Download Gram Positive Bacilli- Non-Spore Formers (Catalase Positive)

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–sporeforming 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–sporeforming 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

Clinical Infections
Septicemia
 Meningitis
 Bacteremia
 Pulmonary disease


Populations Affected
Immunosuppressed
 IV drug users
 Recent invasive procedure

JEKEIUM
C. JEIKEIUM
ISOLATION & IDENTIFICATION




BAP: 48-72 hours @ 35oC in ambient air or 5% CO2 small, graywhite 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)


Superoxide dismutase


damages host cell membrane
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–sporeforming 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. MONOCYTOGENES AND OTHER GRAM POSITIVE
BACTERIA
Hemolysis
Species
Catalase
Motility
At R. T.
Esculin
Hydrolysis
Growth
6.5% NaCl
L. monocytogenes
+
Beta
+
+
+
Corynebacterium sp.
+
None, alpha
=/+
=
+/=
S. agalactiae
=
Beta
=
=
=/+
Enterococcus sp.
=
None, alpha
beta
=
+
+
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.