Transcript staph ppt 1x

Staphylococcus
Kenneth L. Muldrew, MD, MPH
Assistant Professor
Department of Pathology
Medical Director
Clinical Microbiology Laboratory
Molecular Diagnostics Laboratory
[email protected]
Office: 383-6444
Staphylococcus
Objectives:
1) Recognize the epidemiology of staphylococcus
2) Describe the pathogenesis, clinical presentation,
diagnosis, & treatment of common staphylococcal
clinical illnesses
3) Describe the basic microbiology of staphylococcus
a) enzymes/toxins
b) cell structure
c) host defenses
d) lab identification
Staphylococci -General
• Two groups: Coagulase negative Staphylococci
(CoNS) & Coagulase positive Staphylococci (CPS)
• Extremely important genus of bacteria that include
the dreaded Staphylococcus aureus (S. aureus) and
the common less virulent Staphylococcus
epidermidis (S. epidermidis)
• Resistance to methicillin is a worsening problem
(MRSA)
Staphylococci - General
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Members of the family Micrococcaceae
Gram - positive cocci that produce catalase
Facultative anaerobes
CoNS: most are non-hemolytic (γ-hemolysis)
CPS: β-hemolysis
Arranged in clusters, look like grapes- due to irregular and
incomplete division of daughter cells (Staphyle is Greek for
“bunch of grapes”)
• Clustering is usually seen when gram-stain is performed from
an agar colony
• can see clusters, single, paired or chained cocci when gramstain is performed from an agar colony
gram stain-colony
gram stain-pus
Staphylococci - General
• Relatively hardy organisms (not fastidious) and
will withstand heat, drying, higher salt
concentrations
• Grow readily under aerobic and anaerobic
conditions
• Produce catalase (an enzyme that converts
H2O2 into H2O and O2) this enzyme
differentiates them from streptococci
Staphylococci - General
• Medium sized bacteria - @ 1 um/cocci
• agar plates- colonies appear smooth, white or
yellow and ± beta hemolysis
• Age of bacteria, phase of growth, culture
media can all change the characteristic
appearance
• Nonmotile, non-spore forming
• Some strains can also be encapsulated
Staphylococci - General
• Approximately 32 species of Staph - 16 are
associated with humans - 4 are important
– S. aureus
– S. epidermidis
– S. saprophyticus
– S. lugdunensis (more virulent than other CoNS;
severe endocarditis)
• Other staphylococcus species in humans
– S. capitis, S. warneri, S. hominis, S. haemolyticus, S.
caprae, S. pasteuri, S. xylosus, S. cohnii, S. simulans,
S. auricularis, S. schleiferi, S. saccharolyticus
Staphylococci - General
• Staph species are subdivided by their ability to
produce coagulase (2 enzymes that bind to
prothrombin which results in the conversion
of fibrinogen to fibrin) into two major groups
– S aureus produce coagulase (Coagulase Positive
Staphylococci or CPS)
– Most other Staphylococci do not (Coagulase
Negative Staphylococci or CoNS)
Staphylococcus aureus
Staphylococcus aureus
• CPS/Major human pathogen
• Name (aureus) comes from appearance
on agar plates (Au- golden grapes)
– yellow coloration comes from
production of carotenoids (class of
pigments that include the precursors
to vitamin A)
– Non-pigmented experimental strains
are easier for neutrophils to kill and
less likely to form abscesses
– colony morphology affected by
many variables
– Beta hemolytic
S. aureus
S. epidermidis
S. aureus: Beta hemolytic
Staphylococcus aureus
• Virulence is controlled by agr (accessory gene
regulator)
– AgrA to AgrD and other regulatory systems
• Positive and negative controls on this system by other
multi-component gene regulatory systems
• Important cell wall components
– peptidoglycan (common to all GP organisms)
– teichoic acid (common to most GP organisms)
– protein A (most strains of S. aureus)
Peptidoglycan
• Complex polymer - thick
layer is found in GP
organisms and gives
Gram’s stain
characteristics
– backbone of alternating
subunits of Nacetylglucosamine and Nacetylmuramic acid
– side chains are linked to
muramic acid residues and
cross-linked by a glycine
bridge
– peptidoglycan confers
structure and stability
– target for B-lactam and
glycopeptide antibiotics
Peptidoglycan
• Backbone is not species specific
• side chains are species specific
• Causes immune response
– IL-1 production from monocytes
– attracts PMNs
– activates complement
– elicits antibody response
– has some endotoxin-like activity
Teichoic acid
• Phosphate containing polymers
• Two types
– one type linked to cell membrane and one type
with ribitol and phosphate linked to the cell wall
• Constitute major surface antigens
• Function in pathogenesis?
Protein A
• 42,000 dalton protein found in most, but not
all, strains of S aureus
• Binds to the Fc terminus of the IgG molecules
(except IgG3)
• ? Superantigen
• Immunologic tool (eg- monoclonal antibody
purification)
Epidemiology
• Colonization with S. aureus begins at birth
– from humans and surrounding fomites
– skin, perineal area, umbilical stump, occasionally
GI tract
• Children and adults colonized in anterior nares
– 30% chronically colonized, 50% intermittently
colonized, 20% never colonized
• 1.5% colonized with MRSA
Epidemiology
• Vaginal carriage of S. aureus occurs in 10% of
women
• Hospital personnel have high rates of
colonization (50%-90%) - reason why unclear
– also, patients with DM, ESRD, IVDA, chronic skin
conditions have increased rates of carriage
Epidemiology
• CA – MRSA – community acquired MRSA
– Strain(s) of S. aureus which carry resistance to
methicillin and often carry the Panton-Valentine
leukocidin (PVL) toxin
• Association of this toxin in CA-MRSA with skin and soft
tissue infections and severe necrotizing pneumonia
with sepsis
• Prospective study in 2004 of patients with staph
infections (infections NOT colonization) found that 59%
were CA-MRSA, 98% of those had PVL
Epidemiology
• Risk factors for CA-MRSA
– Skin trauma (eg, "turf burns",
lacerations or abrasions)
– Lineman or linebacker
position in football
– A higher body mass index
– Cosmetic body shaving
– Physical contact with a person
who has a draining lesion or is
a carrier of MRSA
– Sharing equipment that is
not cleaned or laundered
between users
– Prison residence
– Military personnel
– Prior skin infection
– Previous antibiotic use
– Illicit drug use
– Tattoo recipients
Clinical Case 1
A 30-year-old woman was transferred to the hospital with hypotension and respiratory
failure. She was well until 4 days before admission, when she slipped and fell and at
that time had severe posterior thoracic pain that radiated to the anterior chest and
lower
back which did not respond to acetaminophen. Physical examination was normal
except for tender paraspinal muscles. Radiographs of the chest and thoracic spine
were
normal, and she was given Ketorlac and sent home. The following day, she went to
another hospital because of persistent pain, and was again sent home, this time with
oxycodone for pain.
One month prior to admission, she was treated for a small right leg abscess due to
methicillin resistant Staphylococcus aureus that was susceptible to clindamycin,
vancomycin, tetracycline, and trimethoprim–sulfamethoxazole. On the day of
admission to the hospital, a friend found her at home, unresponsive and moaning.
She was agitated and aphasic, but responded to painful stimuli.
Clinical Case 1
Physical Exam:
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Rectal temperature 39.6°C, bp 113/53 mm Hg, pulse 156
bpm, RR 46 , O2 Sat= 83%
Obtunded, minimally reactive pupils
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Skin gray, warm, and clammy, with papular and pustular
rash on the face, neck, chest, and abdomen
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1 cm healing ulcer on right leg
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rales in both lungs on inspiration and expiration
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heart exam was normal
Clinical Case 1
• Labs:
– Hg: 10.6 g/dl (normal=12-16)
– HCT: 31.3% (Normal=36-46)
– WBC 1,500 cells/uL (normal=4,500-11,000)
• Differential: 14% Neutrophils, 3% bands, 72% lymphs, 2% Eosinophils, 9%
metamyelocytes
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Plt: 59,000 (normal 150,000-450,000)
D-dimer: 6,895 ng/ml (normal <500)
Prothrombin time and APTT elevated
ABG acidotic
Renal failure (creatinine 4.84 mg/dl; normal=0.6-1.5)
Elevated liver enzymes (LDH, AST, ALT) and elevated CK (muscle)
Urine: blood, protein, increased WBC
Clinical Case 1
Intraparenchymal hemorrhage, edema
Clinical Case 1
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treated with: Ceftriaxone, clindamycin,
meropenem,
gentamicin, bactrim, doxycycline
Blood pressure markedly decreased
(73/40 mm Hg), and she
required significant fluids and pressors.
She further deteriorated, went into
shock, disseminated intravascular
coagulation, and died the next day.
Blood cultures turned positive after
death
Catalase positive
Coagulase positive
Pulsed Field Gel Electrophoresis (PFGE)
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Based on digestion of high MW DNA with a rare cutting restriction endonuclease (SmaI)
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Electrophoresis using a system of alternating current angles
Normal Gel ≈ 2 hrs
0.050-25 kilobase-pairs
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PFGE≈ 18 hrs
25-700 kilobase-pairs
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Pathophysiology
• In order for disease to occur, adherence of
organism to host with invasion and
bypassing of defenses must occur
– S. aureus is in anterior nares of adults in
varying degrees and is found on fomites,
occasionally aerosol spread
– several properties of S. aureus increase its
ability to adhere to skin/mucous membranes
after contact
Adherence
• Three types of adherence
– to mucosal cells/nasal epithelial cells
• teichoic acid (phosphate polymer on cell wall) allows
adherence to mucosal cells
– to traumatized or broken skin
• fibrinogen, fibronectin, laminin, thrombospondin,
collagen
– to endothelial surfaces
• fibronectin, laminin
Invasion
– initial steps of invasion of host after colonization
are incompletely understood
– after bacteremia, S. aureus can invade tissues,
binding to thrombin, fibrin, or endothelial cells
• adherent S. aureus is then phagocytized by endothelial
cells, macrophages
– Staph then release proteolytic enzymes
– host response activated with macrophage
activation, TNF, IL-1, IL-6, IL-8 release
– abscess formation
Toxins and Enzymes
• Severity of disease depends on:
– inoculum size, host immunity, and
virulence
• S. aureus secretes enzymes and toxins:
– catalase, coagulase, hyaluronidase, Blactamase, leukocidins, tsst-1 toxin,
fibrinolysin, Panton-Valentine leukocidin
toxin
Toxins and Enzymes
• H2O2
CATALASE
H2O + ½O2
– PMNs use hydrogen peroxide/free oxygen radicals
to kill staph
– enzyme production is also used to differentiate
staph (+) from strep (-)
Catalase Test
Staph
Strep
Toxins and Enzymes
• Coagulase
– 2 enzymes: (97% of all SA isolates have both)
• Bacterial cell-associated (bound coagulase)
• Extracellular or free coagulase
– converts fibrinogen to fibrin
– activation of the clotting cascade occurs with
sepsis
– Fibrin bound to organism prevents
phagocytosis
Tube Coagulase:
-rabbit plasma & organism
-free and bound coagulase
-read at 2 hours
-if negative read at 18 hours
Rapid Test: Staphaurex (Latex agglutination): Read at 2 minutes
• S. aureus bound coagulase binds fibrinogen that is bound to latex beads
• S. aureus Protein A binds Fc receptor on rabbit IgG that is bound to latex beads
• Latex beads and S. aureus clump together
Bound Coagulase
Fibrinogen
• Staphaurex
Protein A
Rabbit IgG
Rapid Test Staphaurex (Latex agglutination): Read at 2 minutes
Positive
Negative
Toxins and Enzymes
• Hemolysins - proteins which lyse red cells and other cell types
– Panton-Valentin toxin encoded by a mobile phage and regulated by
agr system
• Can transfer easily to other strains
• Not associated with osteomyelitis, endocarditis
• Leukocidin - binds phagocytes and creates pores in membrane
• Hyaluronidase - hydrolyzes hyaluronic acid in connective
tissue; May allow spread of staph in tissues
• Fibrinolysin - dissolves fibrin clots
Toxins and Enzymes
• B-lactamases - confer antibiotic resistance
• Exfoliatins - two serine proteases that bind to
desmosomes and cause SSSS (staph scalded
skin syndrome)
• Cytotoxins - alpha, beta, delta, gamma
– cytolytic activity for a variety of cell types such as
smooth muscle cells, sphingomyelin, and
erythrocytes
Toxins and Enzymes
• Superantigens:
• Toxic shock
syndrome toxin
(TSST-1)
• staph enterotoxins
A- E (SEA-SEE)
Host Defects
• Phagocytosis - major defense
– PMNs and Monocytes/macrophages
• Host defects which predispose to S. aureus
– Chemotaxis defects
• Job’s syndrome
• Chediak-Higashi syndrome
Wiscott-Aldrich syndrome
– PMN killing defects
• chronic granulomatous disease:
– no respiratory burst in phagocytosis (x-linked)
– prone to Staph infections
– life-long antibiotics needed