MICROBIOLOGY LECTURE 5 - Genus Staphylococcus Notes from Lecture USTMED 07 Sec C - AsM o o o o o o o o o Gram positive, spherical cells, usually

arranged in grapelike irregular clusters Grows readily on many types of media and are active metabolically Ferments carbohydrates and produce pigments that vary from white to deep yellow Some are members of the normal flora of the skin and mucous membranes of humans Others cause suppuration, abscess formation, a variety of pyogenic infection and even fatal septicemia Pathogenic staphylococci hemolyze blood, coagulates plasma and produce a variety of extracellular enzymes and toxins Most common type of food poisoning is caused by heat stable staphylococcal enterotoxin. Rapidly develops resistance to many antimicrobial agents. Three main species of clinical importance 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 Coagulase production most important criterion for the recognition of Staphylococcus sp. o coagulase positive - Staphylococcus aureus

b.

complex nutritional requirements grows well on most routine laboratory media such as nutrient agar and trypticase soy agar sheep blood agar- primary isolation media Metabolism Energy is obtained via both respiratory and fermentative pathways. Exists under conditions of both high and low oxidationreduction potential Catalase is produced aerobically. Wide range of sugars and other carbohydrates are used Mannitol fermentation differentiates o Staphylococcus aureus- ferments mannitol o Staphylococcus epidermidis does not ferment mannitol

ANTIGENIC STRUCTURE OF STAPHYLOCOCCUS AUREUS

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ANTIGENIC STRUCTURE of staphylococcus aureus

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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 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 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

coagulase negative Staphylococcus epidermidis and Staphylococcus saprophyticus

b.

STAPHYLOCOCCUS AUREUS 1. MORPHOLOGY

Microscopic morphology o Gram positive, nonmotile coccus, 0.8 to 1.0 um in diameter in irregular grapelike clusters o Smears from pus singly, pairs, clusters or in short chains o Smears from cultures grown on solid media irregular clusters o Broth cultures short chains and diplococcal forms o Few strains produce a capsule or slime layer b. Colonial morphology o 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 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)

a.

c. -

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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 effects o Chemotactic Antiphagocytic o elicits hypersensitivity reactions o platelet injury Teichoic acids Complex, phosphate containing polysaccharides bound to both peptidoglycan and cytoplasmic membrane Species specific o S. aureus ribitol teichoic acid with N-acety Dglucosamine residues(polysaccharide A) o S. epidermidis glycerol teichoic acid with glucosyl residues(polysaccharide B)

o o

Anticomplementary

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2. PHYSIOLOGY

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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

e.

Mediates attachment of staphylococci to mucosal surfaces through their specific binding to fibronectin Antigenic teichoic antibodies are used to detect systemic staphylococcal disease 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 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 c.

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Required for the invasion of staphylococci into the cutaneous and subcutaneous tissues and the formation of superficial skin infections Hyaluronidase (spreading factor) Hydrolyzes the hyaluronic acid present in the intracellular ground substance of connective tissue--facilitating spread of infection Staphylokinase (fibrinolysin) A proteolytic enzyme with fibrinolytic activity Can dissolve fibrin clots- proenzyme plasminogen is converted to the fibrinolytic enzyme plasmin Nucleases A phosphodiesterase with both endonucleolytic and exonucleolytic properties and can cleave either DNA or RNA

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DETERMINANTS OF PATHOGENICITY a. surface receptors o Polysaccharides o Proteins b. extracellular enzymes o Coagulases o Lipases o Hyaluronidase o Staphylokinase(fibrinolysin) o Nuclease c. toxins 1. Cytolytic toxins

Toxins

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cytolytic toxins a group of toxins which includes Streptolysin O and S Various toxins of Clostridium Hemolysins and leukocidin of S. aureus o Proteins

o
o

Extracellular induce the formation of neutralizing antibodies

2.

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 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 Fibronectin o Fibrinogen o Immunoglobulion G o C1 q also binds to components of the extracellular matrix (laminin, collagen, fibronectin) o Fibronectin a glycoprotein ubiquitous in wounds

ii.

Four distinct hemolysins produced by S. aureus 1) alpha toxin o exhibits a wide range of biologic activities including hemolytic, lethal and dermonecrotic o disrupts lysosomes o cytotoxic for a variety of tissue culture cells o human macrophages and platelets are damaged; monocytes resistant o 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

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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 Delta toxin o A relatively thermostable surface active toxin o Detergent like properties have damaging effects on membrane o Exhibits a high degree of aggregation

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 in human 4)

o
o o o

Laminin major glycoprotein basement membrane

High content of hydrophobic amino acids- when localized, becomes amphipathic and strongly surface active Inhibits water absorption by the ileum Stimulates accumulation of adenosine monophosphate Alters ion permeability in the guinea pig ileum

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

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Extracellular enzymes

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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 lipases lipid hydrolyzing enzymes

Influences human polymorphonuclear leukocyte functions and platelet activating factor metabolism Gamma toxin o has pronounced hemolytic activity o 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 leukocidin

o
o

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Attacks polymorphonuclear leukocytes and macrophages but no other cell type Two protein components(S and F) that act synergistically to induce cytolysis

o
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S and F components are bound preferentially by GM1-ganglioside and phosphatidylcholine Primary step in leukocytolysis activation of phospholipase and an increase in membrane phosphatidylcholine binding sites for the F component Unique response of leukocyte to leukocidin altered permeability to cation

b.

Spread of patients 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 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 host o size and virulence of the infective dose Precipitating causes of staphylococcal disease o third degree burns o traumatic wounds o surgical incisions o decubitus or trophic ulcers o certain viral infections

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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 EEnterotoxin 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 shock o Prostaglandin E and other arachidonic acid cascade metabolites- plays a crucial role Chemotactic factors for neutrophil accumulation

2)

Agents that increase vascular permeability and inflammation

Toxic shock syndrome toxin1 (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

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mediates toxic shock syndrome characterized by fever, hypotension, rash followed by desquamation and multiple organ dysfunction Exfoliative toxin o Mediates staphylococcal scalded syndrome o Produced by bacteriophage group II strain o 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 response o Does not primarily cause cell death o Potent mitogen primarily of T cells o A sphingomyelinase different from Beta toxin

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Clinical manifestations 1) Localized skin infections

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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)

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CLINICAL INFECTIONS

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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 transmission

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Debilitated, hospitalized persons being treated with antimicrobials, steroids, cancer chemotherapy or immuno-suppressants necrosis, with formation of multiple abscessescharacteristic of the infection usually patchy and focal Secondary - Results from staphylococcal bacteremia from a focus elsewhere

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

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Metastatic staphylococcal infections o 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 Toxinoses diseases caused by the action of toxin a) o o o toxic shock syndrome Mediated by toxic shock syndrome toxin-1 A multisystem disease that primarily affects young women who use tampons during menstruation Symptomsfever, marked hypotension, diarrhea, conjunctivitis, myalgias and a scarlatiniform rash followed by fine desquamation Food poisoning(gastroenteritis) Due to the ingestion of food that contains the preformed toxin elaborated by enterotoxin producing strains of S. aureus Foods implicated custard or cream filled bakery products, ham, processed meats, ice cream, cottage cheese, hollandaise sauce and chicken salad Onset 2 to 6 hours after ingestion of food Symptoms: severe cramping abdominal pain nausea, vomiting, and diarrhea, sweating and headache; no fever Recovery within 6 to 8 hours Scalded skin syndrome Mediated by staphylococcal exfoliative toxin Three distinct entities

result from the coalescence of furuncles and extend to the deeper subcutaneous tissue With multiple sinus tracts Associated fever and chills

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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

b) o o

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Deep, localized infections a) 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 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

o o o c) o o

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b)

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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 Bullous impetigo a localized form of SSS Produced by phage type 71 Associated with superficial skin blisters Negative Nikolsky sign

3)

Bacteremia and endocarditis o bacteremia may occur with any localized staphylococcal infection o Primary focus infections of the skin, the respiratory tract or the genitourinary tract o 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 Pneumonia a) primary o most often seen in: Patients with impaired host defense Children with cystic fibrosis or measles Influenza patients

iii.

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

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Laboratory diagnosis 1) Microscopic morphology Gram stain

Gram positive cocci in irregular grapelike clusters

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Culture

Blood agar plate primary isolation media - creamy/buff colored colonies surrounded by a zone of complete hemolysis

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Serologic and typing tests

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b)

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Catalase test - differentiates Staphylococci from streptococci a) staphylococci catalase positive b) streptococci catalase negative o o Add H2O2 to a colony in a slide. Add colony paste on a wooden stick to a drop of H2O2 on a slide. Catalase hydrolyzes H2O2 into oxygen and water.

Antibodies to teichoic acid can be detected in prolonged, deep infections (endocarditis) Phage typing used for epidemiologic tracing of infection only in severe outbreaks of S. aureus infections.

e.

[left panel (-) bubbling; right panel (+) bubbling]

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Coagulase test to distinguish pathogenic staphylococci from nonpathogenic staphylococci o Coagulase positive pathogenic; S. aureus o Coagulase negative nonpathogenic o a) Two forms of coagulase bound coagulase (clumping factor) can directly convert fibrinogen to insoluble fibrin and causes the staphylococci to clump together

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 cephalosporins o Duration of treatment 4 to 6 weeks to prevent later emergence of metastatic abscesses 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

Slide coagulase test- detects bound coagulase -a drop of plasma is added to a

4)

drop of bacterial suspension

[left panel (-) no clumping; right panel (+) with clumping]

f.

[right panel (+) with fibrin clot; right panel (-) no fibrin clot]

free coagulase reacts with a globulin plasma factor(coagulase reacting factor-CRF) to form a thrombinlike factor, staphylothrombin--- 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]

b)

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

6)

Susceptibility testing- broth microdilution or disk diffusion susceptibility testing

[Most commonly acquired strains of S. Aureus are resistant to penicillin.]

1.

Identification o Staphylococcus epidermidis characteristically produce white colonies on blood agar.

[blood agar non-hemolytic and white]

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.

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3.

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 patients skin or nasopharynx or from hospital personnel. Resistant to multiple antibiotics including methicillin and penicillin G 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 o 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. 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 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

-finaudsmartinez@gmail.com ustmedc3@yahoogroups.com

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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.