Gram Positive Bacteria

(Bacilli) of Medical
Importance

Dr. Abdul Rahim, SO, NRLPD, ASI,NARC

[email protected]: 03455727072
 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 contaminants of clinical specimens)
– Non motile, no capsule, facultative anaerobic
– Often called ―diphtheroids‖
– Corynebacterium diphtheriae is the most significant
pathogen
– Other species may cause infections in the
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
– Metachromatic granules of
polymetaphosphates
 Strains of C.diphtheria
Depending on generation time and toxin
production C. diphtheria is classified into
following strains i.e
– C. diphtheria gravis generation time 60 min
– C. diphtheria intermidius generation time 100 min
– C. diphtheria mitis generation time 120 min
 Determinants of pathogenicity
• Colonization of mucous membranes of
respiratory tract.
• Toxin production depending on biotype and
lysotype.
 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
– Toxin is produced by certain strains
– Toxin is antigenic
Toxigenic Corynebacterium diphtheriae

• Gene for toxin production is carried by a lysogenic

phage.
• tox gene is regulated by iron concentration.
• Toxin consists of two fragments
– A: Active fragment
• Inhibits protein synthesis by blocking transfer of amino acids
from tRNA to the growing polypeptide chain
• 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
– Skin and cutaneous forms
 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
– Diphtheria is a contagious disease spread by direct
physical contact or breathing aerosolized secretions of
infected individuals.
 Psuedomembrane
• Diphtheria is an upper respiratory tract illness
characterized by sore throat, low fever, and an
adherent membrane (called a pseudomembrane on
the tonsils, pharynx, and/or nasal cavity.

• Diphtheria toxin produced by C. diphtheriae, can

cause myocarditis, polyneuritis, and other systemic
toxic effects. A milder form of diphtheria can be
restricted to the skin.
 Diphtheria
• Diphtheria is a rapidly developing, acute, febrile infection
which involves both local and systemic pathology.
• A local lesion develops in the upper respiratory tract and
involves necrotic injury to epithelial cells.
• As a result of this injury, blood plasma leaks into the area and
a fibrin network forms which is interlaced with rapidly-
growing C. diphtheriae cells.
• This membranous network, called a pseudomembrane, covers
over the site of the local lesion leading to respiratory distress,
even suffocation.
 C. diphtheriae:
Causative Agent of Diphtheria
• C. diphtheriae pseudomembrane
– WBC + organism
 Clinical Infections:
Non-Respiratory Disease
• Systemic infections
– 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
– More prevalent in the tropics

– Infections occur at the site of minor abrasions

– Maybe superinfected with Streptococcus pyogenes and/or

Staphylococcus aureus
 Treatment

• Infected patients treated with anti-toxin and antibiotics

– Anti-toxin produced in horses
– Antibiotics have no effect on circulating toxin, but prevent spread
of the toxin
• Penicillin drug of choice
 Laboratory Diagnosis
• Microscopic morphology
– Gram-positive, non–spore-forming
rods, club-shaped, can be beaded
– Appear in palisades and give
"Chinese letter" arrangement
– Produce metachromatic granules or
―Babes-Ernst‖ bodies (food
reserves) which stain more darkly
than remainder of organism

Corynebacterium diphtheriae
gram stain
 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

Tellurite: tellurium dioxide (TeO2).

 Laboratory Diagnosis:
Cultural Characteristics

• Blood agar plate (BAP) 24-48 hours at 37oC

small, grey translucent colonies
• Small zone of - hemolysis also seen
 Laboratory Diagnosis
• Identification
– Confirm identification by fermentation reactions
(glucose +)
– Catalase positive
– Urease negative
– Non-motile
 Laboratory Diagnosis
• Toxigenicity testing
– Elek test
– Immunodiffusion test to
detect toxin production
• Organisms are streaked at
right angle to the filter
paper dipped in anti toxin
on media with low Fe
content to maximize toxin
production.
• Identification of C.
diphtheriae does NOT
mean the patient has
dipheria. Must show the
isolate produces the toxin.
protease peptone agar + serum (horse or bovine)
1 and 4 positive
 Schick Test
• Test to detect antitoxin level in blood of person (previously
infected or immunized)
• Toxin is injected in intradermally in forearm.
• Same amount of heat treated antitoxin is injected intradermally
in other forearm.
• Observed for 24, 48, 72 hours, upto 6 days for appearance of
redness and swelling.
• Positive test: indicate no enough antitoxin is present to
neutralize anti toxin
• Negative test: anti toxin is present which has neutralized
the toxin
 C. diphtheriae
• Treatment: antitoxin
• Prevention: DPT immunization
( diphtheria, pertussis (whooping cough)
and tetanus)
 Listeria monocytogenes

• Small rod like ―chinese character‖

• No capsule, Facultative aerobic.
• Tumbling movement at 25 C not at 37 C
• Grow at low temperature i.e 4c
• Small and smooth colony on blood with a narrow
zone of beta-hemolysis
• Biochemical tests: Fermentation, Catalase + Oxidase
+
 Listeriosis
• Listeriosis is a serious infection caused by eating
foods contaminated with the bacterium Listeria
monocytogenes.
• This disease affects primarily pregnant women,
new-born, and adults with weakened immune
systems.
• Prenatal transmission occurs when organism
crosses placenta and post natal transmission
occurs through birth canal.
• Intrauterine infections are widely systemic and
results in premature abortion and fetal death.
 Symptoms
• Fever
• Muscle ache
• GI : Nausea, diarrhoea
• Pregnant women: mild flu-like symptoms, miscarriage,
still birth, premature delivery, or infected new-born.
• Lethargy
• Irritability
• If infection spreads to the nervous system: headache,
stiff neck, confusion, loss of balance, or convulsions.
• Listeria can cause Pneumonia, Meningitis, and Sepsis.
 Pathogenesis
• The bacterium is thought to attach to epithelial cells of the GI
tract by means of D-galactose residues on the bacterial surface
which adhere to D-galactose receptors on the host cells.

• The bacteria are then taken up by induced phagocytosis,

analogous to the situation in Shigella. An 80 kDa membrane
protein called internalin probably mediates invasion.

• After engulfment, the bacterium may escape from the

phagosome before phagolysosome fusion occurs mediated by
a toxin, which also acts as a hemolysin, listeriolysin O
(LLO).
 Pathogenesis
• Act A (encoded by actA) promotes the polymerization of
actin, a component of the host cell cytoskeleton, on the
bacterial surface.
• Within the host cell environment, surrounded by a sheet of
actin filaments, the bacteria reside and multiply.
• The growing actin sheet functions as a propulsive force which
drives the bacteria across the intracellular pathways.
• Then, the host cell is induced to form slim, long protrusions
containing living L. monocytogenes. Those cellular projections
are engulfed by adjacent cells, including non-professional
phagocytes such as parenchymal cells.
• By such a mechanism, direct cell-to-cell spread of Listeria in
an infected tissue may occur without an extracellular stage.
 Transmission

• The organism is distributed worldwide in animals, plants and

soil.
• Transmission to human by contact with animals or their feces
• Unpasteurized milk
• Contaminated vegetables.
• Endogenously from gastrointestinal tract
 Diagnosis
• Difficult to isolate
• Cold enrichment procedure: specimen held at
4 C and is periodically cultured
• ELISA
 Treatment/Prevention
• Ampicillin
• Erythromycin

• Infection can be prevented by adequate

cooking, and consuming pasteurized milk