GRAM-POSITIVE BACILLIPART THREE

MLAB 2434: MicrobiologyKeri 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

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. JEIKEIUMISOLATION & 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

Positive CAMP test for Group B Streptococcus

CAMP test with Listeria monocytogenes

DIFFERENTIATING CHARACTERISTICS BETWEEN L. MONOCYTOGENES AND OTHER GRAM POSITIVE BACTERIA

Species Catalase Hemolysis Motility

At R. T.EsculinHydrolysis

Growth6.5% NaCl

L. monocytogenes

Corynebacterium sp.

S. agalactiae

Enterococcus sp.

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Beta

None, alpha

Beta

None, alphabeta

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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.