Gram-positive bacteria of

medical importance

Prof. Dr. Sabiha Pit

Block: General
Medical Microbiology
MD Program Year 2 Semester
1
Session 2014/2015

GRAM-POSITIVE
BACTERIA

Coccus

Coccus in clusters
Catalase positive

Bacillus

Coccus in chains
Catalase negative

Staphylococcus

Aerobic

Streptococcus

Coagulase positive

Corynebacterium
Bacillus
Listeria

Beta-hemolysis

Staphylococcus
aureus

Coagulase negative

Anaerobic

Streptococcus pyogenes
Streptococcus agalactiae

Alpha-hemolysis

Strept pneumoniae
Viridans Streptococcus

Staph epidermidis
Staph saprophyticus

Non-hemolytic

SIMPLIFIED CLASSIFICATION OF GRAM-POSITIVE

BACTERIA

Enterococcus faecalis
Streptococcus milleri

Clostridium
Actinomyces

A: Gram-positive cocci in clusters eg. Staphylococcus aure

B: Gram-positive cocci in chains eg. Streptococcus pyogen
C: Gram-positive diplococci with capsule eg. Streptococcus
D: Gram-positive rods eg. Bacillus sp. and Clostridium sp.

Streptococcus pyogenes

Enterococcus sp.

GRAM-POSITIVE
COCCI

Streptococcus pneumoniae

Staphylococcus aureus

Catalase Test
Catalase acts as a catalyst in the breakdown of hydrogen
peroxide to oxygen and water. This test differentiates
between the catalase-producing (Staphylococcus) and noncatalase producing (Streptococcus) organisms.
Method
Place a drop of hydrogen peroxide on a glass slide using a wire loop.
Using an applicator stick, swipe a colony and touch it onto the drop
of hydrogen peroxide OR
The test can also be performed by pouring hydrogen peroxide
solution over heavy growth of bacteria
on an agar and observing for the
appearance of bubbles.
Results

Active bubbles seen

: Catalase positive
No bubbles seen: Catalase negative

Catalase Test
Organism B

Organism A

Picture
A
Organism
A is catalasenegative
Organism B is catalase
positive.

Picture B

Catalase test performed on

Staphylococcus aureus

Coagulase test
Coagulase test
This is the main differential test. Coagulase is an enzyme that
converts fibrinogen in plasma to fibrin, thus producing
clumping when coagulase-positive staphylococci are mixed with
plasma in a slide coagulase test and clot formation in a tube
coagulase test.
Tube coagulase test
Mechanism: Extracellular free coagulase produced on culture
has thrombin-like activity and converts fibrinogen into fibrin.
Slide coagulase test
Mechanism: Bound coagulase (clumping factor) on the
bacterial cell surface reacts directly with fibrinogen to cause
aggregation of the Staphylococcus.

Picture A

Slide coagulase test: Picture B

Coagulase-positive
Staphylococcus shows
clumping of the cells when
mixed with plasma.

Tube coagulase test: Inoculate diluted human

plasma with Staphylococcus and incubate for 36 hours at 37oC. Clotting (gel formation) is seen
with coagulase-positive strains of
Staphylococcus (Staphylococcus aureus).

Picture

Picture A

Pictures A & B
Gram-stained smear of Staphylococcus: Staphylococcus are
m-positive cocci usually arranged in grape-like irregular clust

Staphylococcus aureus

Picture A

Staphylococcus aureus on
blood agar. Staphylococcus
aureus forms deep golden
yellow colonies. Various
degrees of hemolysis are
produced by Staphylococcus
aureus.

Picture B

Close-up view of
Staphylococcus aureus.

Picture A

A Gram-stained smear of
Staphylococcus aureus
shows
Gram-positive cocci in
Picture B
clusters.
Staphylococcus aureus:
Deep golden yellow, betahemolytic colonies on blood
agar.

Staphylococcus aureus
Laboratory characteristics:
Gram-positive cocci in clusters, -hemolytic golden yellow colonies on
blood agar, catalase positive, coagulase positive.
Normal habitat:
Skin especially the nose and perineum, spread by direct contact and air
borne routes.
Diseases
a. Toxin-mediated diseases
Staphylococcal scalded skin syndrome (SSSS is also called Ritters
disease) is due to the exfoliative toxins.
Bullous impetigo: a localized form of SSSS caused by specific strains of
toxin-producing Staphylococcus aureus (phage type 71).
Staphylococcal food poisoning : intoxication by the enterotoxin
present in the food.
Toxic shock syndrome: The disease is initiated with the localized
growth of toxin-producing strains in the vagina or a wound, followed by
the release of TSST-1 into the blood stream.

Staphylococcus aureus infections

b. Pyogenic infections
Impetigo, folliculitis, furunculosis, carbuncles and
wound infections.
c. Systemic infection (frequently associated with
bacteraemia).
Pneumonia, pyemia, septic arthritis, osteomyelitis
and endocarditis (esp. in drug addicts).
Treatment
Antibiotics of choice: Cloxacillin / flucloxacillin.
MRSA (methicillin-resistant Staphylococcus
aureus): Vancomycin, teicoplanin, or fusidic acid.

Staphylococcus epidermidis

Staphylococcus epidermidis on blood agar:

Staphylococcus epidermidis colonies on blood
agar are usually grey to white. Generally,
there is no hemolysis.

Staphylococcus epidermidis
Laboratory characteristics:
Gram-positive cocci in clusters, white colonies on blood
agar, catalase positive, coagulase negative.
Natural Habitat:
Skin, spread by contact.
Diseases:
Opportunistic pathogen associated with device-related
sepsis (eg. catheter-related sepsis, prosthetic valve
endocarditis, infection of artificial joints, shunt
infections, sternal wound osteomyelitis (This organism
produces extracellular slime that aids in the
colonization of plastic implants).
Treatment:
Similar to Staphylococcus aureus. This organism is
often multi-resistant to antibiotics.

Organism A
Organism B

Comparison between the colonial morphology of

Staphylococcus aureus and Staphylococcus
epidermidis.
Organism A is Staphylococcus aureus and organism
B is Staphylococcus epidermidis.

Picture A
A

Bacitracin disc

Streptococcus pyogenes :
Pin-point, beta-hemolytic
colonies on blood agar and is
sensitive to bacitracin.
STREPTOCOCCUS
PYOGENES
Gram-stained
smears of blood
cultures :
Gram-positive
cocci

Picture B

Streptococcus pyogenes
Laboratory characteristics:
Gram-positive cocci in chains, -hemolytic
colonies on blood agar, sensitive to bacitracin.
Normal habitat:
Human upper respiratory tract and skin. Spread by
air-borne droplets and by contact.
Diseases
Pharyngitis
- sore throat
Scarlet fever
- It is a complication of streptococcal pharyngitis
that
occurs when an infecting strain is
lysogenized by a temperate bacteriophage which
stimulates the
production of a pyrogenic
exotoxin.

Streptococcus pyogenes
Pyoderma (Impetigo)
- Pyoderma is a confined purulent infection of the
skin
that primarily affects exposed areas (face,
arms and
legs). Vesicles develop and become
pustules
(pus-filled vesicles) which then rupture
and form
crust.
Primarily seen in young
children.
Erysipelas
- Acute infection of the skin, local pain,
inflammation (erythema, warmth) lymph node
enlargement and systemic signs (chills, ever,
leukocytosis).

Streptococcus pyogenes
Cellulitis
- Unlike erysipelas, cellulitis typically involves
the
skin and deeper subcutaneous
tissues.
Necrotizing fasciitis
- An infection that occurs deep in the
subcutaneous
tissues, spreads along the
fascial planes and is characterized by
extensive destruction of muscle and fat.
Streptococcal toxic shock syndrome
- Multi-system involvement, progresses to
shock and multi-organ failure (kidneys,
lungs, liver and
heart).

Streptococcus pyogenes
Non-suppurative streptococcal disease (late sequelae)
a.Rheumatic fever
Rheumatic fever is characterized by inflammatory changes
involving the heart, (endocarditis, pericarditis, myocarditis)
joints
(arthralgia to frank arthritis with multiple joint
involvement in
migratory pattern), blood vessels and subcutaneous tissues.
b.Acute glomerulonephritis
It is characterized by acute inflammation of the renal
glomeruli with edema, hypertension, hematuria and
proteinuria.

Treatment
Penicillin.
Erythromycin or an oral cephalosporin can be used in patients allergic
to penicillin.

Picture A

Latex agglutination

Picture B

Close-up view of
Pictures A & B: Lancefield grouping
. There are six
agglutination
Lancefield grouping antisera: Lancefield groups A, B, C,
D, F and G. One drop of each antiserum is placed on
the card and a colony of the test Streptococcus is
mixed with the respective antisera eg.
When the test Streptococcus agglutinates when mixed
with antiserum group A, thus confirmed that the test
Streptococcus belongs to Lancefield group A.

Streptococcus agalactiae
Laboratory characteristics:
Streptococcus agalactiae is the only species that carries the group B
antigen (group-specific cell wall polysaccharide antigen) and
has type-specific capsular polysaccharides (Ia, Ib and II to VII). It
produces a narrow zone of -hemolysis, CAMP positive.
Habitat : It colonizes the lower gastrointestinal tract and the
genitourinary tract. Transient vaginal carriage of this organism has
been observed in 10-30% of pregnant women.
Clinical diseases
Most infections in newborns are acquired from the mother during
pregnancy or at time of delivery.
Neonates are at higher risk for infections if:
i. There is premature rupture of membranes, prolonged labour,
preterm delivery or disseminated maternal group B
streptococcal disease.
ii. Mother is without type-specific antibodies and has low
complement levels.

Streptococcus group B (Streptococcus

agalactiae) on blood agar.
Group B Streptococcus can be found as normal flora of
female
genital tract and is an important cause of neonatal
sepsis and
meningitis. This organism is -hemolytic and produces
zones of

CAMP TEST
B

A: Streptococcus agalactiae
B: Staphylococcus aureus
C: Streptococcus pyogenes

CAMP test is a test to id

Streptococcus agalactiae
(group B) based on the
formation of a substance
(CAMP factor) that enlarg
the area of hemolysis for
by Staphylococcus aureu
It is also frequently used
identify Listeria sp.

Streptococcus agalactiae
Early onset neonatal disease
A disease in neonates younger than 7 days of age.
It is characterized by bacteremia, pneumonia or meningitis.
Late-onset neonatal disease
A disease appearing between 1 week to 3 months of life, is
acquired from an exogenous source (mother or from other
infants).
The predominant manifestation is bacteremia with
meningitis.
Other group B Streptococcus infections include:
Urinary tract infection
Wound infection
Bacteremia
Treatment
Penicillin alone or a combination of penicillin and
aminoglycoside is frequently used in the management of
serious infections.

Streptococcus
pneumoniae is
sensitive to optochin

Viridans
Streptococcus is
resistant
to optochin

Sensitivity of Viridans streptococci and Streptococcus

pneumoniae
to optochin. Streptococcus pneumoniae shows a zone
of inhibition Zone of inhibition of growth P Optochin disc
around the optochin disc and Viridans streptococci are

Streptococcus pneumoniae vs Viridans

Streptococcus
Streptococcus
pneumoniae

Viridans
Streptococcus

hemolytic
colonies on blood
agar
Sensitive to optochin
Soluble in bile
Quellung reaction
positive

-hemolytic colonies
on blood agar
Resistant to
optochin
Insoluble in bile
Quellung reaction
negative

Picture A

Picture B

Picture A:
Direct Gram-stained smear of sputum showing
numerous Gram-positive diplococci, likely to be
Streptococcus pneumoniae
Picture B:
Close-up view of microscopic appearance of
Streptococcus pneumoniae.

Optochin
P

Streptococcus pneumoniae:
Alpha-hemolytic colonies on
blood agar and sensitive to
optochin.

Picture

Picture A

Quellung reaction:
Capsular swelling of
Streptococcus
pneumoniae.

P Optochin disc

Sensitivity to optochin:
Streptococcus
pneumoniae is sensitive to
optochin, there is a zone of

Streptococcus pneumoniae
Laboratory characteristics
Gram-positive coccus characteristically appearing in pairs
(diplococci).
Growth may be enhanced in CO2.
On blood agar, -hemolytic draughtsman colonies
that may
autolyse within 48hr at 35oC.
Catalase negative, sensitive to optochin and bile
solubility test positive.
Capsule will swell in the presence of specific antiserum
(Quellung reaction).
Habitat
Human respiratory tract. Transmission via droplet spread.
Diseases
Pneumonia, septicemia and meningitis, otitis media,
sinusitis
Treatment
Penicillin remains the antibiotic of choice but resistance is
increasing rapidly. Vaccine is available.

Viridans Streptococcus
Alpha hemolytic colonies on the blood agar.
Most species are commensals in the mouth.
Streptococcus mutans is strongly
associated with dental caries. Several
species are capable of causing infective
endocarditis.
Most strains are sensitive to penicillin.
Moderately resistant isolates may be treated
with penicillin + aminoglycoside while
highly resistant strains require a broad
spectrum cephalosporin or vancomycin.

Viridans Streptococcu

Picture B:

Alpha-hemolytic
colonies, resistant to opto

Picture A:

A direct Gram-stained
smear shows Gram-positive cocci in
chains

Gram-positive bacilli
Non-spore forming
bacilli
Corynebacterium
diphtheriae
Listeria
monocytogenes
Corynebacterium diphtheriae

Spore forming grampositive bacilli

Bacillus sp (aerobic)
Clostridium sp
(anaerobic)

Bacillus s

A Gram-stained smear of B
anthracis. Spores are seen.

Picture A

Gram-stained smear of Bacillus

cereus: Gram-positive bacilli in long
chains and no spores seen in
young cultures.

Bacillus cereus
Laboratory characteristics:
Bacillus cereus is a large aerobic Gram-positive sporeforming rod. It produces hemolysis on blood agar.
Transmission:
Bacillus cereus spores are found on many foods, especially rice
and vegetables. Infections occur following ingestion of the
organisms or toxin.
Diseases
Food poisoning: emetic and diarrheal forms.
Ocular infections following trauma to the eye.
Other opportunistic infections : intravenous catheterrelated sepsis.
Treatment
Majority of the illness is short-lived and self-limiting and
antibiotic treatment is not indicated. Bacteremia should be
treated with gentamicin, vancomycin, ciprofloxacin or
clindamycin.

Close-up view of the spore

vegetative cells.

Picture A

Spore stain: The

endospores are stained
green (malachite green)
and the vegetative (carbol
fuchsin) cells are stained
red.

Picture B

Pictures A and B

acillus cereus on blood agar: spreading, gray and

beta-hemolytic colonies.

A Gram-stained smear of Clostridium

sp: large Gram-positive bacilli but older
cultures may stain irregularly.

Clostridium botulinum produ

subterminal spores.
Picture

Fig 19b

Picture A

The spores (as indicated by

arrows) are usually wider than
the diameter of the bacillus in
which they formed. Clostridium tetani
form round terminal spores giving
characteristic drum-stick

Clostridium perfringens
Laboratory characteristics:
Anaerobic Gram-positive rods and spore-forming. Hemolytic
colonies on blood agar. Toxin is detected by Naglers test.
Natural habitat:
It is found widespread in soil and normal gut flora of
humans and animals. Infection acquired by contact, may be
endogenous (eg. from patients own flora) or exogenous
(wound contaminated with soil, ingestion of contaminated food).
Disease:
Gas gangrene and food poisoning (results from ingestion of
large numbers of vegetative cells, sporulate in the gut and
release the enterotoxin).
Treatment:
Extensive debridement of wounds. Penicillin is the
antibiotic of choice (alternatively metronidazole). Food
poisoning does not require specific treatment.

Pictures A and B:

Clostridium
perfringens: Double zone h
sensitive to metronidazole
MT
Z

CLOSTRIDIUM
PERFRINGENS

M
T
Z

Picture C:

Gram-stained sme
shows Gram-positive bacillus w
spores.

Naglers reaction

Presence of
antitoxin

No antitoxin

When Clostridium perfringens is grown on a medium

containing egg yolk (lecithin), the enzyme(lecithinase)
activity can be detected as opacity around the line of
growth. On the left side of the plate, no opacity develops,
as antitoxin previously applied to this half of the plate
has neutralized the toxin.

Clostridium tetani
Laboratory characteristics:
Gram-positive spore-forming rod with terminal round
spore (drumstick). It is a strict anaerobe and grows on
blood agar as fine spreading colonies.
Natural habitat:
The organism is widespread in soil. Humans acquire the
infection by implantation of contaminated soil into wound.
Disease:
Tetanus (lockjaw) It is due to the neurotoxin
(tetanospasmin), inhibiting the release of inhibitory
neurotransmitters, causes convulsive contraction of
voluntary muscles. Severe disease is characterized by
tonic muscle spasms and hypereflexia, trismus,
opisthotonus and convulsions.
Treatment:
Antitoxin is available (hyperimmune human gamma
globulin, tetanus immune globulin). Immunization with
toxoid.

Clostridium botulinum
Laboratory characteristics
Anaerobic Gram-positive spore forming rods,
produces the most potent toxins known to man. Seven
immunologically distinct toxins (A,B, C, C, D, E and
F).
Serotypes A, B and E (sometimes F) are most
commonly associated with human disease. Serotypes
A and B are linked to a variety of foods (eg. meat)
serotype F is especially associated with fish.
Natural habitat
Soil is the normal habitat. Intoxication is by
ingestion of toxins in food that have not been
adequately sterilized or improperly processed canned
food.

Clostridium botulinum
Disease:
Botulism acquired by ingesting preformed toxins.
Infant botulism results from ingestion of organisms
and production of toxin in the infants gut,
associated with feeding honey contaminated with
spores. Toxin- acts at the neuromuscular junctions
by inhibiting acetylcholine release, results in
muscle paralysis and death from respiratory failure.

Treatment:
Supportive therapy. Trivalent vaccine is available

Clostridium difficile
Laboratory characteristics:
Slender Gram-positive spore-forming anaerobic rods.
Selective medium CCFA (cycloserine-cefoxitinfructose agar)-mere presence of organism is not
indicative of infection. Diagnosis by detection of
toxin in feces.
Habitat:
Component of normal gut flora, flourishes under
selective pressure of antibiotics, may spread from
person to person by fecal-oral route.
Disease:
Pseudomembranous (antibiotic-associated
diarrhea).
Treatment:
Oral vancomycin or metronidazole.

Picture A : The coloni

morphology of Clostr
difficile on CCFA agar

CLOSTRIDIUM DIFFICILE

Picture B: A Gram-stained smear

of Clostridium difficile

Corynebacterium diphtheriae
Laboratory characteristics:
- Gram-positive rods arranged as Chinese
characters or pallisades. Characteristic
black colonies form within 48 hours on
potassium tellurite.
- Four biotypes identified : mitis,
intermedius,
gravis and belfanti.
Natural habitat:
- Usually nasopharynx, occasionally the
skin.
Infection is usually spread by aerosol.
Patient may carry toxigenic strain for 2-3
months after
infection.

Plate A

Plate B

assium tellurite agar: Corynebacterium diphtheriae appea

black colonies (Plate A).

CORYNEBACTERIUM
DIPHTHERIAE
Picture A

Picture

Pictures A & B
.
Methylene
blue-stained smear of Corynebacterium diph
pleomorphic Gram-positive club-shaped bacilli, arranged in

Gram-stained smear: Gram-positive

bacilli arranged like Chinese characters
Picture A:

Picture

Picture A

Alberts stain: The

metachromatic granules
are stained bluish-black.
Picture B:

Negative control

Test organism

Positive control

Elek test for the demonstration of toxin production by

nebacterium diphtheriae. The toxin combines with antitox
produce antigen-antibody complexes which form visible lines
of precipitation (precipitin lines) in the agar.

Corynebacterium diphtheriae
Disease:
- Diphtheria (is due to the production
of diphtheria toxin, which inhibits
protein
synthesis by inactivating
an elongation factor).
Treatment:
Antitoxin &
antibiotics
Immunization.

Listeria monocytogenes
Laboratory characteristics:
- It appears as short Gram-positive rod often
coccobacillary in clinical specimens. It motile at 25oC
with characteristic tumbling movement and hemolytic
on blood agar.
Natural habitat:
Widely distributed in nature, survive in cold.
Excreted in large numbers in cows milk.
Human may carry Listeria in gut as normal flora.
Infection may be acquired by ingestion or
transplacentally
to the baby in-utero.
Diseases
Meningitis and sepsis in neonates.
Infection in the immunocompromised (particularly
meningitis)
and in pregnant women.
Treatment
Treatment with penicillin or ampicillin often in
combination
with gentamicin.

Picture B: Colonial morphology

Picture A: A Gram-stained smear
blood agar
Picture A

A Gram-stained smear of Listeria monocytogenes:

Gram-positive
coccobacilli frequently arranged in pairs resembling
enterococci or pneumococci
Picture B
Colonial morphology of Listeria monocytogenes on
blood agar:

A 56-year-old lady complained of cough

productive of purulent sputum, associated with
high grade fever with chills and rigors.
Picture 1: A Gram-stained smear of blood culture.
Picture 1
1 Describe briefly what you see in Picture 1.
.
Gram-positive diplococci

2 If the organism is cultured on blood agar, describe

. your expected colonial morphology of the organism.
Alpha hemolytic colonies on blood agar

3 Name ONE laboratory test that helps in the

. identification of the causative organism.

Sensitivity to Optochin, Quellung reaction, bile solubility test

4 State ONE antibiotic that is active against the

. causative organism.
Penicillin, cephalosporins