominal sepsis; liver abscesses; aspiration pneumonia; brain abscesses; wound infections. Infections often mixed with aerobic and microaerophilic bacteria. • Transmission: • Endogenous infection arising from contamination by gut contents or feces is most common route of acquisition. • Pathogenesis: • Little is known about the virulence factors of B. fragilis. A polysaccharide capsule and production of extracellular enzymes (e.g. enterotoxin) are important features. An anaerobic environment is essential and in mixed infections growth of aerobic organisms probably helps the growth of Bacteroides by using up available oxygen. • Treatment and prevention: • Metronidazole, imipenem, or beta-lactam-beta-lactamase inhibitor combinations used in therapy. Many strains produce beta-lactamases and thus susceptibility to penicillin and ampicillin is unreliable. Prevention of endogenous infection difficult; good surgical technique and appropriate use of prophylactic antibiotics important in abdominal surgery.

34. Dental Plaque &Odontogenic Infections

35. Dental Plaque &Odontogenic Infections A unifying hypothesis demonstrating a microbial shift from a plaque-free tooth surface and progression to supragingival and subgingival plaque organisms.