1. Corynebacterium, Listeria, Erysipelothrix
Aerobic Gram-Positive Bacilli
(Non-Spore-Forming)

2. Corynebacterium: Characteristics
“club” “small rod”
Gram(+), small, club shaped rod, diphtheroid
Related to Mycobacterium, Nocardia – diaminopinelic acid, mycolic acid in cell wall
Some saprophyte, plants, animals; some disease in animals, humans
Majority NF of animals, humans

3. Corynebacterium: Genera - Human Disease
C. diphtheriae
Toxigenic strains – diphtheria; respiratory, cutaneous
Nontoxigenic strains – pharyngitis, endocarditis
Human pathogen
C. ulcerans
Respiratory, diphtheria
Veterinary pathogen
C. jeikeium (group JK)
Septicemia, endocarditis, wound infection, foreign body (catheter, shunt, prosthesis) infection
Human skin NF
C. pseudotuberculosis
Lymphadentitis, ulcerative lyphangitis, abscess formation

4. Corynebacterium: Lab Culture
370 C, 24 hours, pH , require oxygen
CBA – raised, translucent, gray colonies
Cystine Tellurite Blood Agar CTBA)
Enriched, selective, differential media
SRBC, bovine serum, cystine, tellurite
Tellurite inhibits RT normal flora
Corynebacterium colonies black, brownish due to tellurite reduction
Loeffler Coagulated Serum slant
Enriched media; serum, egg
Enhances formation metachromatic granules characteristic of Corynebacterium, visualized  by methylene blue stain

5. C. diphtheriae : Virulence Factors - Exotoxin
Must be  produced to cause diphtheria
Lysogenic bacteriophage carries "tox” gene
Trypsin  cleaves toxin - fragment A (toxic  activity), fragment B (tissue binding)
Toxin  inhibits protein  synthesis  by  ADP-ribosylating host cell ribosome elongation factor-2 (EF-2)
Systemic effects - heart failure, paralysis, adrenal hypofunction leading to Addison’s-like disease
C. ulcerans, C. pseudotuberculosis – some strains also make diphtheria-like toxin

6. C. diphtheriae: Exotoxin

7. Elek Plate DiphtheriaToxin Test
Immunodiffusion test in agar plate
Streak MO isolate on plate, place filter strip of antitoxin (antibody) perpendicular to streak
If MO makes toxin, at zone of equivalence (antigen + antibody) precipitate forms
Newer rapid tests:
Enzyme-linked immunosorbent assay (ELISA)
PCR DNA amplification to detect tox gene

8. C. diphtheriae : Virulence Factors
Capsule – protein, antiphagocytic.
Phospholipase D – dermonecrotic toxin, spreading factor
Antibiotic resistance – select for resistance during antibiotic therapy

9. Diphtheria: Respiratory
“leather” “skin”
MO in throats of healthy carriers
MO infects only humans, limited capacity to invade host
Disease starts as local infection of mucous membranes, causing pharyngitis
Local toxin results in degeneration epithelial cells
Inflammation, edema, pseudomembrane (fibrin clots, leukocytes, dead epithelial cells, MO) in throat

10. Diphtheria: Respiratory
Membrane obstructs airway, may result in suffocation
Recovery ~1 week, membrane dislodged, expectorated
Dangerous when toxin spreads systemic via blood:
heart (heart failure)
peripheral nerves (paralysis)
adrenal glands (hypofunction)

11. Diphtheria: Cutaneous
More  common tropical, subtropical areas
Skin contact with infected person
MO colonizes skin surface, enters subcutaneous tissue through break on skin (e.g. insect bite)
Papule, chronic non-healing ulcer
Exotoxin with systemic signs

12. C. diphtheriae: Treatment and Prevention
Treat by giving specific antitoxin (antibody) binds and neutralizes toxin
Penicillin or erythromycin to eliminate MO, stop toxin production
DTaP vaccine - immunize with toxoid (inactivate toxin by formalin) to elicit neutralizing antibody
Remains epidemic in developing countries due to inadequate vaccination

13. Other Corynebacterium: Infection and Disease
Normal flora of skin, URT
May occasionally cause disease, particularly immunocompromised
C.  ulcerans ­  toxigenic strains produce disease similar to diphtheria, , but less severe
C. jeikeium ­ those with underlying disease; bacteremia, meningitis, peritonitis, wound infection
C. pseudotuberculosis ­ those with exposure to animals (cattle, sheep, horses, goats, deer); pneumonia, lymphadenitis

14. Listeria monocytogenes: Characterisitcs
“blood cell” “produce” monocytosis in rabbits
G(+) short rods; singly, pairs, chains
Isolated from soil, water, vegetation, animals (bird, fish, insect)
Disease in wild, domestic animals; uncommon human infection
Facultative intracellular pathogen in humans; grows in macrophage, epithelial cells

15. L. Monocytogenes: Lab Culture
Aerobic, microaerophilic
Grows well on ordinary lab media
CBA – weak beta hemolysis
Able to grow slowly in cold (1°C)
Motile:
Peritrichous flagella, RT (umbrella motility)
Polar flagella, 370 C

16. L. monocytogenes: Lab ID
Catalase(+)
Oxidase (-)
TSI= A/A, H2S(-)
Esculin hydrolysis(+)
CAMP(+) reaction - enhanced “block” type hemolysis with Staphylococcus aureus
Grows in 6.5% NaCl
Serogroups based on O, H antigen

17. L. monocytogenes: Virulence Factors
Listeriolysin O (LLO) – hemolysin, pore forming toxin; escapes from phagocytic endosome to cytosol; required for intracellular growth
Phospholipase – also involve in escape of MO from endosome to cytosol
Listeric polysaccharide – capsule component

18. L. monocytogenes: Virulence Factors
Internalins – surface associated proteins; uptake MO into epithelial cells
ActA – surface protein; rearrangement of actin, propel MO through cell into adjacent cell (very invasive)
LPS-like substance – high fever in host
CNS Tropism - invasive

19. L. monocytogenes: Listeriosis
Found in environment - soil, decaying vegaetable, animal feces
Ingest contaminated animal products (milk, cheese, undercooked meat & poultry), unwashed vegetables (especially cabbage)
Disease usually mild, flu-like or GI distress
Individuals underlying chronic primary disorder
Disease - widely disseminated abscesses, granulomas; lesions may be found in liver, spleen, adrenals, respiratory tract, CNS, skin
Also meningitis with septicemia, pneumonia
High mortality may occur

20. Listeriosis
Uncommon disease - restricted to elderly, pregnant women, immunocompromised
Healthy children and adults – AS carriage
Pregnant moms
AS carriage, septicemia, neonatal disease
Pregnancy renders mom more susceptible (immune suppressed)
Effect on mom usually minimal, can be devastating for fetus or newborn
Immunocompromosed:
AS carriage, meningitis, septicemia, other infections

21. Listeriosis: Neonates
Early onset - infected transplacentally:
Septicemia, granulomatous foci in many organs
May result in abortion, stillbirth, premature delivery, death soon after birth
Baby born with cardio and respiratory distress, vomiting, diarrhea, meningitis, hepatosplenomegaly, skin lesions
Fatality rate 70-90% in untreated cases
Late onset – infected from genital tract during delivery:
Usually 1-4 weeks after birth
Manifested as meningitis
High fatality rate, but less than Early onset infection

22. L. monocytogenes: Treatment and Prevention
Poor prognosis in neonates
Infected moms treated as soon as disease is diagnosed to prevent transmission to fetus/neonate
Most drugs only bacteriostatic with Listeria - treatment of choice is combination penicillin and gentamycin
At risk individuals, avoid eating raw or partially cooked foods (soft cheese, turkey franks, cold cuts, vegetables)

23. Erysipelothrix rhusiopathiae
“red” “skin” “hair”; “red” “disease”
G(+) slender, pleomorphic, small bacilli; form filaments
Worldwide wild, domestic animals; swine main reservoir
Survives well in environment – water, soil, plant material
Animal disease widely recognized, human disease uncommon

24. Erysipelothrix rhusiopathiae: Lab Culture
Growth on CBA – alpha or gamma hemolysis, two types of colonies:
Smooth – rod, coccobacilli
Rough – long, thin filamentous rod
Usually 48 hours for growth
Microaerophilic, better growth in CO2 or AnO2

25. Erysipelothrix: Virulence Factors
Polysaccharide capsule – protect from phagocytosis
Adherence – especially heart valves
Neuraminidase – spreading
Hyaluronidase – spreading

26. Erysipelothrix: Clinical Significance
Primarily pathogen swine, turkey, fresh water fish
Swine - cutaneous, reddish rash; occasional complications of septicemia, endocarditis, arthritis
Humans - uncommon pathogen, zoonotic spread; erysipeloid most common form of disease

27. Erysipeloid Resembles Streptococcus erysipelas skin infection
Reddish-blue, edematous lesion at site of inoculation, following trauma (abrasion, wound) to hands
Occasionally disseminates -septicemia, endocarditis, arthritis
Occupation associated disease:
Butchers - handle contaminated meat, poultry,fish animals
Farmer, veterinarian – contact with infected animals

28. Erysipelothrix: Treatment and Prevention
Penicillin, tetracycline, erythromycin can be used
At risk workers - should cover exposed skin when handling animals or animal products
Swine herds - should be vaccinated

29. Class Assignment
Textbook Reading: Chapter 16 Aerobic Gram-Positive Bacilli
Corynebacterium
Listeria
Erysipelothrix
Key Terms
Learning Assessment Questions

30. Case Study - Gram(+) Coccobacilli (Listeria)
A 35-year-old man was hospitalized because of headache, fever, and confusion.
He had received a kidney transplant 7 months before, after which he had been given immunosuppressive drugs to prevent organ rejection.

31. Case Study - Gram(+) Coccobacilli (Listeria)
CSF was collected, which revealed a white-blood cell count of 36 cells/mm3 with 96% polymorphonuclear leukocytes, a glucose concentration of 40 mg/dl, and a protein concentration of 172 mg/dl.
A Gram stain preparation of CSF was negative for organisms, but gram-positive coccobacilli grew in cultures of the blood and CSF.

32. Case Study - Questions
1. What is the most likely cause of this patient’s meningitis?
2. What are the potential sources of this organism?
3. What virulence factors are associated with this organism?
4. How would this disease be treated? Which antibiotics are effective in vitro?