STAPHYLOCOCCI

Staphylococci are typical Gram-positive bacteria forming irregular clusters of cocci.

Staphylococci are widespread in nature, although they are mainly found on the skin, skin
glands and mucous membranes of mammals and birds, but can cause infection under certain
circumstances. 1S. aureus is more pathogenic than the other common members of the genus,
S. epidermidis and S. saprophyticus. S. epidermidis has been known to cause various
hospital-acquired infections (such as prosthetic or indwelling devices), whereas S.
saprophyticus is mainly associated with urinary tract infections in young females who are
sexually active. Disease processes with S. aureus are numerous. The portal of entry is
variable, since they gain access to the body via the skin, the respiratory tract or the genito-
urinary tract. Staphylococcus aureus expresses many potential virulence factors:

1. surface proteins - promote colonization of host tissues

2. leukocidin, kinases, hyaluronidase - invasins that promote bacterial spread in tissues
3. capsule, Protein A - surface factors that inhibit phagocytic engulfment
4. carotenoids, catalase - enhance staphylococcal survival in phagocytes
5. protein A, coagulase - immunological disguises
6. hemolysins, leukotoxin, leukocidin - membrane-damaging toxins that lyse eucaryotic cell
membranes
7. 2TSST, 3ET - exotoxins that damage host tissues or otherwise provoke symptoms of
disease
8. inherent and acquired resistance to antimicrobial agents.

Fig. 1 Virulence determinants of Staphylococcus aureus.

1
S. - Staphylococcus
2
TSST - Toxic Shock Syndrome Toxin
3
ET - Exfoliatin Toxin
Staphylococci can cause many forms of infection:

1. S. aureus causes superficial skin lesions (boils) and localized abscesses in other sites.
2. S. aureus causes deep-seated infections, such as osteomyelitis and endocarditis and more
serious skin infections (furunculosis).
3. S. aureus is a major cause of hospital acquired (nosocomial) infection of surgical wounds
and, with S. epidermidis, causes infections associated with indwelling medical devices.
4. S. aureus causes food poisoning by releasing enterotoxins into food.
5. S. aureus causes toxic shock syndrome by release of superantigens into the blood stream.
6. S. saprophyticus causes urinary tract infections, especially in girls.
7. Other species of staphylococci (S. lugdunensis, S. haemolyticus, S. warneri, S. schleiferi, S.
intermedius) are infrequent pathogens.

Fig. 2 Sites of infection and diseases caused by Staphylococcus aureus.

Although strains of Staphylococcus aureus resistant to penicillin have caused infections for
many years, isolates resistant to methicillin, oxacillin, and other ß-lactams have become
predominant-primarily in the last 20 years.
Diagnosis of staph infections begins with attempting to culture the bacteria from an infected
site. Any area with pus, crusty drainage, or blisters should be cultured. Blood from patients
with sepsis, toxic shock syndrome, or pneumonia should be cultured. Standard
microbiological techniques include a positive coagulase test to identify staph. S. aureus lyses
red blood cells in blood agar plates (hemolytic staphylococci) while S. epidermidis does not
(nonhemolytic staphylococci). All staph should be further tested to see if the bacteria are
resistant to the antibiotic methicillin (and other antibiotics) and thus determine if the
organisms are 4MRSA.

GRAM STAIN
Staphylococcus is a genus of bacteria that is characterized by a round shape (coccus or
spheroid shaped), Gram-positive (purple), and found as either single cells, in pairs, or more
frequently, in clusters that resemble a bunch of grapes. The genus name Staphylococcus is
derived from Greek terms (staphyle and kokkos) that mean "a bunch of grapes,"

Fig. 3 S. aureus (left), S. epidermidis (right) - Gram stain.

STAPHYLOCOCCI - BLOOD AGAR CULTURE

Blood agar is both differential and enriched medium. The blood that is incorporated into this
medium is an enrichment ingredient for the cultivation of fastidious organisms. On blood
agar, S. aureus usually displays a light to golden yellow pigment, whereas S. epidermidis has
a white pigment and S. saprophyticus either a bright yellow or white pigment. However,
pigmentation is not always a reliable characteristic. On blood agar, S. aureus is usually beta-
hemolytic, S. epidermidis and S. saprophyticus are almost always nonhemolytic.

Fig. 4 S. aureus (left) and S. epidermidis (right) - colonies on blood agar.

4
MRSA – methicillin resistant Staphylococcus aureus
S. aureus - individual colonies on agar are round, convex, and 1-4 mm in diameter with a
sharp border. On blood agar plates, colonies of Staphylococcus aureus are frequently
surrounded by zones of clear beta-hemolysis. The golden appearance of colonies of some
strains is the etymological root of the bacteria's name; aureus meaning "golden" in Latin.
Staphylococcus epidermidis - showing γ-haemolytic, porcelin-white colonies as compared to
S. aureus on the same medium. This clear distinction in colony color is not seen at all times.

CATALASE TEST
Some bacteria contain flavoproteins that reduce oxygen (O2), resulting in the production of
hydrogen peroxide (H2O2) and, in some cases, an extremely toxic superoxide (O2–).
Accumulation of these substances will result in death of the organism as they are powerful
oxidizing agents and destroy cellular constituents very rapidly unless they can be
enzymatically degraded. These substances are produced when aerobes, facultative anaerobes,
and microaerophiles use the aerobic respiratory pathway, in which oxygen is the final electron
acceptor, during degradation of carbohydrates for energy production. A bacterium must be
able to protect itself against such O2 products or it will be killed. Many bacteria possess
enzymes that afford protection against toxic O2 products. Facultative anaerobes and obligate
aerobes usually contain the enzymes superoxide dismutase, which has the ability to catalyze
the destruction of superoxide, and either catalase or peroxidase, which catalyze the
destruction of hydrogen peroxide as follows:

The inability of strict anaerobes to synthesize catalase, peroxidase, or superoxide dismutase

may explain why oxygen is poisonous to these microorganisms. In the absence of these
enzymes, the toxic concentration of H2O2 cannot be degraded when these organisms are
cultivated in the presence of oxygen. Organisms capable of producing catalase rapidly
degrade hydrogen peroxide which is a tetramer containing four polypeptide chains, which are
usually 500 amino acids long. It also contains four porphyrin heme groups(ie., iron
groups) that will allow the enzyme to react with the hydrogen peroxide.
The enzyme catalase is present in most cytochrome-containing aerobic and facultative
anaerobic bacteria. Catalase is the enzyme which has one of the highest turnover numbers
compared to all other enzymes; one molecule of catalase has the ability to convert millions
of molecules of hydrogen peroxide to water and oxygen in each second. Catalase production
and activity can be detected either by adding the substrate H2O2 to an appropriately incubated
(18- to 24-hour) tryptic soy agar slant culture or by slide test. Organisms which produce the
enzyme break down the hydrogen peroxide, and the resulting O2 production produces bubbles
in the reagent drop, indicating a positive test. Organisms lacking the cytochrome system also
lack the catalase enzyme and are unable to break down hydrogen peroxide, into O2 and water
and are catalase negative.
The catalase test is primarily used to distinguish among Gram-positive cocci. Members of the
genus Staphylococcus are catalase-positive, and members of the genera Streptococcus and
Enterococcus are catalase-negative.

Procedure of catalase test (Slide Test)

1. Transfer a small amount of bacterial colony to a surface of clean, dry glass slide using
a loop or sterile wooden stick
2. Place a drop of 3% H2O2 on to the slide and mix.
3. A positive result is the rapid evolution of oxygen (within 5-10 sec.) as evidenced by
bubbling (Fig. 5).
4. A negative result is no bubbles or only a few scattered bubbles.

Fig. 5 Catalase - slide test.

MANNITOL SALT AGAR CULTURE

Mannitol salt agar (MSA) is both a selective and differential media used for the isolation of
Staphylococci from mixed cultures.

MSA components

7,5% NaCl – selects for species of Staphylococcus. This concentration of salt is too high for
most other bacteria to withstand and, therefore, inhibits their growth.
Mannitol – alcohol of the carbohydrate mannose. Mannitol fermentation produces acid end
products which turn the medium yellow. Yellow indicates mannitol positive and no color
change indicates mannitol negative.
Phenol red pH indicator – yellow in acid pH (the same indicator that is used in phenol red
carbohydrate fermentation broths).

Fig. 6 Mannitol Salt Agar.

On MSA, only pathogenic Staphylococcus aureus produces small colonies surrounded by

yellow zones. The reason for this color change is that S. aureus have the ability to ferment
the mannitol, producing an acid, which changes the indicator color from red to yellow. The
growth of other types of bacteria is usually inhibited. This growth differentiates S. aureus
from S. epidermidis, which forms colonies with red zones.

Expected Results

1. On MSA, pathogenic Staphylococcus aureus ferments mannitol, thereby changing the

colour of the medium from red to yellow.

Fig. 7 Staphylococcus aureus on Mannitol Salt Agar.

2. Staphylococcus epidermidis grows on MSA, but does not ferment mannitol (media remains
light pink in color, colonies are colorless).
Fig. 8 Staphylococcus epidermidis on Mannitol Salt Agar.

COAGULASE TEST
Coagulases are enzymes that clot blood plasma by a mechanism that is similar to normal
clotting. The coagulase test identifies whether an organism produces this exoenzyme. This
enzyme clots the plasma component of blood. The only significant disease causing bacteria of
humans that produce coagulase enzyme are Staphylococcus aureus. Thus this enzyme is a
good indicator of the pathogenic potential of S. aureus.
In human host, the action of coagulase enzyme produces clotting of the plasma by converting
fibrinogen to fibrin in the immediate vicinity of the bacterium as a means of protection by
itself. The fibrin meshwork that is formed by this conversion surrounds the bacterial cells or
infected tissues, protecting the organism from non-specific host resistance mechanisms such
as phagocytosis and the anti staphylococcal activity of normal serum. This enables the
bacterium to persist in the presence of a host immune response, which can lead to the
establishment of infection. Thus, coagulase is described as a virulence factor( disease-
causing factor) of Staphylococcus aureus. Citrate and EDTA (Ethylenediaminetetraacetic
acid) are usually added to act as anticoagulants and prevent false-positive results. Most
strains of S.aureus produce one or two types of coagulase; free coagulase and bound
coagulase. Bound coagulase is localized on the surface of the cell wall and reacts with α- and
β-chains of the plasma fibrinogens to form a coagulate. Free coagulase is an enzyme that is
secreted extracellularly and bound coagulase is a cell wall associated protein. Free coagulase
can be detected in tube coagulase test and bound coagulase can be detected in slide coagulase
test.
Slide coagulase test may be used to screen isolates of S.aureus and tube coagulase may be
used for further confirmation. There are seven antigenic types of free coagulase, but only one
antigenic type of bound coagulase exists. Free coagulase is always heat labile while bound
coagulase is heat stable.
In the test, the sample is added to rabbit plasma and held at 37° C for a specified period of
time. Clot formation occurs within 4 hours is interpreted as a positive result and indicative of
a virulent Staphylococcus aureus strain. The absence of coagulation after 24 hours of
incubation is a negative result, indicative of an avirulent strain.

Detection of bound coagulase - Slide Test

This method measures bound coagulase. The bound coagulase is also known as clumping
factor. It cross-links the α and β chain of fibrinogen in plasma to form fibrin clot that deposits
on the cell wall. As a result, individual coccus stick to each other and clumping is observed.

1. Divide the slide into two sections with grease pencil. One should be labeled as „test” and
the other as „control“.
2. Place a small drop of distilled water on each area.
3. Emulsify one or two colonies of Staphylococcus on blood agar plate on each drop to make
a smooth suspension.
4. The test suspension is treated with a drop of citrated plasma and mixed well with a needle.
5. Do not put anything in the other drop that serves as control. The control suspension serves
to rule out false positivity due to auto agglutination.
6. Clumping of cocci within 5-10 seconds is taken as positive (Fig. 9).

Fig. 9 Slide Coagulase Test.

Some strains of S.aureus may not produce bound coagulase, and such strains must be
identified by tube coagulase test

Detection of free coagulase - Tube Coagulase Test

Most strains of S.aureus produce one or two types of coagulase; free coagulase and bound
coagulase. Free coagulase is an extracellular enzyme which reacts with prothrombin and its
derivatives. This method helps to measure free coagulase. The free coagulase secreted by
S.aureus reacts with coagulase reacting factor (CRF) in plasma to form a complex, which is
thrombin. This converts fibrinogen to fibrin resulting in clotting of plasma (Fig. 10).

1. Three test tubes are taken and labeled “test”, “negative control” and “positive control”.
2. Each tube is filled with 1 ml of 1 in 10 diluted rabbit plasma.
3. To the tube labeled test, 0.2 ml of overnight broth culture of test bacteria is added.
4. To the tube labeled positive control, 0.2 ml of overnight broth culture of known S.
aureus is added.
5. To the tube labeled negative control, 0.2ml of sterile broth is added.
 6. All the tubes are incubated at 37°C.
7. Positive result is indicated by gelling of the plasma, which remains in place even after
inverting the tube.
8. If the test remains negative until four hours at 37°C, the tube is kept at room
temperature for overnight incubation.

Fig. 10 Tube Coagulase Test.

The coagulase test is used to distinguish between pathogenic and nonpathogenic members of
the genus Staphylococcus. All pathogenic strains of S. aureus are coagulase positive whereas
the nonpathogenic species (S. epidermidis) are coagulase negative.
While slide coagulase test is useful in screening, tube coagulase test is useful in confirmation
of coagulase test. Samples must be observed for clotting within 24 hours. This is because
some strains that produce coagulase also produce an enzyme called fibrinolysin, which can
dissolve the clot. Therefore, the absence of a clot after 24 hours is no guarantee that a clot
never formed. The formation of a clot by 12 hours and the subsequent disappearance of the
clot by 24 hours could produce a so-called false negative if the test were only observed at the
24-hour time.

Sources:

Samuel Baron Medical Microbiology, 4th edition, University of

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