Staphylococci microbiology

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Frances Angelique A. Tequillo, MD Cebu Doctors’ University Staphylococci & Micrococci Monday, June 27, 2011
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Transcript of Staphylococci microbiology

  • Frances Angelique A. Tequillo, MDCebu Doctors University

    Staphylococci& Micrococci

    Monday, June 27, 2011

  • The Staphylococci: An Overview

    gram-positive spherical cells

    arranged in grape-like irregular clusters

    grow readily on many types of media

    active metabolically

    ferment carbohydrates

    produce pigments from white to deep yellow

    Monday, June 27, 2011

  • members of the normal flora of the skin & mucous membranes

    may cause suppuration, abscess formation, and fatal septicemia

    pathogenic types often hemolyze blood, coagulate plasma, & produce extracellular enzymes and toxins

    rapidly develop resistance to antimicrobials

    35 species

    3 most important (clinically):

    1.Staphylococcus aureus2.Staphylococcus epidermidis3.Staphylococcus saprophyticus

    Monday, June 27, 2011

  • 1. Coagulase positive Staphylococci

    S. aureus

    2.Coagulase negative Staphylococci

    S. epidermidis

    S. saprophyticus

    Grouping for Clinical Purposes

    Monday, June 27, 2011

  • Staphylococcus aureus

    major human pathogen

    habitat: part of normal flora in humans and animals

    usual site: skin, nasopharynx & perineum

    can enter underlying tissue when there is a breach in mucosal barriers

    forms characteristic abscesses

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  • Diseases caused by S. aureus

    due to direct effect of organism

    local lesions of skin

    deep abscesses

    systemic infections

    osteomyelitis, septic arthritis, infective endocarditis

    toxin-mediated

    food poisoning

    toxic shock syndrome

    scalded skin syndrome

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  • Staphylococcus epidermidis

    skin commensal

    has predilection for plastic material

    associated with infections of IV lines, prothetic heart valves, AV shunts

    causes urinary tract infection in catheterized patients

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  • Staphylococcus saprophyticus

    skin commensal

    important cause of urinary tract infection in sexually active young women

    usually sensitive to a wide range of antibiotics

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  • & IdentificationMorphology

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  • Typical Organisms

    spherical cells

    about 1 um in diameter

    arranged in irregular clusters

    young cocci stain strongly Gm+

    on aging, many cells become Gm-

    nonmotile, non-sporeforming

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  • grow readily on most media

    under aerobic or microaerophilic conditions

    grow most rapidly at 37C

    form pigment best at room temperature (20-25C)

    form round, smooth, raised, & glistening colonies on solid media

    Culture

    Monday, June 27, 2011

  • S. aureus form gray to deep golden yellow colonies

    S. epidermidis form gray to white colonies

    many colonies develop pigment only upon prolonged incubation

    Peptostreptococcus sp. resemble staphylococci morphologically

    S. aureus

    S. epidermidis

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  • produce catalase (unlike streptococci)

    slowly ferment carbohydrates

    producing lactic acid

    resistant to drying, heat, and 9% NaCl

    readily inhibited by some chemicals (3% hexachlorophene)

    variably sensitive to many antimicrobials

    Growth Characteristics

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  • Resistance:

    1. penicillins

    2.nafcillin, methicillin, & oxacillin

    3.vancomycin

    4.tetracyclines, erythromycins, & aminoglycosides

    Resistance

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  • Variation

    colony size, pigment, & hemolysis

    enzyme elaboration

    drug resistance

    pathogenicity

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  • Antigenic Structures

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  • polysaccharide polymer w/c provides the rigid exoskeleton of the cell wall

    destroyed by strong acid or exposure to lysozyme

    elicits interleukin-1 production to cause fever and opsonic antibodies by monocytes

    serves as chemoattractant for PMN leukocytes

    have endotoxin-like activity

    activates complement

    Peptidoglycan

    Monday, June 27, 2011

  • Teichoic acid

    polymers of glycerol or ribitol phosphate

    linked to the peptidoglycan layer

    can be antigenic

    antiteichoic acid antibodies detected in patients with active endocarditis (S. aureus)

    Monday, June 27, 2011

  • Protein A

    cell wall component of many S. aureus strains

    binds to the Fc portion of IgG molecules, except IgG3

    important reagent in immunology and diagnostic laboratory technology

    protein A with attached IgG directed against a specific bacterial antigen will agglutinate bacteria that have that antigen (coagglutination)

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  • other antigenic structures

    capsules - inhibit phagocytosis by PMN leukocytes unless specific antibodies are present

    coagulase (clumping factor) - binds nonezymatically to fibrinogen, leading to aggregation of bacteria

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  • Enzymes & Toxins

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  • Catalase

    converts H2O2 into H2O and O2

    catalase test differentiates staphylococci (+) from streptococci (-)

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  • Coagulase & Clumping Factor

    enzyme-like protein that clots oxalated or citrated plasma

    binds to thrombin to initiate fibrin polymerization

    deposits fibrin on the surface of bacteria and alters their ingestion by phagocytes

    essential in invasive pathogenic potential

    clumping factor: responsible for adherence of organisms to fibrinogen & fibrin

    S. aureus tends to form clumps when mixed with plasma

    Monday, June 27, 2011

  • Other Enzymes

    hyaluronidase (spreading factor)

    staphylokinase

    proteinases

    lipases

    -lactamase

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  • Exotoxins

    -toxin: potent hemolysin-toxin: degrades sphingomyelin; toxic to many cells, including erythrocytes

    -toxin: disrupts biologic membranes; plays a role in S. aureus diarrheal disease

    -toxin: lyses white blood cells by causing pore formation in the cellular membranes that increase cation permeability

    Monday, June 27, 2011

  • Leukocidin

    S. aureus

    has 2 components

    act synergistically on the white blood cell membrane

    important virulence factor in community associated methicillin resistant S. aureus infections

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  • Exfoliative Toxins

    epidermolytic toxins of S. aureus (superantigens)

    toxin A: heat-stable (resists boiling for 20 mins)

    toxin B: heat-labile

    yield the generalized desquamation of the staphylococcal scalded skin syndrome by dissolving the mucopolysaccharide matrix of the epidermis

    Monday, June 27, 2011

  • Toxic Shock Syndrome Toxin

    TSST-1: produced by most strains of S. aureus isolated from patients with toxic shock syndrome; prototypical superantigen

    binds to MHC class II molecules, stimulates T cells, leading to symptoms of TSS

    fever, shock, multisystem involvement; desquamative rash

    enterotoxin F

    Monday, June 27, 2011

  • Enterotoxin

    (A-E, G-I, K-M); superantigens

    heat-stable and resistant to the action of gut enzymes

    important cause of food poisoning

    produced when S. aureus grows in carbohydrate and protein foods

    ingestion of 25 g of enterotoxin B leads to vomiting and diarrhea

    emetic effect is due to CNS stimulation (vomiting center) after the toxin acts on neural receptors in the gut

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  • Pathogenesis

    Monday, June 27, 2011

  • members of the normal flora of human skin and respiratory & GI tracts

    also found in clothing, bed linens & other fomites in human environments

    pathogenicity is due to combined effect of extracellular factors and toxins together with the invasive properties of the strain

    S. aureus (pathogenic, invasive) produce coagulase and forms a yellow pigment;hemolytic

    S. epidermidis (nonpathogenic, non-invasive) are coagulase negative & non-hemolytic.

    Monday, June 27, 2011

  • Pathology

    Monday, June 27, 2011

  • prototype lesion: furuncle

    S. aureus in hair follicles cause tissue necrosis

    coagulase coagulates fibrin around the lesion & within the lymphatics

    formation of a wall that limits the process, plus presence of inflammatory cells & fibrous tissue

    liquefaction of necrotic tissue occurs at the center of the lesion

    organisms may spread via lymphatics & bloodstream

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  • Clinical Findings

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  • localized infection appears as a pimple, hair follicle infection, or abscess

    intense, localized, painful inflammatory reaction that undergoes central suppuration & heals quickly when the pus is drained

    wall of fibrin & inflammatory cells around the core limits the spread of organisms

    should not be broken down by manipulation or trauma

    Monday, June 27, 2011

  • infection can result from direct contamination of a wound (e.g.) postoperative infections

    secondary localization within or organ or system is accompanied by signs & symptoms of organ dysfunction and intense focal suppuration

    food poisoning - violent nausea, vomiting & diarrhea; rapid convalescence; (-) fever

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  • TSS - abrupt onset of high fever, vomiting, diarrhea, myalgia, scarlatiniform rash (erythematous skin rash which desquamates); hypotension with cardiac & renal failure (severe); can recur

    seen in women who use tampons (5 days ff onset of menses)

    also in men & children with staphylococcal wound infections

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  • Monday, June 27, 2011

  • Staphylococcal Scalded Skin Syndrome

    disease of young children

    mediated through minor staphylococcal infection by epidermolytic toxin-producing strains

    mild erythema and blistering of skin followed by shedding of sheets of epidermis

    children are relatively healthy and most eventually recover

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  • Diagnostic Laboratory Tests

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  • Specimens

    surface swab pus

    blood

    tracheal aspirate

    spinal fluid for culture

    depends on localization of the process

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  • Smears

    gram positive cocci in clusters in Gram-stained smears

    (pus or sputum)

    impossible to distinguish saprophytic from pathogenic staphylococci on smears

    Monday, June 27, 2011

  • Smears

    gram positive cocci in clusters in Gram-stained smears

    (pus or sputum)

    impossible to distinguish saprophytic from pathogenic staphylococci on smears

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  • Culture

    specimen placed on blood agar plates give rise to colonies in 18 hours at 37C

    hemolysis & pigment color takes days (RT)

    S. aureus ferments mannitol

    Monday, June 27, 2011

  • Culture

    specimen placed on blood agar plates give rise to colonies in 18 hours at 37C

    hemolysis & pigment color takes days (RT)

    S. aureus ferments mannitol

    Monday, June 27, 2011

  • Catalase Test

    used to detect presence of cytochrome oxidase enzymes

    a drop of 3% H2O2 is placed on a slide + small amount of bacteria

    presence of bubbles (release of O2) indicates a (+) test

    Monday, June 27, 2011

  • Catalase Test

    used to detect presence of cytochrome oxidase enzymes

    a drop of 3% H2O2 is placed on a slide + small amount of bacteria

    presence of bubbles (release of O2) indicates a (+) test

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  • Coagulase Test

    Citrated rabbit (or human) plasma diluted 1:5 is mixed with broth culture / growth

    incubated at 37C

    clot formation in 1-4 hours is (+)

    indicates pathogenicity in humans

    Monday, June 27, 2011

  • Coagulase Test

    Citrated rabbit (or human) plasma diluted 1:5 is mixed with broth culture / growth

    incubated at 37C

    clot formation in 1-4 hours is (+)

    indicates pathogenicity in humans

    Monday, June 27, 2011

  • Susceptibility Testing

    broth microdilution or disk diffusion

    should be done routinely for isolates of clinically significant lesions

    resistance to penicillin G can be predicted by a (+) test for -lactamase (produced by 90%)resistance to nafcillin (& oxacillin & methicillin) occurs in 35% of S. aureus & 75% of S. epidermidis isolates

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  • Serologic & Typing Tests

    little practical value

    molecular typing techniques used to document spread of epidemic disease-producing clones of S. aureus

    pulsed-field gel electrophoresis & multilocus sequence typing are highly discriminatory

    Monday, June 27, 2011

  • Clues

    in all pus-forming lesions

    Gram stain and culture of pus

    in all systemic infections

    blood culture

    in infections of other tissues

    culture of relevant tissue or exudate

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  • Treatment

    Monday, June 27, 2011

  • tetracyclines - used for long term treatment of multiple skin infections (acne, furunculosis)

    drainage - for abscess & other closed suppurating lesions; + antimicrobial therapy

    Penicillin G - drug of choice for non--lactamase producing S. aureus

    Vancomycin - for nafcillin-resistant staphylococci (eg S. epidermidis infection in patients with prosthetic devices)

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  • Epidemiology & Control

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  • chief sources of infection: shedding human lesionsfomites contaminated from shed lesionsrespiratory tractskin

    cleanliness, hygiene & aseptic management can control spread of lesionsin hospitals: NICU, ICU, OR, and cancer chemotherapy units are at highest risk for severe staphylococcal infectionsrecent development: dissemination of CA-MRSA

    Monday, June 27, 2011

  • So, if we were to answer...

    species coagulase catalase Gram stain -hemolysis

    S. ____ + + ? ?

    S. ____ - - ? ?

    S. ____ - + ?

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  • The Micrococci

    Monday, June 27, 2011

  • comprise the normal flora of the upper layers of the epidermis & hair follicles (w/ S. epidermidis)

    Gram+ spherical cells that appear in tetrads

    cell wall comprises ~50% of cell mass

    rich in guanine & cytosine (GC)

    M. luteus, M. roseus

    produce yellow & pink colonies when grown on mannitol salt agar

    Monday, June 27, 2011

  • thought to be saprophytic or commensal organisms

    may be opportunistic pathogens, esp. in immunocompromised hosts

    pulmonary infections (in immunocompromised), recurrent bacteremia, septic shock, septic arthritis, endocarditis, meningitis

    difficult to identify as cause of infection

    part of normal skin flora

    not usually identified with disease

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  • M. lutea

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  • Monday, June 27, 2011

  • Blessed is the man who, having nothing to say,abstains from giving us wordy evidence of the fact.

    GEORGE ELIOT, Impressions of Theophrastus Such

    Monday, June 27, 2011