MICROBIOLOGY LECTURE 5 - Genus StaphylococcusNotes from LectureUSTMED ’07 Sec C - AsM

o Gram positive, spherical cells, usually arranged in grapelike irregular clusters

o Grows readily on many types of media and are active metabolically

o Ferments carbohydrates and produce pigments that vary from white to deep yellow

o Some are members of the normal flora of the skin and mucous membranes of humans

o Others cause suppuration, abscess formation, a variety of pyogenic infection and even fatal septicemia

o Pathogenic staphylococci hemolyze blood, coagulates plasma and produce a variety of extracellular enzymes and toxins

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 importanceo 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 common cause of urinary tract infection in young women

o Coagulase production – most important criterion for the recognition of Staphylococcus sp.

o coagulase positive - Staphylococcus aureus

o coagulase negative - Staphylococcus epidermidis and Staphylococcus saprophyticus

STAPHYLOCOCCUS AUREUS

1. MORPHOLOGY

a. Microscopic morphologyo Gram positive, nonmotile coccus, 0.8 to 1.0

um in diameter in irregular grapelike clusterso Smears from pus – singly, pairs, clusters or in

short chainso Smears from cultures grown on solid media –

irregular clusterso Broth cultures – short chains and diplococcal

formso Few strains produce a capsule or slime layer

b. Colonial morphologyo Agar plates

o colonies are smooth, opaque, round, low convex, 1 to 4 mm. In diameter

o most strains produce golden yellow colonies on primary isolation due to carotenoid pigments – ranging from deep orange to pale yellow

o Blood agar – zone of β hemolysis surrounds colonies of organisms that produce soluble hemolysins

microscopic morphology (Gram positive cocci in Irregular grapelike clusters); right panel – colonial morphology (Blood agar - Zone of beta hemolysis around the colonies)

2. PHYSIOLOGY

a. cultural characteristics

- facultative anaerobe but growth more abundant under aerobic conditions

- some strains require an increased CO2 tension- 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 - complex nutritional requirements- grows well on most routine laboratory media such

as nutrient agar and trypticase soy agar- sheep blood agar- primary isolation media

b. Metabolism- Energy is obtained via both respiratory and

fermentative pathways.- Exists under conditions of both high and low

oxidation-reduction potential- Catalase is produced aerobically.- Wide range of sugars and other carbohydrates are

used- Mannitol fermentation – differentiates

o Staphylococcus aureus- ferments mannitolo Staphylococcus epidermidis – does not

ferment mannitol

ANTIGENIC STRUCTURE OF STAPHYLOCOCCUS AUREUS

3. ANTIGENIC STRUCTURE of staphylococcus aureus

a. Capsule- A loose fitting polysaccharide layer- Protects the bacteria by inhibiting chemotaxis and

phagocytosis by polymorphonuclear leukocytes and proliferation of mononuclear cells following mitogen exposure

- Facilitates adherence of bacteria to catheters and other synthetic material (graft, prosthetic valves and joints and shunts)

- Interferes with the interaction between the underlying teichoic acid-peptidoglycan complex and complement

b. Peptidoglycan Layer- Elicits the production of interleukin-1 (endogenous

pyrogen) and opsonic antibodies by monocytes- Chemoattractant for polymorphonuclear leukocytes- Has endotoxin like activity- Produces a localized Shwartzman phenomenon- Activates complement- Elicits both humoral and cellular immune responses- Increased antipeptidoglycan IgG level in infections

accompanied by a bacteremic phase c. Protein A- A group specific antigen unique to S. aureus- Consists of a single polypeptide chain - Has five regions:

o four highly homologous domains- Fc-binding

o fifth, C terminal domain – bound to the cell wall and does not bind Fc

- Binds to the Fc portion of the IgG molecules except Ig3

- Provokes a variety of biologic effectso Chemotactico Anticomplementaryo Antiphagocytico elicits hypersensitivity reactionso platelet injury

d. Teichoic acids- Complex, phosphate containing polysaccharides

bound to both peptidoglycan and cytoplasmic membrane

- Species specifico S. aureus – ribitol teichoic acid with N-

acety D-glucosamine residues(polysaccharide A)

o S. epidermidis – glycerol teichoic acid with glucosyl residues(polysaccharide B)

- Mediates attachment of staphylococci to mucosal surfaces through their specific binding to fibronectin

- Antigenic – teichoic antibodies are used to detect systemic staphylococcal disease

e. Clumping factor- Component in the cell wall that results in the

clumping of whole staphylococci in the presence of plasma

- Protein which binds fibrinogen and differs from free coagulase in both its mechanism of action and its antigenic properties

f. Cytoplasmic membrane- A complex of protein, lipids and a small amount of

carbohydrate forming an osmotic barrier for the cell

- Provides an anchor site for the cellular biosynthetic and respiratory enzymes

4. DETERMINANTS OF PATHOGENICITY

a. surface receptorso Polysaccharideso Proteins

b. extracellular enzymeso Coagulaseso Lipaseso Hyaluronidaseo Staphylokinase(fibrinolysin)o Nuclease

c. toxins1. Cytolytic toxins2. Pyrogenic protein toxins

Enterotoxins Exfoliative toxin Toxic shock syndrome toxin -1

a. Surface receptors

i. Polysaccharides- Surface components that possess antiphagocytic

activity are advantageous to the staphylococcus in its initial establishment in the host.

- Encapsulated staphylococci are able to spread rapidly through tissue by protecting the organisms from the complement mediated attack of polymorphonuclear leukocytes.

- adhesion of the organisms to a biosurface-essential initiating event for colonization to occur

ii. Protein receptors - specific binding sites on the staphylococcal cell

surface- provide the organism with an adhesion mechanism

by which infective foci become established - plasma proteins that bind specifically to S. aureus

o Fibronectino Fibrinogeno Immunoglobulion Go C1 q

- also binds to components of the extracellular matrix (laminin, collagen, fibronectin)

o Fibronectin a glycoprotein ubiquitous in

wounds mediates the adherence of vital

cells such as fibroblasts, epithelial cells, and monocytes to an injured site

may serve as a bridge between the organism and the host wound tissue

o Laminin major glycoprotein in human

basement membrane Metastasis like potential of

staphylococci to breach the normal barriers between host tissues may be related to its ability to bind specifically to basement membrane

b. Extracellular enzymes

i. Coagulase- An enzyme which clots plasma- Used as a marker for virulence of S. aureus- May cause the formation of a fibrin layer around a

staphylococcal abscess thus localizing the infection and protecting the organism from phagocytosis

ii. lipases – lipid hydrolyzing enzymes- Required for the invasion of staphylococci into the

cutaneous and subcutaneous tissues and the formation of superficial skin infections

iii. Hyaluronidase (spreading factor)- Hydrolyzes the hyaluronic acid present in the

intracellular ground substance of connective tissue---àfacilitating spread of infection

iv. Staphylokinase (fibrinolysin) - A proteolytic enzyme with fibrinolytic activity- Can dissolve fibrin clots- proenzyme plasminogen is

converted to the fibrinolytic enzyme plasminv. Nucleases- A phosphodiesterase with both endonucleolytic and

exonucleolytic properties and can cleave either DNA or RNA

c. Toxins

i. cytolytic toxins – a group of toxins which includes

- Streptolysin O and S- Various toxins of Clostridium- Hemolysins and leukocidin of S. aureus

o Proteinso Extracellularo induce the formation of neutralizing

antibodies

- Four distinct hemolysins produced by S. aureus1) alpha toxin

o exhibits a wide range of biologic activities including hemolytic, lethal and dermonecrotic

o disrupts lysosomeso cytotoxic for a variety of tissue culture

cellso human macrophages and platelets are

damaged; monocytes resistanto causes injury to the circulatory system,

muscle tissue and tissue to the renal cortex

o contributes to pathogenicity by producing tissue damage after the establishment of a focus of infection

2) beta toxin (sphingomyelinase C)o a heat labile protein that is toxic for a

variety of cells, including erythrocytes, macrophages and fibroblasts

o catalyzes the hydrolysis of membrane phospholipids in susceptible cells

o with alpha toxin – responsible for the tissue destruction and abscess formation characteristic of staphylococcal diseases and the ability of Staphylococcus aureus to proliferate in the presence of a vigorous inflammatory response

3) Delta toxino A relatively thermostable surface active

toxino Detergent like properties – have damaging

effects on membraneo Exhibits a high degree of aggregationo High content of hydrophobic amino acids-

à when localized, becomes amphipathic and strongly surface active

o Inhibits water absorption by the ileumo Stimulates accumulation of adenosine

monophosphateo Alters ion permeability in the guinea pig

ileumo Influences human polymorphonuclear

leukocyte functions and platelet activating factor metabolism

4) Gamma toxino has pronounced hemolytic activityo contains two protein components that act

synergistically both essential for hemolysis and toxicity

o elevated specific neutralizing antibodies in human staphyloccal bone disease – suggestive of its role in the disease state

- leukocidin- Panton-Valentine leukocidino Attacks polymorphonuclear leukocytes

and macrophages but no other cell typeo Two protein components(S and F) that act

synergistically to induce cytolysis

o S and F components are bound preferentially by GM1-ganglioside and phosphatidylcholine

o Primary step in leukocytolysis – activation of phospholipase and an increase in membrane phosphatidylcholine binding sites for the F component

o Unique response of leukocyte to leukocidin –altered permeability to cation

ii. Pyrogenic protein toxins – - all are pyrogenic and immunosuppressive as a

result of their ability to induce nonspecific T lymphocyte mitogenicity and enhance host susceptibility to lethal endotoxin shock

1) Enterotoxins o unique feature – ability to provoke

vomiting and diarrhea in humans after oral ingestion

o Six serological types, A,B,C,C2,D and E- Enterotoxin A – most frequently associated with 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 shocko 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

syndromeo Produced by bacteriophage group II straino Two distinct forms

a) ETA – gene is chromosomal b) ETB – gene is plasmids

o Ultrastructural studies – splitting of the intercellular bridges(desmosomes) in the stratum granulosum

o Does not elicit an inflammatory responseo Does not primarily cause cell death o Potent mitogen primarily of T cellso A sphingomyelinase different from Beta

toxin

5. CLINICAL INFECTIONS

a. epidemiology- Habitat(reservoir)

o normal flora of human anterior nares, nasopharynx, perineal area, and skin

o can colonize various epithelial or mucosal surfaces

- Mode of transmissiono Spread of patient’s endogenous strain to

normally sterile site by traumatic introduction

o Also may be transmitted person to person by fomites, air, or unwashed hands of health care workers.

o May be transmitted from infected lesion of health care worker to patient

b. Pathogenesis- Typical staphylococcal skin infection – organisms

penetrate a sebaceous gland or hair shaft where the environment is suitable for growth

- Likelihood of infection is determined by:o defense mechanisms of the hosto size and virulence of the infective dose

- Precipitating causes of staphylococcal diseaseo third degree burnso traumatic woundso surgical incisionso decubitus or trophic ulcerso certain viral infections

c. Clinical manifestations

1) Localized skin infections

a) Folliculitis Superficial folliculitis – raised,

domed pustules form around hair follicles (left)

Deep folliculitis – micro-organisms invades the deep portion of the follicle and dermis (right)

b) Furuncle or boils – an extension into the subcutaneous tissue resulting in the formation of a focal suppurative lesion

c) Carbuncles result from

the coalescence of furuncles and extend to the deeper subcutaneous tissue

With multiple sinus tracts Associated fever and chills

d) Impetigo A superficial infection affecting

mostly young children Manifested primarily on the face

and limbs Starts initially as a small macule

that develops into a pus filled vesicle on an erythematous base

Crusting when pustules rupture

2) Deep, localized infectionsa) osteomyelitis

Follows hematogenous spread from a primary focus, usually a wound or furuncle

Organisms localize at the diaphysis of long bones

Acute osteomyelitis – fever, chills, pain over the bone and muscle spasm around the area of involvement

Secondary osteomyelitis – associated with a penetrating trauma or surgery and frequent in patients with diabetes mellitus and peripheral vascular disease

b) Pyoarthrosis May occur after orthopedic

surgery in conjunction with osteomyelitis or local skin infections

May result from direct inoculation of staphylococci into the joint during intra-articular injections, especially in patients with rheumatoid arthritis

Destroys the articular cartilage resulting to permanent joint deformity

3) Bacteremia and endocarditiso bacteremia may occur with any localized

staphylococcal infectiono Primary focus – infections of the skin, the

respiratory tract or the genitourinary tracto commonly seen in persons with diabetes

mellitus, cardiovascular disease, granulocyte disorders and immunologic deficiency

o Symptoms – fever, shaking chills, and systemic toxicity

o frequent complication- endocarditis with heart valve destruction

4) Pneumonia a) primary o most often seen in:

Patients with impaired host defense

Children with cystic fibrosis or measles

Influenza patients 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 focalb) Secondary - Results from staphylococcal

bacteremia from a focus elsewhere

5) Metastatic staphylococcal infectionso production of metastatic abscesses –

characteristic feature of staphylococcal bacteremia

o most frequent sites – skin, subcutaneous tissues and the lungs; also kidneys, brain and spinal cord

6) Toxinoses – diseases caused by the action of toxin

a) toxic shock syndromeo Mediated by toxic shock syndrome toxin-1o A multisystem disease that primarily

affects young women who use tampons during menstruation

o Symptoms- fever, marked hypotension, diarrhea, conjunctivitis, myalgias and a scarlatiniform rash followed by fine desquamation

b) Food poisoning(gastroenteritis)o Due to the ingestion of food that contains

the preformed toxin elaborated by enterotoxin producing strains of S. aureus

o Foods implicated – custard or cream filled bakery products, ham, processed meats, ice cream, cottage cheese, hollandaise sauce and chicken salad

o Onset – 2 to 6 hours after ingestion of foodo Symptoms: severe cramping abdominal

pain nausea, vomiting, and diarrhea, sweating and headache; no fever

o Recovery within 6 to 8 hours

c) Scalded skin syndromeo Mediated by staphylococcal exfoliative

toxino Three distinct entities

i. Generalized exfoliative dermatitis (Ritters disease, toxic epidermal necrolysis)

Most severe form Characterized by generalized

painful erythema and dramatic bullous desquamation of large areas of the skin

Positive Nikolsky sign – skin is displaced under slight pressure

ii. Bullous impetigo – a localized form of SSS

Produced by phage type 71 Associated with superficial skin

blisters Negative Nikolsky sign

iii. Staphylococcal scarlet fever – a mild generalized form of the scalded skin syndrome, clinically similar to streptococcal scarlet fever.

d. Laboratory diagnosis

1) Microscopic morphology – Gram stain

Gram positive cocci in irregular grapelike clusters

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 positiveb) 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 staphylococcio Coagulase positive – pathogenic; S. aureuso Coagulase negative – nonpathogenic

o Two forms of coagulasea) 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 acquired strains of S. Aureus are resistant to penicillin.]

7) Serologic and typing testsa) 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. Treatment1) Localized staphylococcal infections

o adequate drainageo debridemento 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 infectionso 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 diseaseo parenteral administration of nafcillin or

oxacillino alternative drugs – vancomycin or

cephalos-porinso 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. Prevention1) Staphylococcal infection will never be controlled

because of the carrier state in humans.2) Home and hospital setting

o proper hygienic careo disposal of contaminated materials

3) Hospital settingo 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. Identification2. Epidemiology3. Pathogenesis4. Clinical Infections5. Treatment

1. Identificationo 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.

2. Epidemiology- Host specific for humans which serve as an:

o endogenous sourceo 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

Glycocalyxo Facilitates adhesion to the smooth

prosthetic surfaces o Protects them from antibiotics and natural

host 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 novobiocino 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.- 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.

-fin-

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