1. Gram-Positive Bacilli Part three
MLAB 2434: Microbiology
Keri Brophy-Martinez

2. Corynebacteria Significant Corynebacterium species C. diphtheriae
C. xerosis
C. pseudodiphtheriticum
C. pseudotuberculosis
C. jekeium
C. ulcerans

3. Corynebacterium Species: General Characteristics
Found as free-living saprophytes in fresh and salt water, in soil and in the air
Members of the usual flora of humans and animals (often dismissed as contaminants)
Often called “diphtheroids” or “corneforms”
Corynebacterium diphtheriae is the most significant pathogen
Other species may cause infections in immunocompromised hosts

4. Corynebacterium Species: General Characteristics
Morphology
Gram-positive, non–spore-forming rods
Arrange in palisades: “L-V” shape; “Chinese characters”
Pleomorphic: “club-ends” or coryneform
Beaded, irregular staining

5. C. diphtheriae: Agent of Diphtheria
Toxigenic Corynebacterium diphtheriae
Worldwide distribution but rare in places where vaccination programs exist
Exotoxin, Diphtheria toxin, as the virulence factor
Not all C. diphtheriae strains produce toxin
Disrupts protein synthesis
Triggers cell lysis

6. Toxigenic Corynebacterium diphtheriae
Toxin consists of two fragments
A: Active fragment
Inhibits protein synthesis
Leads to cell/tissue death
B: Binding
Binds to specific cell membrane receptors
Mediates entry of fragment A into cytoplasm of host cell

7. Clinical Forms of Diphtheria
Respiratory
Acquired by droplet spray or hand to mouth contact
Non-immunized individuals are susceptible
Non-respiratory
Systemic form
Toxin is absorbed in the blood stream and carried systemically
Affects the kidneys, heart, and nervous system
Death occurs due to cardiac failure
Cutaneous form
Seen in tropical geographic areas
Infections occur at the site of abrasions
Associated with animal contact & unpasteurized dairy products

8. C. diphtheriae: Causative Agent of Diphtheria
Respiratory disease–diphtheria
Incubation period–2 to 5 days
Symptoms: sore throat, fever, malaise
Toxin is produced locally, usually in the pharynx or tonsils
Toxin causes tissue necrosis, can be absorbed to produce systemic effects
Forms a tough grey to white pseudomembrane which may cause suffocation

9. C. diphtheriae: Causative Agent of Diphtheria
C. diphtheriae pseudomembrane
WBC + organism

10. C. diphtheriae: Treatment
Infected patients treated with anti-toxin and antibiotics
Anti-toxin produced in horses
Binds the circulating toxin
Antibiotics have no effect on circulating toxin, but prevent spread of the toxin
Penicillin drug of choice
DPT Immunization

11. Laboratory Diagnosis: Cultural Characteristics
Loeffler's slant or Pai's slant— Used to demonstrate pleomorphism and metachromatic granules ("Babes’ Ernst bodies“)
Growth on Serum Tellurite or modified Tinsdale exhibits brown or grayish→ to black halos around the colonies

12. Corynebacterium diphtheriae gram stain
Laboratory Diagnosis
Microscopic morphology
Gram-positive, non–spore-forming rods, club-shaped
Appear in palisades and give "Chinese letter" arrangement
Can be beaded
From the production of metachromatic granules
Corynebacterium diphtheriae gram stain

13. Laboratory Diagnosis: Corynebacterium diphtheria
Identification
Confirm identification by fermentation reactions (glucose +)
Catalase positive
Urease negative
Non-motile

14. Laboratory Diagnosis Toxigenicity testing Elek test
Immunodiffusion test
Organisms are streaked on media with lox Fe content to maximize toxin production.
Identification of C. diphtheriae does NOT mean the patient has dipheria. Must show the isolate produces the toxin.

15. Corynebacterium jekeium
Clinical Infections
Septicemia
Meningitis
Bacteremia
Pulmonary disease
Populations Affected
Immunosuppressed
IV drug users
Recent invasive procedure

16. C. jeikeium Isolation & Identification
BAP: oC in ambient air or 5% CO2 small, gray- white colony, nonhemolytic
Gram stain: pleomorphic, club-shaped GPR arranged in V forms or palisades
Key Biochemicals
Catalase= positive
Nitrate reduction= negative
Urea= negative
Sucrose= negative
Glucose= positive
Resistant to most antibiotics
Susceptible to vancomycin

17. Listeria monocytogenes: General Characteristics
Gram-positive, non–spore-forming rods
Only human pathogen in genus
Widespread in nature
Known to infect a wide variety of animals
Human exposure is limited; direct or indirect
Transient colonization occurs without disease

18. Listeria monocytogenes: Clinical Infections
Adults
Septicemia/meningitis in the compromised/elderly
Mild flu-like syndrome in pregnant women could be fatal to fetus
Ingestion of contaminated food (cottage cheese, coleslaw, chicken, hot dogs, lunch meat)
Neonatal
Early onset from intrauterine transmission results in sepsis; high mortality rate
Late onset manifests as meningitis; lower mortality rate

19. Listeria monocytogenes: Virulence Factors
Hemolysin ( Listeriolysin O)
damages host cell membrane
Superoxide dismutase
Resists toxic effects of the host
P60 surface protein
Induces phagocytosis thru adhesion and penetration

20. Laboratory Diagnosis: L. monocytogenes
Identification
Microscopic morphology
Gram Positive non–spore-forming coccobacillary, pairs or short chains
Colony Morphology
Grows well on blood agar; colonies produce a narrow zone of hemolysis similar to Group B Streptococcus
Small, round and translucent

21. Laboratory Diagnosis: L. monocytogenes
Grows well at 0.5° C to 45° C
Because of this temperature range, especially the cooler end of the range, this organism grows well in refrigerated products, such as cream, cheese, deli meats, etc.
Can sometimes be isolated after “cold enrichment” (hold broth at 4° C for several weeks and subculture)

22. Laboratory Diagnosis: L. monocytogenes
Identification
Catalase positive
Motility:
Motile at 25o C; "umbrella" type →
Tumbling motility in hanging drop preparations (this can be seen on Gram Stain Tutor at
“Umbrella” motility pattern (Left) typical for L. monocytogenes

23. Laboratory Diagnosis: L. monocytogenes
Identification
CAMP test
Produces a “block” type of hemolysis in contrast to “arrow”-shape produced by Group B Streptococcus
CAMP test with Listeria monocytogenes
Positive CAMP test for Group B Streptococcus

24. Differentiating Characteristics between L
Differentiating Characteristics between L. monocytogenes and Other Gram Positive Bacteria

25. References
Engelkirk, P. G., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams & Willkins.
Kiser, K. M., Payne, W. C., & Taff, T. (2011). Clinical Laboratory Microbiology: A Practical Approach . Upper Saddle River, NJ: Pearson Education, Inc.
Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.