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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

C. jeikeium Isolation & Identification BAP: 48-72 hours @ 35oC 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

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

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

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

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

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)

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 www.medtraining.org) “Umbrella” motility pattern (Left) typical for L. monocytogenes

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

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

References Engelkirk, P. G., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams & Willkins. http://en.wikipedia.org/wiki/Lactobacillus http://www.thefullwiki.org/Corynebacterium_diphtheriae http://quizlet.com/10262287/print/ 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.