MICROBIOLOGY LECTURE 5 - Genus Staphylococcus - complex nutritional requirements

Notes from Lecture - grows well on most routine laboratory media such as
USTMED ’07 Sec C - AsM nutrient agar and trypticase soy agar
- sheep blood agar- primary isolation media
o Gram positive, spherical cells, usually arranged in
grapelike irregular clusters b. Metabolism
o Grows readily on many types of media and are active - Energy is obtained via both respiratory and fermentative
metabolically pathways.
o Ferments carbohydrates and produce pigments that vary
from white to deep yellow - Exists under conditions of both high and low oxidation-
o Some are members of the normal flora of the skin and reduction potential
mucous membranes of humans - Catalase is produced aerobically.
o Others cause suppuration, abscess formation, a variety of - Wide range of sugars and other carbohydrates are used
pyogenic infection and even fatal septicemia - Mannitol fermentation – differentiates
o Pathogenic staphylococci hemolyze blood, coagulates o Staphylococcus aureus- ferments mannitol
plasma and produce a variety of extracellular enzymes o Staphylococcus epidermidis – does not ferment
and toxins mannitol
o Most common type of food poisoning is caused by heat
stable staphylococcal enterotoxin.
o Rapidly develops resistance to many antimicrobial
agents.
o Three main species of clinical importance
o Staphylococcus aureus – most significant
pathogen for man; infection ranges from food
poisoning or minor skin infection to severe life
threatening infection
o Staphylococcus epidermidis – normal human
flora but can cause infection often associated
with implanted appliances and devices
especially in very young, old and
immunocompromised patients
o Staphylococcus saprophyticus – relatively ANTIGENIC STRUCTURE OF STAPHYLOCOCCUS AUREUS
common cause of urinary tract infection in
young women 3. ANTIGENIC STRUCTURE of staphylococcus aureus
o Coagulase production – most important criterion for the
recognition of Staphylococcus sp. a. Capsule
o coagulase positive - Staphylococcus aureus - A loose fitting polysaccharide layer
o coagulase negative - Staphylococcus - Protects the bacteria by inhibiting chemotaxis and
epidermidis and Staphylococcus saprophyticus phagocytosis by polymorphonuclear leukocytes and
proliferation of mononuclear cells following mitogen
exposure
STAPHYLOCOCCUS AUREUS - Facilitates adherence of bacteria to catheters and other
synthetic material (graft, prosthetic valves and joints
1. MORPHOLOGY
and shunts)
a. Microscopic morphology - Interferes with the interaction between the underlying
o Gram positive, nonmotile coccus, 0.8 to 1.0 um in teichoic acid-peptidoglycan complex and complement
diameter in irregular grapelike clusters b. Peptidoglycan Layer
o Smears from pus – singly, pairs, clusters or in short - Elicits the production of interleukin-1 (endogenous
chains pyrogen) and opsonic antibodies by monocytes
o Smears from cultures grown on solid media –
irregular clusters - Chemoattractant for polymorphonuclear leukocytes
o Broth cultures – short chains and diplococcal forms - Has endotoxin like activity
o Few strains produce a capsule or slime layer - Produces a localized Shwartzman phenomenon
b. Colonial morphology - Activates complement
o Agar plates - Elicits both humoral and cellular immune responses
o colonies are smooth, opaque, round,
low convex, 1 to 4 mm. In diameter
- Increased antipeptidoglycan IgG level in infections
accompanied by a bacteremic phase
o most strains produce golden yellow c. Protein A
colonies on primary isolation due to - A group specific antigen unique to S. aureus
carotenoid pigments – ranging from - Consists of a single polypeptide chain
deep orange to pale yellow - Has five regions:
o Blood agar – zone of β hemolysis surrounds o four highly homologous domains- Fc-binding
colonies of organisms that produce soluble o fifth, C terminal domain – bound to the cell
hemolysins wall and does not bind Fc
- Binds to the Fc portion of the IgG molecules except Ig3
microscopic morphology - Provokes a variety of biologic effects
(Gram positive cocci in o Chemotactic
Irregular grapelike clusters);
right panel – colonial o Anticomplementary
morphology (Blood agar - Zone
of beta hemolysis around the
o Antiphagocytic
colonies) o elicits hypersensitivity reactions
o platelet injury
d. Teichoic acids
- Complex, phosphate containing polysaccharides bound to
2. PHYSIOLOGY both peptidoglycan and cytoplasmic membrane
- Species specific
a. cultural characteristics o S. aureus – ribitol teichoic acid with N-acety D-
- facultative anaerobe but growth more abundant under glucosamine residues(polysaccharide A)
aerobic conditions o S. epidermidis – glycerol teichoic acid with
- some strains require an increased CO2 tension
glucosyl residues(polysaccharide B)
- wide temperature range 6.5-4 oC;optimum of 30-37 oC
- pH range 4.2 to 9.3; optimum of 7.0 to 7.5
 - Mediates attachment of staphylococci to mucosal - Required for the invasion of staphylococci into the
surfaces through their specific binding to fibronectin cutaneous and subcutaneous tissues and the formation of
- Antigenic – teichoic antibodies are used to detect superficial skin infections
systemic staphylococcal disease iii. Hyaluronidase (spreading factor)
e. Clumping factor
- Component in the cell wall that results in the clumping of
- Hydrolyzes the hyaluronic acid present in the
intracellular ground substance of connective tissue---
whole staphylococci in the presence of plasma
àfacilitating spread of infection
- Protein which binds fibrinogen and differs from free
iv. Staphylokinase (fibrinolysin)
coagulase in both its mechanism of action and its
- A proteolytic enzyme with fibrinolytic activity
antigenic properties
f. Cytoplasmic membrane - Can dissolve fibrin clots- proenzyme plasminogen is
- A complex of protein, lipids and a small amount of converted to the fibrinolytic enzyme plasmin
v. Nucleases
carbohydrate forming an osmotic barrier for the cell
- Provides an anchor site for the cellular biosynthetic and
- A phosphodiesterase with both endonucleolytic and
exonucleolytic properties and can cleave either DNA or
respiratory enzymes
RNA
4. DETERMINANTS OF PATHOGENICITY
c. Toxins
a. surface receptors
o
o
Polysaccharides
Proteins
i. cytolytic toxins – a group of toxins which includes
b. extracellular enzymes - Streptolysin O and S
o Coagulases - Various toxins of Clostridium
o Lipases - Hemolysins and leukocidin of S. aureus
o Hyaluronidase o Proteins
o Staphylokinase(fibrinolysin)
o Nuclease o Extracellular
c. toxins o induce the formation of neutralizing antibodies
1. Cytolytic toxins
2. Pyrogenic protein toxins - Four distinct hemolysins produced by S. aureus
 Enterotoxins 1) alpha toxin
 Exfoliative toxin o exhibits a wide range of biologic activities
 Toxic shock syndrome toxin -1
including hemolytic, lethal and dermonecrotic
o disrupts lysosomes
a. Surface receptors
o cytotoxic for a variety of tissue culture cells
i. Polysaccharides o human macrophages and platelets are
damaged; monocytes resistant
- Surface components that possess antiphagocytic activity o causes injury to the circulatory system, muscle
are advantageous to the staphylococcus in its initial tissue and tissue to the renal cortex
establishment in the host. o contributes to pathogenicity by producing
- Encapsulated staphylococci are able to spread rapidly tissue damage after the establishment of a
through tissue by protecting the organisms from the focus of infection
complement mediated attack of polymorphonuclear 2) beta toxin (sphingomyelinase C)
leukocytes. o a heat labile protein that is toxic for a variety
- adhesion of the organisms to a biosurface-essential of cells, including erythrocytes, macrophages
initiating event for colonization to occur and fibroblasts
ii. Protein receptors o catalyzes the hydrolysis of membrane
phospholipids in susceptible cells
- specific binding sites on the staphylococcal cell surface
o with alpha toxin – responsible for the tissue
- provide the organism with an adhesion mechanism by
destruction and abscess formation
which infective foci become established characteristic of staphylococcal diseases and
- plasma proteins that bind specifically to S. aureus the ability of Staphylococcus aureus to
o Fibronectin proliferate in the presence of a vigorous
o Fibrinogen inflammatory response
o Immunoglobulion G 3) Delta toxin
o C1 q o A relatively thermostable surface active toxin
- also binds to components of the extracellular matrix o Detergent like properties – have damaging
(laminin, collagen, fibronectin) effects on membrane
o Fibronectin o Exhibits a high degree of aggregation
 a glycoprotein ubiquitous in wounds o High content of hydrophobic amino acids-à
 mediates the adherence of vital cells when localized, becomes amphipathic and
such as fibroblasts, epithelial cells, strongly surface active
and monocytes to an injured site o Inhibits water absorption by the ileum
 may serve as a bridge between the o Stimulates accumulation of adenosine
organism and the host wound tissue monophosphate
o Laminin o Alters ion permeability in the guinea pig ileum
 major glycoprotein in human o Influences human polymorphonuclear leukocyte
basement membrane functions and platelet activating factor
metabolism
 Metastasis like potential of
4) Gamma toxin
staphylococci to breach the normal o has pronounced hemolytic activity
barriers between host tissues may be
o contains two protein components that act
related to its ability to bind
synergistically both essential for hemolysis and
specifically to basement membrane
toxicity
b. Extracellular enzymes o elevated specific neutralizing antibodies in
human staphyloccal bone disease – suggestive
of its role in the disease state
i. Coagulase
- An enzyme which clots plasma
- Used as a marker for virulence of S. aureus - leukocidin- Panton-Valentine leukocidin
- May cause the formation of a fibrin layer around a o Attacks polymorphonuclear leukocytes and
staphylococcal abscess thus localizing the infection and
macrophages but no other cell type
protecting the organism from phagocytosis
o Two protein components(S and F) that act
ii. lipases – lipid hydrolyzing enzymes synergistically to induce cytolysis
 o S and F components are bound preferentially by o Spread of patient’s endogenous strain to
GM1-ganglioside and phosphatidylcholine normally sterile site by traumatic introduction
o Primary step in leukocytolysis – activation of o Also may be transmitted person to person by
phospholipase and an increase in membrane fomites, air, or unwashed hands of health care
phosphatidylcholine binding sites for the F workers.
component
o Unique response of leukocyte to leukocidin – o May be transmitted from infected lesion of
altered permeability to cation health care worker to patient
b. Pathogenesis
ii. Pyrogenic protein toxins – - Typical staphylococcal skin infection – organisms
- all are pyrogenic and immunosuppressive as a result of penetrate a sebaceous gland or hair shaft where the
their ability to induce nonspecific T lymphocyte environment is suitable for growth
mitogenicity and enhance host susceptibility to lethal - Likelihood of infection is determined by:
endotoxin shock o defense mechanisms of the host
o size and virulence of the infective dose
1) Enterotoxins - Precipitating causes of staphylococcal disease
o unique feature – ability to provoke vomiting o third degree burns
o traumatic wounds
and diarrhea in humans after oral ingestion
o surgical incisions
o Six serological types, A,B,C,C2,D and E- o decubitus or trophic ulcers
Enterotoxin A – most frequently associated with o certain viral infections
staphylococcal food poisoning
o Emetic receptor sites – abdominal viscera from
which site the sensory stimulus reaches the
vomiting center via the vagus and sympathetic
nerves
o Enterotoxin induced diarrhea – due to inhibition
of water absorption from the lumen of the
intestine and to increased transmucosal fluid
flux into the lumen
o Biologic response modifiers which affect host
immune defense mechanisms – SUPERANTIGENS
o Powerful T cell mitogens whose activity leads
to the activation of T lymphocytes which
requires the involvement of MHC Class II
molecules
o Directly stimulates macrophages to produce
tumor necrosis factor
o Associated with endotoxin induced shock
o Prostaglandin E and other arachidonic acid
cascade metabolites- plays a crucial role
 Chemotactic factors for neutrophil
accumulation
 Agents that increase vascular
permeability and inflammation

2) Toxic shock syndrome toxin–1 (formerly pyrogenic

exotoxin C and enterotoxin F)
a) an exotoxin with pronounced and diverse
immunologic effects
 Induction of interleukin –2
 Receptor expression
 Interleukin synthesis
 Proliferation of human T lymphocytes
 Stimulation of interleukin-l synthesis
• by human monocytes
b) mediates toxic shock syndrome – characterized
by fever, hypotension, rash followed by
desquamation and multiple organ dysfunction
3) Exfoliative toxin
o Mediates staphylococcal scalded syndrome
o Produced by bacteriophage group II strain
o Two distinct forms
a) ETA – gene is chromosomal
c. Clinical manifestations
b) ETB – gene is plasmids
o Ultrastructural studies – splitting of the
1) Localized skin infections
intercellular bridges(desmosomes) in the
stratum granulosum
o Does not elicit an inflammatory response a) Folliculitis
o Does not primarily cause cell death  Superficial folliculitis – raised, domed
o Potent mitogen primarily of T cells pustules form around hair follicles
o A sphingomyelinase different from Beta toxin (left)
5. CLINICAL INFECTIONS  Deep folliculitis – micro-organisms
invades the deep portion of the
a. epidemiology follicle and dermis (right)
- Habitat(reservoir)
o normal flora of human anterior nares,
nasopharynx, perineal area, and skin
o can colonize various epithelial or mucosal
surfaces
- Mode of transmission
 Debilitated, hospitalized persons

being treated with antimicrobials,
steroids, cancer chemotherapy or
immuno-suppressants
o necrosis, with formation of multiple abscesses-
characteristic of the infection
o usually patchy and focal
b) Secondary - Results from staphylococcal
bacteremia from a focus elsewhere

b) Furuncle or boils – an
5) Metastatic staphylococcal infections
extension into the
subcutaneous tissue o production of metastatic abscesses –
resulting in the formation characteristic feature of staphylococcal
of a focal suppurative bacteremia
lesion o most frequent sites – skin, subcutaneous tissues
and the lungs; also kidneys, brain and spinal
c) Carbuncles cord

 result from the coalescence of 6) Toxinoses – diseases caused by the action of toxin
furuncles and extend to the deeper
subcutaneous tissue a) toxic shock syndrome
 With multiple sinus tracts o Mediated by toxic shock syndrome toxin-1
 Associated fever and chills o A multisystem disease that primarily affects
young women who use tampons during
menstruation
o Symptoms- fever, marked hypotension,
diarrhea, conjunctivitis, myalgias and a
d) Impetigo scarlatiniform rash followed by fine
 A superficial infection affecting desquamation
mostly young children
b) Food poisoning(gastroenteritis)
 Manifested primarily on the face and
o Due to the ingestion of food that contains the
limbs
preformed toxin elaborated by enterotoxin
 Starts initially as a small macule that
producing strains of S. aureus
develops into a pus filled vesicle on
o Foods implicated – custard or cream filled
an erythematous base
bakery products, ham, processed meats, ice
 Crusting when pustules rupture
cream, cottage cheese, hollandaise sauce and
chicken salad
o Onset – 2 to 6 hours after ingestion of food
2) Deep, localized infections
o Symptoms: severe cramping abdominal pain
a) osteomyelitis
nausea, vomiting, and diarrhea, sweating and
 Follows hematogenous spread from a
headache; no fever
primary focus, usually a wound or
o Recovery within 6 to 8 hours
furuncle
 Organisms localize at the diaphysis of
c) Scalded skin syndrome
long bones
o Mediated by staphylococcal exfoliative toxin
 Acute osteomyelitis – fever, chills, o Three distinct entities
pain over the bone and muscle spasm
around the area of involvement
i. Generalized exfoliative dermatitis
(Ritters disease, toxic epidermal
 Secondary osteomyelitis – associated
necrolysis)
with a penetrating trauma or surgery
 Most severe form
and frequent in patients with
diabetes mellitus and peripheral  Characterized by generalized painful
vascular disease erythema and dramatic bullous
b) Pyoarthrosis desquamation of large areas of the
skin
 May occur after orthopedic surgery
in conjunction with osteomyelitis or  Positive Nikolsky sign – skin is
local skin infections displaced under slight pressure
ii. Bullous impetigo – a localized form of
 May result from direct inoculation of
SSS
staphylococci into the joint during
intra-articular injections, especially  Produced by phage type 71
in patients with rheumatoid arthritis  Associated with superficial skin
 Destroys the articular cartilage blisters
resulting to permanent joint  Negative Nikolsky sign
deformity

3) Bacteremia and endocarditis

o bacteremia may occur with any localized iii. Staphylococcal scarlet fever – a mild
staphylococcal infection generalized form of the scalded skin
o Primary focus – infections of the skin, the syndrome, clinically similar to
respiratory tract or the genitourinary tract streptococcal scarlet fever.
o commonly seen in persons with diabetes
d. Laboratory diagnosis
mellitus, cardiovascular disease, granulocyte
disorders and immunologic deficiency
1) Microscopic morphology – Gram stain
o Symptoms – fever, shaking chills, and systemic
toxicity Gram positive cocci
o frequent complication- endocarditis with heart in irregular
valve destruction grapelike clusters

4) Pneumonia
a) primary
o most often seen in:
 Patients with impaired host defense
 Children with cystic fibrosis or
measles
 Influenza patients 2) Culture
Blood agar plate – primary isolation media
- creamy/buff colored colonies surrounded by a zone of complete hemolysis
3) Catalase test - differentiates Staphylococci from
streptococci
a) staphylococci – catalase positive
b) streptococci – catalase negative
o Add H2O2 to a colony in a slide. Add colony
paste on a wooden stick to a drop of H2O2 on a
slide.
o Catalase hydrolyzes H2O2 into oxygen and
water.
[left panel (-)
bubbling; right panel
(+) bubbling]
4) Coagulase test – to distinguish pathogenic
staphylococci from nonpathogenic staphylococci
o Coagulase positive – pathogenic; S. aureus
o Coagulase negative – nonpathogenic
o Two forms of coagulase
a) bound coagulase (clumping factor) – can
directly convert fibrinogen to insoluble fibrin
and causes the staphylococci to clump together
 Slide coagulase test- detects bound
coagulase -a drop of plasma is added
to
a
drop of bacterial suspension
[left panel (-) no clumping; right panel (+)
with clumping]
[right panel (+) with fibrin clot;
right panel (-) no fibrin clot]
b) free coagulase – reacts with a globulin plasma
factor(coagulase reacting factor-CRF) to form a
thrombinlike factor, staphylothrombin---à
catalyzes the conversion of fibrinogen to
insoluble fibrin
 Tube coagulase test – detects free
coagulase
• Microorganisms are incubated in plasma for 2 to 4 hours.
5) Mannitol fermentation- differentiates S. aureus from
other catalase positive gram-positive cocci
[left panel (-) pink colonies, no
fermentation; right panel (+)
yellow colonies, mannitol
fermented]
6) Susceptibility testing- broth microdilution or disk
diffusion susceptibility testing
[Most commonly
strains of S. Aureus are
resistant to penicillin.]
7) Serologic and typing tests
a) Antibodies to teichoic acid can be detected in
prolonged, deep infections (endocarditis)
b) Phage typing – used for epidemiologic tracing of
infection only in severe outbreaks of S. aureus
infections.
e. Treatment
1) Localized staphylococcal infections
o adequate drainage
o debridement
o antibiotics – may control the spread of the
organisms from the abscess but less effective
on bacteria within the abscess and do not
facilitate its resolution
o initial drug of choice – penicillinase-resistant
drugs since most isolates are resistant to
penicillin G, penicillin V and ampicillin
o If sensitivity testing shows staphylococcus to be
sensitive to penicillin, continue treatment with
penicillin because it is more active and less
expensive
2) Cutaneous infections
o Oral therapy with a semisynthetic penicillin
such as cloxacillin or dicloxacillin; not nafcillin
and oxacillin- not well absorbed orally
o Erythromycin if allergic to penicillin
3) Serious systemic staphylococcal disease
o parenteral administration of nafcillin or
oxacillin
o alternative drugs – vancomycin or cephalos-
porins
o Duration of treatment – 4 to 6 weeks to prevent
later emergence of metastatic abscesses
4) Methicillin resistant staphylococci – staphylococci that
are resistant to the B lactam antibiotics
o methicillin – drug used in testing the resistance
of these organisms
o If resistant to methicillin, also resistant to
nafcillin , oxacillin and all B lactam
antibiotics; also to gentamicin, tobramycin and
clindamycin
o Recommended treatment for MRSA –
vancomycin alone or in combination with
rifampin
f. Prevention
1) Staphylococcal infection will never be controlled because
of the carrier state in humans.
2) Home and hospital setting
o proper hygienic care
o disposal of contaminated materials
3) Hospital setting
o Segregate persons with staphylococcal lesions
from newborn infants and from highly
susceptible adults
o Avoid indiscriminate use of antibiotics to
prevent establishment and spread of resistant
strains.
o Perform all surgical procedures and
instrumentation observing aseptic techniques.
o In the newborn infant
 Proper care of the umbilical stump
 Screen personnel in the nursery for
staphylococcal carriers.
o The infection committee should provide
effective surveillance and follow through of
problems encountered.
STAPHYLOCOCCUS EPIDERMIDIS
1. Identification
2. Epidemiology
3. Pathogenesis
4. Clinical Infections
5. Treatment
acquired
1. Identification
o Staphylococcus epidermidis characteristically
produce white colonies on blood agar.
[blood agar – non-hemolytic and white]
o It may be distinguished from S. aureus and from
other coagulase negative staphylococci in
biochemical properties.
- Common cause of urinary tract infections in sexually
active young women second to Escherichia coli - upper
urinary tract is involved
- Shows tropism for the epithelial lining of the urinary
tract
- Selectively adheres to urothelial cells via specific
oligosaccharide receptors on the cell membrane
- Certain strains are able to suppress growth of other
bacteria such as Neisseria gonorrheae and S. aureus
attributed to an extracellular enzyme complex.

2. Epidemiology
- Host specific for humans which serve as an:
o endogenous source
o exogenous source of contamination for
infection to others
- Most frequent sites – axillae, head, arms, nares and legs
- All infections are hospital acquired and result from
contamination of a surgical site by organisms from the
patient’s skin or nasopharynx or from hospital personnel.
- Resistant to multiple antibiotics including methicillin and
penicillin G

3. Pathogenesis
- In the normal host, S. epidermidis is an organism with
low virulence, but when host defenses are breached, it
may cause serious often life threatening infections.
- has a distinct predilection for foreign bodies like artificial
heart valves, indwelling intravascular cathethers, central
nervous system shunts and hip prostheses
- initiating step for infection - adhesion of organisms to
the surface of the prosthetic device
- some produce a viscous extracellular substance that
facilitates colonization on smooth surfaces
• Glycocalyx -fin-
o Facilitates adhesion to the smooth prosthetic
surfaces audsmartinez@gmail.com
o Protects them from antibiotics and natural host ustmedc3@yahoogroups.com
defenses
- Adherence of S. epidermidis causes erosive changes in
the inert surface of polyethylene catheters.

4. Clinical Infection
- single most common isolate from infections associated
with cardiac valve or total hip replacement and central
nervous system shunt insertion
- Causes infections of pacemakers, vascular grafts and
prosthetic joints and also peritonitis in patients
undergoing peritoneal dialysis
- Single most common organism infecting intravenous
catheters
- Bacteremia
- Urinary tract infections especially in elderly hospitalized
men
- Natural valve endocarditis in intravenous drug abusers
- Produce toxins involved in Toxic shock syndrome

5. Treatment
- multiple antibiotic resistance, including methicillin
- Choice of appropriate therapy – based on the local
antibiogram
- Initial regimen – if no antibiogram
o aminglycoside(gentamicin or tobramycin) with
cephalothin
o rifampin or vancomycin alone

STAPHYLOCOCCUS SAPROPHYTICUS

- This coagulase negative staphylococci can be

distinguished from S. epidermidis by its:
o resistance to novobiocin
o failure to ferment glucose anaerobically
- It is nonhemolytic and does not contain Protein A.
- Most strains have the ability to agglutinate sheep
erythrocytes.
- Occurs on the normal skin and in the periurethral and
urethral flora.