Microbio Lec 5 - Staphylococcus

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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 7) Serologic and typing tests
- creamy/buff colored colonies surrounded by a zone of complete hemolysis a) Antibodies to teichoic acid can be detected in
prolonged, deep infections (endocarditis)
b) Phage typing – used for epidemiologic tracing of
3) Catalase test - differentiates Staphylococci from infection only in severe outbreaks of S. aureus
streptococci infections.
a) staphylococci – catalase positive
b) streptococci – catalase negative e. Treatment
1) Localized staphylococcal infections
o Add H2O2 to a colony in a slide. Add colony o adequate drainage
paste on a wooden stick to a drop of H2O2 on a o debridement
slide. o antibiotics – may control the spread of the
o Catalase hydrolyzes H2O2 into oxygen and organisms from the abscess but less effective
water. 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
[left panel (-) penicillin G, penicillin V and ampicillin
bubbling; right panel o If sensitivity testing shows staphylococcus to be
(+) bubbling]
sensitive to penicillin, continue treatment with
penicillin because it is more active and less
expensive
2) Cutaneous infections
4) Coagulase test – to distinguish pathogenic o Oral therapy with a semisynthetic penicillin
staphylococci from nonpathogenic staphylococci such as cloxacillin or dicloxacillin; not nafcillin
o Coagulase positive – pathogenic; S. aureus and oxacillin- not well absorbed orally
o Coagulase negative – nonpathogenic o Erythromycin if allergic to penicillin

o Two forms of coagulase 3) Serious systemic staphylococcal disease


a) bound coagulase (clumping factor) – can o parenteral administration of nafcillin or
directly convert fibrinogen to insoluble fibrin oxacillin
and causes the staphylococci to clump together o alternative drugs – vancomycin or cephalos-
porins
 Slide coagulase test- detects bound
o Duration of treatment – 4 to 6 weeks to prevent
coagulase -a drop of plasma is added
later emergence of metastatic abscesses
to
4) Methicillin resistant staphylococci – staphylococci that
a
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 –
drop of bacterial suspension vancomycin alone or in combination with
rifampin
[left panel (-) no clumping; right panel (+)
with clumping] 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
[right panel (+) with fibrin clot; o Segregate persons with staphylococcal lesions
right panel (-) no fibrin clot] from newborn infants and from highly
susceptible adults
b) free coagulase – reacts with a globulin plasma o Avoid indiscriminate use of antibiotics to
factor(coagulase reacting factor-CRF) to form a prevent establishment and spread of resistant
thrombinlike factor, staphylothrombin---à strains.
catalyzes the conversion of fibrinogen to o Perform all surgical procedures and
insoluble fibrin instrumentation observing aseptic techniques.
 Tube coagulase test – detects free o In the newborn infant
coagulase  Proper care of the umbilical stump
• Microorganisms are incubated in plasma for 2 to 4 hours.  Screen personnel in the nursery for
5) Mannitol fermentation- differentiates S. aureus from staphylococcal carriers.
other catalase positive gram-positive cocci o The infection committee should provide
effective surveillance and follow through of
problems encountered.
[left panel (-) pink colonies, no
fermentation; right panel (+)
yellow colonies, mannitol
fermented]
STAPHYLOCOCCUS EPIDERMIDIS

1. Identification
6) Susceptibility testing- broth microdilution or disk 2. Epidemiology
3. Pathogenesis
diffusion susceptibility testing 4. Clinical Infections
5. Treatment
[Most commonly acquired
strains of S. Aureus are
1. Identification
resistant to penicillin.] o Staphylococcus epidermidis characteristically
produce white colonies on blood agar.
[blood agar – non-hemolytic and white] - 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
o It may be distinguished from S. aureus and from - Certain strains are able to suppress growth of other
other coagulase negative staphylococci in bacteria such as Neisseria gonorrheae and S. aureus
biochemical properties. 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.

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