BACTERIOLOGY LECTURE

S.Y. ‘23 - ‘24 | BY HUEN MINGARINE MODULE 4

MODULE ● Habitat is the human nose and skin

- S. aureus are found in the anterior
1.0 Staphylococcus species nares of 20% to 40% of adults,
carried by healthy individuals
intermittently rather than
● classified the family Staphylococcaceae chronically.
● Historically, the genus Staphylococcus was - Those on the skin are believed to be
included in the family Micrococcaceae which contaminants from the nose.
also includes the Genus Micrococcus - Other sites of colonization include
● Several of the Micrococcus species are now the nasopharynx, perineum, the
reclassified into 4 other genera. These axillae, and the vagina.
genera have now reorganized into 2 families ● Transmission is via contaminated hands
Micrococcaceae, and Dermococcaceae - Staphylococcal infections are most
● There are, currently several species and commonly acquired endogenously
subspecies (45 species and 21 subspecies) from colonized anterior nares or by
within the genus Staphylococcus direct contact with someone
● The coagulase-positive Staphylococcus carrying S. aureus.
aureus, two coagulase-negative species, S. - From the nasal site, the bacteria are
epidermidis and S. saprophyticus are seen shed to the exposed skin and
frequently in human infections clothing of the carrier and others
● S. hyicus – causes an infectious dermatitis with whom they are in direct
in swine contact.
● S. chromogenes (formerly a subspecies of S. - Spread is augmented by touching
hyicus) – causing infection in swine, cattle, the face and, of course, nose
and goat picking.
● S. intermedius – has been isolated from
several types of infections in dogs
● S. delphini – dolphins 1.3 Pathogenesis and Clinical
● S. felis – domesticated cats Manifestations
● S. schleiferi subsp. Coagulans – dogs
● S. lutrae – sea otters
● S. schleiferi subsp coagulans – cause ● Although this organism is frequently a part
external otitis in dogs of the normal human microflora, it can cause
● S. pseudointermedius – in dogs, cats, horse, significant opportunistic infections under the
parrots appropriate conditions.
● Factors which may predispose an individual
to serious S. aureus infections include:
1.1 General Characteristics 1. breaks in the continuity and
integrity of mucosal and cutaneous
● Gram-positive cocci, 0.5 - 1.5 um in diameter, surfaces
and typically in grape-like clusters. 2. presence of foreign bodies or
● Non-motile implants (e.g., sutures, intravenous
● Non–spore-forming lines, prosthetic devices)
● Facultative anaerobes 3. prior infection with other agents,
● Grow most rapidly at 37°C particularly viruses (e.g., influenza)
● Halotolerant 4. underlying diseases with defects in
● Catalase(+) cellular or humoral immunity, either
● Fermentative congenital or acquired, (e.g.,
● Nitrate reduction(+) defects in leukocyte chemotaxis,
● Coagulase(+) defects in opsonization by
● DNase(+) antibodies,
● Mannitol fermenter hypogammaglobulinemia or
complement component
deficiencies and/or defects)
1.2 Habitat and Transmission

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S.Y. ‘23 - ‘24 | BY HUEN MINGARINE MODULE 4

5. chronic underlying diseases such

skin or clothing
as malignancy, alcoholism, and
heart disease
6. therapeutic or prophylactic Carbuncle – a larger and deeper lesion created
antimicrobial administration by aggregation and interconnection
of a cluster of furuncles
– extremely painful and can even be
2.0 Diseases fatal in elderly patients when they
give rise to systemic disease
– usually found in areas of which
tough skin such as on the back of
2.1 Cutaneous diseases the neck

● Staphylococcal infections of the skin Impetigo – a staphylococcal skin infection

(through wounds, follicles, or skin glands) that is not confined to follicles and
are pyogenic (pus-forming), and often skin glands
presents as an inflamed, fibrous lesion – characterized by bubble-like
enclosing a core of pus called an abscess epidermal sweeping that can break
and peel away like a localized form
of scalded skin syndrome
Folliculitis – mild inflammation of the – usually present on exposed areas,
superficial dermis that is restricted especially the face, and may spread
to ostia (opening) of the hair to surrounding areas by
follicles and is characterized by the autoinoculation (by the infected
presence of small, reddish, painful person himself, to other sites of his
lesions and the absence of body)
systemic symptoms
– Stye – eyelid Non-bullous impetigo
– begins as a single red macule
Hidradenitis – chronic or relapsing inflammatory
(patch) or papule that quickly
suppurativa disease of the skin, involving the
becomes a vesicle.
apocrine gland-bearing areas
– The vesicle rupture and forms an
axillae, groin, perineal (perineum —
erosion, and the lesion and its
an area between the thighs) and
contents dry to form a
perianal regions
characteristic honey-colored crusts
– characterized by presence of
with erythematous (reddish)
multiple lesions associated with
margins that may be pruritic (itchy).
blocked and infected apocrine
sweat glands
Bullous impetigo
– While local pain, swelling and
– is a localized form of
erythema are present, systemic
staphylococcal scalded skin
symptoms such as fever is usually
syndrome that commonly affects
absent
neonates but can also occur in older
children and adults.
Furuncle or – a deeper-seated infection of the – Superficial vesicles progress to
boil hair follicles enlarging bullae (singular; bulla
– results when the inflammation of plural; vesicle or blister) with sharp
single hair follicle or sebaceous margins with no surrounding
gland progresses into a large, red, erythema. When the bullae ruptures,
extremely tender abscess or pustule yellow crust with oozing results.
– Furuncles often appear in clusters – Bullous impetigo is usually found
(known as furunculosis) in parts of in moist, intertriginous areas (e.g.,
the body such as the buttock, the diaper area, axillae, neck folds).
breasts, axillae, and back of the
neck where skin rubs against other

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BACTERIOLOGY LECTURE
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a sunburn, and can involve any

2.2 Toxigenic diseases region of the body, including the
lips, mouth, eyes, palms and soles
● Toxigenic disease caused by S. aureus can - patients who survive the initial
present itself as a toxemia due to the phase of the infection, the rash is
production of toxins in the body or as food followed by desquamation 1 – 2
intoxication, the ingestion of preformed weeks after onset
toxin. - involvement of multiple (three or
1. Staphylococcal food poisoning more) organ systems with varying
- a gastrointestinal illness caused by symptoms of vomiting, diarrhea,
eating foods contaminated with renal failure, headache, chills, sore
toxins produced by the S. aureus, throat and conjunctivitis
thus it is a food intoxication - Initially, the disease was noted
- characterized by acute symptoms most frequently in women, with
that appear in 2 to 6 hours after onset mainly occurring during
ingestion of the toxin-contaminated menstruation; association between
food the onset of disease and the use
- Emesis (vomiting) is often high-absorbency tampons during
projectile, and diarrhea is less menses
frequent - non-menstrual-associated TSS was
- Recovery is rapid, usually within 24 reported in males and females as
hours complication of staphylococcal
- associated with eating foods such abscesses or systemic infections
as custards, sauces, cream
pastries, processed meats, chicken 2.3 Miscellaneous Systemic Infections
salad, or ham that have been
contaminated by handling and then
left unrefrigerated for a few hours ● Most systemic staphylococcal infections
- Because of the high salt tolerance have a focal pattern, spreading from a local
of S. aureus, even foods that cutaneous infection to other sites. These
contain salt as a food preservative include:
are not exempt - osteomyelitis (bone infection)
- The toxins produced by the - pneumonia which usually occurs
multiplying bacteria does not alter after influenza viral infection
the food’s taste or smell - bacteremia with consequences
2. Staphylococcal Scalded Skin Syndrome such as, but not limited to,
(SSSS) (AKA Ritter's disease) endocarditis, arthritis, and
- usually seen in neonates (newborns meningitis
or children less than a month old)
and infants less than 5 years
- characterized by widespread 3.0 Virulence Factors
erythema and the appearance of
bullous lesions over large areas of
● S. aureus expresses many potential virulence
the body, with subsequent
factors that promote colonization of, spread
sloughing of the superficial skin
in, or damage to host tissues, or enhance
layers
their survival within the host.
- exposure of large areas of d e n u d
● These include a variety of cellular structures
e d (stripped of surface layers) and
and extracellular substances:
raw skin
- most cases, desquamation occurs
and symptoms wane over 5-7 days
3. Staphylococcal toxic shock syndrome (TSS)
- initially a flu-like illness Microcapsule – consists of polysaccharides
characterized by fever, hypotension, – can be visualized only by
and rash on the skin that resembles

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S.Y. ‘23 - ‘24 | BY HUEN MINGARINE MODULE 4

electron microscopy (hyaluronic acid) in tissue, thus

– mediates attachment to host spreads the organism to
cells or tissues, and inhibits adjacent areas in tissues
phagocytosis by
polymorphonuclear leukocytes Lipase – hydrolyzes lipids and help
unless specific antibodies are the spread of the organism in
present cutaneous and subcutaneous
– At least 11 serotypes have tissues
been identified, with types 5
and 8 responsible for the Staphylokinase – plasminogen activator. This
majority of infections factor dissolves fibrin clot (a
fibrinolysin)
Protein A – a bacterial surface protein in – Localized fibrinolysis might
some S. aureus strains aid in the spread of infection to
– binds to the Fc portion of IgG, contiguous tissues
which, therefore, will no longer
be capable of binding to Fc Phosphatidylino – affects tissues of the host
receptor on phagocytes, thus sitol-specific making them more susceptible
preventing opsonization phospholipase C to damage and destruction by
– blocks complement fixation complement components and
– S. aureus with Protein A products during complement
bound to the Fc portion of IgG activation
molecules are used as reagents
for identification of certain β-lactamase – under plasmid-control
organisms transmitted by transduction
and perhaps also by
Catalase – inactivates toxic hydrogen conjugation and provides
peroxide and free radicals resistance to β-lactam
formed by the myeloperoxidase antibiotics such as penicillin,
system within the phagocytic penicillin derivatives (penams),
cells, which are the primary and cephalosporins
killing mechanisms within the – These antibiotics all have a
phagolysosome common element in their
molecular structure: a
Coagulase – promotes conversion of four-atom ring known as a
fibrinogen to fibrin causing β-lactam
plasma to clot. Fibrin coat the – Through hydrolysis, the
bacterial cells, hiding their β-lactamase breaks the
antigenic surface, thus β-lactam ring open,
protecting them from deactivating the molecule's
phagocytosis and immune antibacterial properties
response
Toxic Shock – a superantigen
Deoxyribonuclea – hydrolyzes DNA Syndrome Toxin – promotes the manifestations
se (DNase) – facilitates the spread of (TSST-1) of toxic shock syndrome. The
bacteria by liquefying and gene for TSST-1 is found in
decreasing the viscosity of about 20% of S. aureus isolates
abscess materials – Superantigens are protein
toxins that stimulate T cells
Hyaluronidase – also known as the non-specifically without normal
“spreading factor” antigenic recognition
– hydrolyzes the intercellular
matrix of acid Exfoliative – epidermolytic toxins that
mucopolysaccharides toxins (or facilitate dissolution of the

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S.Y. ‘23 - ‘24 | BY HUEN MINGARINE MODULE 4

exfoliatins) mucopolysaccharide matrix of in hospitals or other health care settings, such as

the stratum granulosum in the nursing homes and dialysis centers. When it occurs
epidermis resulting in the in these settings, it's known as health
intraepithelial splitting of care-associated MRSA (HA-MRSA). HAMRSA
cellular linkages seen in infections usually are associated with invasive
Staphyloccoccal Scalded Skin procedures or devices, such as surgeries,
Syndrome (SSSS). intravenous tubing or artificial joints. HA-MRSA can
– There are two distinct spread by health care workers touching people with
proteins of the same molecular unclean hands or people touching unclean
weight: surfaces. Another type of MRSA infection has
a. Exfoliative toxin A occurred in the wider community — among healthy
(ET-A) is produced by people. This form, community-associated MRSA
lysogenized strains of (CA-MRSA), often begins as a painful skin boil. It's
S. aureus usually spread by skin-toskin contact.
b. Exfoliative toxin B
(ET-B) is
plasmid-mediated. Blood Cell Toxins (Hemolysins and Leukocidins)
Both have
superantigen – hemolysins lyse red blood cells by disrupting
activities. their cell membranes
– Leukocidins damage cell membranes of
Enterotoxins A – heatstable exotoxins which neutrophils and macrophages, causing them to lyse
through E, G-J, are responsible for the clinical – help incapacitate the host’s phagocytic line of
K-R and U, V features of staphylococcal defense
food poisoning
– Heating the food may not ⍺ (alpha)-Hemolysin
prevent the disease because – heterogeneous protein that facilitates formation
inactivation of staphylococcal of pores on the target eukaryotic cell membrane
enterotoxin requires 100oC for through which monovalent cations can pass,
at least 30 minutes leading to osmotic swelling and rupture of the cell.
– S. aureus enterotoxins are – This potent toxin lyses red blood cells of various
superantigens. Approximately mammals and damages monocytes, macrophages,
50% of S aureus strains can lymphocytes, skeletal muscle, heart, and renal
produce one or more of them tissue as well.
– It is also active against thrombocytes (platelets)
and may contribute to septic thrombotic events
NOTE during S. aureus bacteremia.

MRSA are strains of the S. aureus that are resistant β (beta)-Hemolysin

to the action of methicillin, and related beta-lactam – a sphingomyelinase that degrades sphingomyelin
antibiotics (e.g., penicillin, oxacillin, amoxicillin). in the cell membrane and therefore is toxic for
MRSA has evolved resistance not only to many kinds of cells. It is cytotoxic not only to
beta-lactam antibiotics, but to several classes of human erythrocytes but also to monocytes.
antibiotics. – β-hemolysin is said to be inactive against
granulocytes, lymphocytes, and fibroblasts. It is a
Resistance to methicillin is due to the presence of protein exotoxin described as a “hot-cold”
an altered penicillin-binding protein called PBP2a hemolysin; its hemolytic properties are enhanced
(or PBP2′) that results from acquisition of a by subsequent exposure to red blood cells to low
chromosomal gene called mecA which is located temperatures. A lysogenic bacteriophage is known
within the large mobile staphylococcal to encode the toxin.
chromosomal element known as the SCCmec
(staphylococcal cassette chromosome). δ (delta)-Hemolysin
– is a small peptide that acts primarily as a
Most MRSA infections occur in people who've been surfactant or a detergent-like molecule that

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interacts with cell membranes and forms channels 5.0 Laboratory Diagnosis
resulting in slow leakage of cellular contents. It
may have a role in S. aureus diarrheal diseases.
5.1 Staphylococcus epidermidis
γ (gamma)-Hemolysin
– a leukocidin that lyses white blood cells and is
composed of two proteins designated S and F. ● Gram-positive cocci, 0.5 - 1.5 um in diameter,
– It is capable of efficiently lysing white blood cells and typically in grape-like clusters.
by causing pore formation in the cellular ● Non-motile
membranes that increase cation permeability. ● Non–spore-forming
– Affected cells undergo degranulation of the ● Facultative anaerobes
cytoplasm, cell swelling, and lysis. This leads to ● Grow most rapidly at 37°C
massive release of inflammatory mediators which ● Halotolerant
are responsible for necrosis and severe ● Catalase(+)
inflammation. ● Fermentative
● Nitrate reduction(+)
Panton-Valentine Leukocidin (PVL) ● Coagulase(-)
– is produced by lysogenized strains of S. aureus. ● DNase(-)
This pore-forming toxin is active against ● Non-mannitol fermenter
neutrophils and causes tissue necrosis. It consists ● most commonly encountered staphylococcal
of two components designated as S and F that act species
synergistically on the white blood cell membrane as ● Comprises 99% of the normal flora of the
described for γ toxin. skin
● Considered opportunistic pathogens, but are
substantially less virulent than S. aureus
4.0 Prevention and Control ● Associated with nosocomial infections
related to use of contaminated medical
devices
● Infection and spread thereof may be - Endocarditis - associated with
prevented by cleanliness, hygiene, and prosthetic heart valves; also
aseptic management of lesions. pacemaker wires, implanted
● Application of topical antiseptics such as defibrillators, and vascular grafts
mupirocin to nasal or perineal carriage sites - Arthritis - associated with
may diminish shedding of dangerous prosthetic joint, or hip implant
organisms. - Urinary tract infection (UTI) —
● In health care facilities, surveillance of associated with indwelling urinary
anterior nares colonization among staff in catheters
health facilities particularly those who man - Bacteremia - associated with IV
the Intensive Care Unit by periodic swabbing. catheters
● Health care workers are advised to strictly ● Pathogenesis is related to production of
adhere to infection control policies by adherent slime resulting in biofilm formation
wearing gloves and washing hands before on the surface of a prosthetic device
and after patient contact.
● Clothes and bedding that may cause
reinfection should be dry-cleaned or washed 5.2 Staphylococcus saprophyticus
at a sufficiently high temperature (70°C or
higher) to destroy staphylococci. ● Gram-positive cocci, 0.5 - 1.5 um in diameter,
● Chemoprophylaxis is effective in surgical and typically in grape-like clusters.
procedures such as hip and cardiac valve ● Non-motile
replacements, in which infection with ● Non–spore-forming
staphylococci can have devastating ● Facultative anaerobes
consequences. ● Grow most rapidly at 37°C
● Halotolerant
● Catalase(+)

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● Fermentative stain pink to red in properly gram

● Nitrate reduction(+) stained specimens.
● Coagulase(-) B. Cultural method
● DNase(-) - Staphylococcus species grow well
● Urease (+) on routine laboratory media:
● Mannitol fermentation (variable)
Blood – Enriched medium that supports the
● Primarily a saprophyte
agar growth of staphylococci and permits
● Gastrointestinal tract (primarily the rectum)
medium observation of the pattern of
as the primary site of colonization in
(BAM) hemolysis of blood
humans; also part of the normal flora of
female genitourinary tract (urethra, cervix),
perineum Mannitol – MSA is both a selective and
● Second most common cause of salt agar differential medium for staphylococci.
community-acquired UTI in young, sexually (MSA) Uninoculated MSA appears light to
active females (most common is Escherichia medium rose red in color
coli) – The high salt (7.5% NaCl)
● Pathogenesis is related to production of concentration inhibits the growth of
adherent slime and urease (an enzyme that other organisms (except for
hydrolyzes urea in urine resulting in the enterococci), and selectively allows
formation of ammonia and ammonium the growth of halotolerant
carbonate, making the urine alkaline and staphylococci
favoring bacterial growth) – Mannitol fermentation results in
acids indicated by a change in the
phenol red indicator to yellow, and
6.0 Laboratory Diagnosis of aids in the differentiation of
staphylococcal species
Staphylococcus species
Columbia – Sheep blood supports the growth of
CNA Agar fastidious bacteria and allows
6.1 Specimens detection of hemolytic reactions.
– Antimicrobial agents, colistin and
● Pus from wound and other pyogenic nalidixic acid, render the medium
infections selective for G+ bacteria by inhibiting
● Blood from septicemia G- bacteria.
● CSF from meningitis
● Sputum from respiratory infections Phenyleth – Sheep blood supports the growth of
● Urine from UTI yl Alcohol fastidious bacteria particularly G+
● Nasal swab from suspected carriers (PEA) cocci.
Agar – The medium should not be used for
determination of hemolytic reactions
6.2 Procedure Notes since atypical reactions may be
observed.
– PEA inhibits G- bacteria.
A. Microscopy
- G+ cocci, 0.5-1.5 µm in diameter, Vogel-Jo – Selectivity of the medium is
and grape-like clusters hnson achieved by potassium tellurite,
- A direct smear, one that is prepared (VJ) Agar lithium chloride and glycine which
from a clinical material such as pus inhibit nonstaphylococcal organisms,
or CSF, usually reveals G+ cocci both G+ and G- bacteria.
arranged as single cells, pairs, – Tellurite is reduced to metallic
tetrads, and short chains. tellurium by S. aureus producing
- The direct smears should also be colonies that are black or gray-black in
examined for the presence of color; mannitol is degraded into acid
inflammatory cells. These cells, which is indicated by phenol red,
background tissue debris and which changes the color of the
proteinaceous material generally

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medium to yellow. (BAM) slightly raised, low convex, opaque,

– Small, gray-black, not surrounded by and butyrous in consistency
yellow zones (may become deeper red – Colonies of some S. aureus are
in color) presumptively identifies S. usually large (4-6 mm in diameter).
epidermidis – Some strains (b) produce golden
yellow pigments "lipochrome" so
Chapman – It is selective due to relatively high appear creamy yellow or
Stone salt content, although it is reduced to yellow-orange (hence, the name
Agar 5.5%. "aureus", meaning "golden")
– Gelatin serves as a substrate for – Others (c) may have a distinct or
gelatinase activity; ammonium sulfate hazy zone of β-hemolysis around the
allows detection of gelatin hydrolysis colonies; this hemolytic property may
which is characterized by clear zone become apparent only after prolonged
around the colonies. incubation
– differential due to mannitol – S. epidermidis produce small to
fermentation. To determine mannitol, medium, opaque, gray-white colonies;
add few drops of bromcresol purple to most colonies nonhemolytic;
areas on the medium; yellow color slime-producing strains are extremely
indicates mannitol fermentation sticky and adhere to the agar surface
– Yellow colonies surrounded by clear – S. saprophyticus colonies are large,
halo presumptively identifies S. aureus entire, very glossy, smooth, opaque,
– White nonpigmented colonies with butyrous, convex; usually white but
or without clear zone probably is S. colonies can be yellow or orange
epidermidis
Mannitol – Mannitol-fermenting staphylococci
Baird-Par – Tellurite, lithium chloride and glycine salt agar (a) produce growth of yellow colonies
ker Agar have inhibitory action for organisms (MSA) with yellow discoloration of the
other than staphylococci medium. This is typical of S. aureus.
– Sodium pyruvate is incorporated in However, other staphylococci
order to stimulate the growth of S. (particularly S. saprophyticus) may
aureus without destroying the also ferment mannitol and thus
selectivity resemble S. aureus on this medium
– Reduction of tellurite to tellurium – Non-mannitol fermentation (b) by S.
produces a black coloration epidermidis and several other species
– Addition of egg yolk is used to produce small red colonies with no
demonstrate proteolysis and lipolysis color change to the medium
by lecithinase and lipase respectively
producing clear zones around the
colonies 7.0 Identification Tests
– S. aureus: Black, shiny, convex
colonies 1-5 mm in diameter with a
narrow, white edge.
– S. epidermidis: Black, shiny, irregular 7.1 Catalase Test
shape. Opaque zone develop around
the colonies after 24 hours. ● When performed with bacterial isolates that
are G+ cocci, this test distinguishes
● Interpretation of colonial morphologies is staphylococci which are strongly
usually performed after 24 to 48 hours of catalase-positive from streptococci
incubation at 35-37 oC (catalase-negative)
● Colonial characterization: ● Based on the ability of the bacteria to p r o d
u c e t hecatalaseenzymethat
Blood – Staphylococcal colonies (a) are decomposes hydrogen peroxide into water
agar medium to large (1-2 mm in diameter), and oxygen. The evolution of oxygen results
medium off-white or gray, smooth, entire, in effervescence (i.e.,rapid bubble formation)

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coagulase-positive. Other
phenotypic tests distinguish them
7.2 Coagulase test from S. aureus.
- When observing for any degree of
● single most reliable characteristic for clot formation (partial or complete)
identifying S. aureus The conventional after incubation, gently tilt the tube;
coagulase test may be performed by the do NOT agitate as a small clot
slide or tube procedures formed may be dislodged and will
● The coagulase enzyme produced by S. not reform anymore.
aureus binds plasma fibrinogen and - Tube coagulase tests that are
activates a cascade of reactions causing negative after 4 hours incubation at
plasma to clot. 35°C should be held at room
a. Slide coagulase test temperature and read again after 18
- screening test for S. aureus. Strains to 24 hours, because some strains
that are negative with the slide will produce fibrinolysin
coagulase test must be confirmed (staphylokinase) on prolonged
with a tube coagulase test incubation at 35°C, causing
- Based on the presence of bound dissolution of the clot during the
coagulase or “clumping factor” on incubation period.
the surface of the cell wall of most
strains of S. aureus. This factor NOTE
reacts directly with and converts
fibrinogen in plasma into fibrin, – The medium for both slide coagulase and tube
causing rapid cell agglutination coagulase procedures is rabbit plasma with EDTA,
- Some human coagulase-negative which is commercially available in lyophilized form
species (e.g., S. lugdunensis and S. – Rabbit plasma has high amounts of
schleiferi subsp schleiferi) produce coagulase-reacting factor (CRF); and, EDTA is the
clumping factor and may be slide preferred anticoagulant.
coagulase-positive – Human plasma must not be used because it
- The test can be performed with contains variable amounts of CRF and may contain
growth from blood agar, or other anti-staphylococcal antibodies.
nonselective medium, but should – Citrated plasma should not be used as some
not be performed from media bacteria that are able to utilize citrate (such as
having a high salt content (e.g., enterococci) will yield positive results if they are
mannitol salts agar) since the high mistaken for staphylococci; always perform a
salt causes some strains of S. catalase test first.
aureus to autoagglutinate.
b. Tube coagulase test
- confirmatory test for S. aureus
7.3 Novobiocin test
since free coagulase is secreted by
virtually all strains of S. aureus
- Based on the detection of free ● one of the methods to distinguish between
coagulase which is secreted coagulase-negative staphylococci, S.
extracellularly by S. aureus. The free epidermidis and S. saprophyticus
coagulase reacts with a substance ● S. epidermidis as an agent involved in
in the plasma called several infectious processes and the
coagulase-reacting factor (CRF), recognized clinical significance of S.
which in turn reacts with fibrinogen saprophyticus in urinary tract infections
in plasma to form fibrin (clot ● performed as a disk susceptibility test using
formation). a novobiocin disk (NB,5 µg)
- Some animal isolates (S. ● some human staphylococcal species (S.
intermedius, S. hyicus, S. delphini, cohnii subspecies, S. hominis subsp.
S. schleiferi subsp. coagulans and novobiosepticus, S. xylosus, some S.
strain of S. schleiferi subsp. pseudolugdunensis) are resistant to
schleiferi ) may be tube novobiocin

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they are rarely implicated in human

infections. When infections occur,
7.4 Deoxyribonuclease (DNase) test particularly in hosts with compromised
immune systems, such as HIV patients, they
● DNase test detects bacterial likely involve endogenous strains (those that
deoxyribonuclease which is an extracellular are part of the host’s normal flora).
enzyme that depolymerizes (breaks down) ● rarely clinically significant
DNA into subunits composed of nucleotides ● typically appear as gram-positive cocci in
● Inoculate, either by streak inoculation or spot tetrads, rather than in clusters and usually
inoculation, a DNase test agar plate larger (0.5 -3.5 µm) than staphylococci
(containing deoxyribonucleic acid), and ● Some micrococci are pigmented bacteria
incubate at 35 oC for 24-48 h; after - M. luteus produces yellow colonies
incubation, flood with 1N hydrochloric acid and M. roseus produces reddish
(HCl) which will precipitate DNA. colonies
● A clear area surrounding the growth after - The yellow colonies of M. luteus
addition of 1N HCL indicates a positive may be mistaken for the pigmented
reaction for DNase activity. A negative colonies of S. aureus.
reaction is indicated by no clearing and a
cloudy precipitate around the colonies. 9.0 Methods
● Alternative method: The test may also be
carried out in agar plates with dye, such a s
m e t h y l g r e e n a n d toluidine blue. This
eliminates the need to add 1N HCl. 9.1 Oxidation-Fermentation (O-F) test

7.5 Thermostable Endonuclease Test ● O-F test determines whether bacteria can
utilize glucose in the presence or absence of
oxygen.
● The same DNase test agar is used; 3-mm ● A semisolid medium containing glucose as
holes are cut into the agar with a sterile cork the sole carbohydrate in the medium and
borer, and the wells are filled with a 24-hour bromothymol blue as pH indicator is used.
broth culture of the test organism that has ● Two tubes are inoculated by stabbing; one
been boiled in a water bath for 15 minutes. tube (designated as the "closed tube") is
The plate is incubated overnight at 35°C. overlaid with sterile mineral oil which shields
● S. aureus which is thermostable it from oxygen in air, while the other tube
endonuclease positive will show a pink zone ("open tube") is exposed to oxygen.
surrounding the well containing the boiled ● Micrococci are oxidative because they are
suspension. Negative test is when the blue aerobic bacteria; they produce acids from
color in the medium remains glucose only in the presence of oxygen, not
in its absence. Medium in the open tube
turns yellow but not in the closed tube.
8.0 Micrococcus species ● Staphylococci are fermentative, utilizing
glucose, and produce acids in the presence
● often resemble and ,therefore, are easily of oxygen as well as in its absence. This is
confused with staphylococci related to the fact that staphylococci are
● Most species are found free-living in the facultative anaerobes. This is evidenced by
environment including air, soil, various C. bound coagulase by yellow color change
extreme environments (e.g., marine of the medium in both open and closed
sediment, deep sea mud, activated sludge), tubes.
and food (usually fermented seafood).
● In humans, Micrococcus species are 9.2 Susceptibility to Bacitracin, 0.04 U
considered part of the normal microbiota of
the skin, mucosa, and oropharynx. Their and Furazolidone, 100 µg
mode of transmission is still uncertain, and

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BACTERIOLOGY LECTURE
S.Y. ‘23 - ‘24 | BY HUEN MINGARINE MODULE 4

● This test is performed as an agar disk

susceptibility procedure using commercially 9.5 Production of acids from glycerol
available disks: in the presence of erythromycin
- Taxo A disk contains 0.04 U
bacitracin
● The test uses a medium containing glycerol
- FX disk contains 100 µg
(1%) and erythromycin (0.4 µg/mL). It is
furazolidone
prepared with an enriched agar base
● Micrococci are susceptible to bacitracin
containing bromocresol purple indicator and
producing zones of 10 m m o r g r e a t e r, b
poured into Petri plates. Several colonies of
u t a r e furazolidone-resistant and shows
the isolate are streaked as a single line on
zones of inhibition up to 9 mm.
the medium and the plate is incubated for up
● Staphylococci are bacitracin-resistant and
to 3 days at 35°C.
grow to the edge of the Taxo A disk; but,
● Micrococci will not produce acids from
furazolidone-susceptible, thus are inhibited
glycerol, so the medium will remain purple in
by furazolidone and show zones of 15 mm or
color.
more.
● Staphylococci will produce acids from
glycerol causing a yellow color change in the
9.3 Lysostaphin test medium.

● Lysostaphin is an endopeptidase enzyme

that can lyze the cell wall of staphylococci
but not micrococci.
● Lysostaphin solution is added to a bacterial
suspension and incubated at 35-37 oC for 2
hours.
● Micrococci are lysostaphin resistant —
bacterial suspension remains turbid; it does
not clear.
● Staphylococci are lysostaphinsusceptible —
there is clearing of the bacterial suspension.

9.4 Modified oxidase test

● Modified oxidase test is also known as the

microdase test; it is recommended for
gram-positive, catalase-positive cocci only.
● uses filter paper disks impregnated with
tetramethyl-p phenylenediamine
dihydrochloride in dimethyl sulfoxide
(DMSO). A colony from the growth medium
is removed with an applicator stick and
rubbed onto the disk
● Micrococcus species are modified
oxidase-positive; the test is indicated by
development of a blue-purple color within 30
seconds.
● Staphylococcus species are generally
modified oxidase-negative (no change in
color).

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