1. Ken B, Waites, M.D. F(AAM) Professor of Pathology Director of Clinical Microbiology Gram-positive Bacilli

2. Objectives To review and discuss: microbiological characteristics epidemiology virulence factors associated diseases laboratory detection of clinically important gram-positive bacilli. Organism groups to be discussed include: –Bacillus –Listeria –Erysipelothrix –Corynebacterium –Rhodococcus –Nocardia –Tropheryma

3. Bacillus Ubiquitous in soil worldwide Endospore-forming Aerobic/facultative anaerobic Gram-positive bacilli Can be gram-variable Often  hemolytic & motile – (except B. anthracis) Usually catalase positive B. cereus - Gray, spreading  -hemolytic colonies on SBA

4. Bacillus Endospores Thick walled structures formed by vegetative cells Resistant to radiation, chemicals, heat, desiccation (dipicolinic acid) Steam autoclaving necessary for destruction

5. Bacillus: Clinical Significance Common environmental commensals Occasional opportunistic pathogens & culture contaminants B. cereus group B. anthracis Others

6. Bacillus cereus Group Gastroenteritis –Heat-stable enterotoxin (emetic form) –Heat-labile enterotoxin (diarrheal form) Similar to V. cholera – stimulates cAMP watery diarrhea Contaminated rice, meat, vegetables Other Clinical conditions –Ocular infections after trauma Necrotic toxin, hemolysin, phospholipase C –IV-catheter-related sepsis, endocarditis, meningitis Immunocompromised persons and drug abusers

7. B. anthracis: Anthrax Rare in US due to control in animals Enzootic in middle east Transmitted by contact with animal products Spores remain infectious for years Usually cutaneous inoculation - slow healing ulcer, bacilli spread to lymphatics and bloodstream –20% mortality if untreated –Respiratory anthrax is usually fatal even if treated –GI anthrax also occurs in some Asian countries Excellent biological warfare agent Gram stain with spores eschar

8. Anthrax Pathogenesis Poly-D-glutamic acid (protein) capsule – antiphagocytic 3 component exotoxin Protective antigen (PA) - binds to cells, forms channel that permits EF and LF to enter Edema factor (EF) – adenyl cyclase causes fluid to accumulate at the site of infection and inhibit immune function Lethal factor (LF) -disrupts cell's functions, stimulates TNF-alpha and IL-1- beta, kills infected cells

9. Anthrax Vaccine Made from avirulent, nonencapsulated B. anthracis strain Requires series of injections and annual boosters Used in military

10. Listeria monocytogenes Gram-positive bacilli/coccobacilli Catalase-positive Motile Esculin positive  hemolytic Multiplies at 4 o C Umbrella motility

11. Habitat & Sources of Infection –Intestines of mammals, birds –Cheese, other dairy products –Vegetables –Undercooked meat Epidemiology & Disease –Asymptomatic carriage –Meningitis/sepsis in immunocompromised host –Foodborne illness – Neonatal infections (transplacental)

12. Listeria: Pathogenesis Organism adapted to grow at low temperatures Multiplies in cytoplasm of macrophages and epithelial cells Listeriolysin O (hemolysin) Asymptomatic carriage reservoir

13. Erisipelothrix rhusopathiae Gram-positive bacillus Common in animals Transmitted to humans by skin wound Occupational hazard in butchers, farmers, veterinarians Self-limited skin lesions with erythema & eruption May spread to bloodstream

14. Aerobic Actinomycetes Gram-positive, catalase-positive rods Occur in soil and decaying vegetation + mycolic acid- mycolic acid Mycobacterium Corynebacterium Nocardia Streptomyces Rhodococcus Tropheryma Others

15. Corynebacterium Gram-positive, curved pleomorphic rods “Chinese Letters” Aerobic/facultatively anaerobic Grow readily on Sheep blood agar Catalase & oxidase positive Usually non-motile Commensals on skin Some species pathogenic

16. Diphtheria (C. diphtheriae) Spreads by droplet Phospholipase D increases vascular permeability & promotes spread Phage-coded exotoxin (A&B subunits) acts on respiratory mucous membranes interfering with protein synthesis by inactivating EF-2 Pseudomembrane of fibrin, bacteria, epithelial & phagocytic cells impairs breathing Toxin spreads to heart, CNS, & adrenals Selective media (cysteine-tellurite) Identify in Public Health Labs Protect by toxoid vaccination - DaPT < 5 cases/yr in US

17. Other Pathogenic Corynebacteria C. jeikeium (JK) –Opportunistic bloodstream infections in bone marrow transplant recipients –Multiple antibiotic resistance - except vancomycin and tetracycline –Commonly carried on skin of hospitalized persons C. urealyticum –Occasional cause of UTI & stones (splits urea)

18. Nocardia Epidemiology –Ubiquitous in environment Inhalation Cutameous inoculation Pathogenesis –Cord factor – prevents phagolysosome fusion & intracellular killing in phagocytes –Catalase –Superoxide dismutase Filamentous gram-positive bacilli Partial Acidfast Smear

19. Nocardia Diseases Opportunist (HIV, Malignancy) –Pneumonia –Lung Abscess –Brain Abscess/Meningitis –Cellulitis/ulceration –Mycetoma Suppurative granulomas Fibrosis Necrosis Sinus tracts

20. Nocardia 6 year-old boy with HIV with 2-month-history of fever & cough.

21. Nocardia Lab Diagnosis Stained smears Culture - slow growth ~ 7 days in 5% CO 2 Several species associated with human disease Identify by PCR Nocardia colonies Middlebook 7H11 agar Aerial hyphae

22. Rhodococcus equi Gram-positive weakly acidfast bacilli that sometimes revert to coccoid forms Common in animals & environment Opportunistic lung infections in AIDS & transplant patients Facultative intracellular pathogen that survives in macrophages, leading to granuloma & abscess Slow-growing, mucoid salmon-colored colonies of R. equi on chocolate agar

23. Tropheryma whippeli Actinomycete etiologic agent of Whipple’s Disease – a malabsorptioin syndrome affecting the small bowel Organism cannot be cultured except in tissue culture Diagnose by histopathology & PCR