mlab 2434: microbiology keri brophy-martinez
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MLAB 2434: MICROBIOLOGYKERI BROPHY-MARTINEZ
Staphylococci
TAXONOMY
Family: MicrococcaceaeGenus:
StaphylococcusCoagulase positiveCoagulase negative
Micrococcus
GENUS STAPHYLOCOCCUS
S. aureus S. epidermidis S. saprophyticus S. haemolyticus
Coagulase positive Coagulase negative
STAPHYLOCOCCUS: GRAM REACTION AND MORPHOLOGY
Gram-positive spherical cells (0.5-1.5 m) in singles, pairs, and clusters
Appear as “bunches of grapes”
Gram-stained smear of staphylococci from colony
Scanning electron micrograph of staphylococci
STAPHYLOCOCCUS: GENERAL CHARACTERISTICS
Nonmotile
Non–spore-forming
Nonencapsulated
Catalase-producing
Oxidase: negative
Glucose fermenters
Primarily aerobic, some facultatively anaerobic
STAPHYLOCOCCUS:GENERAL CHARACTERISTICS (CON’T)
Bacitracin resistant Grow on agar that contains peptone Inhibited by media that has high bile salt
concentration Some are ß-hemolytic Colony morphology: buttery looking, cream or
white colored
STAPHYLOCOCCUS AUREUS
Primary pathogen of the genus Habitat:
Anterior nares (carriers) Colonization: axilla, vagina, pharynx
Produce superficial to systemic infections Skin Bacterial sepsis Hospital acquired infections
STAPHYLOCOCCUS AUREUS
Mode of transmission Traumatic introduction Direct contact with infected person Inanimate objects
Predisposing conditions Chronic infections Indwelling devices Skin injuries Immune response defects
STAPHYLOCOCCUS AUREUS
Infection will elaborate inflammatory response with GPC accumulating as pus Pus: mix of active and inactive neutrophils, bacterial
cells and extravascular fluid
VIRULENCE FACTORS OF S. AUREUS
Enterotoxins Cytolytic toxins Enzymes Protein A
VIRULENCE FACTORS: ENTEROTOXINS
Enterotoxins: Heat-stable exotoxins that cause diarrhea and
vomiting Exotoxin: protein produced by a bacteria and released into
environment Heat stable @ 100o C for 30 minutes
Implications Food poisoning Toxic shock syndrome Pseudomembranous enterocolitis
TYPES OF ENTEROTOXINS
Exfoliatin Epidermolytic toxin
TSST-1: Toxic shock syndrome toxin-1 Multisystem disease Stimulates T cell production & cytokines
Cytolytic Toxins Affects RBCs and WBCs Hemolytic toxins: alpha, beta, gamma, delta Panton-Valentine leukocin, lethal to WBCs
VIRULENCE FACTORS: EXTRACELLULAR ENZYMES
Hyaluronidase: Hydrolyzes hyaluronic acid in connective tissue allowing spread
of infection
Staphylokinase: Fibrinolysin which allows spread of infection
Coagulase: Virulence marker
Lipase: Allows colonization by acting on lipids present on the surface of
the skin.
VIRULENCE FACTORS: EXTRACELLULAR ENZYMES (CON’T) Penicillinase:
Confers resistance
DNase: Degrades DNA
Beta-lactamase: Cuts the beta lactam wall of certain antibiotics
VIRULENCE FACTORS: PROTEIN A
Protein A:Found in cell wallBinds to Fc part of IgGBlocks phagocytosis
STAPHYLOCOCCUS AUREUS: CLINICAL INFECTIONS
Skin and wound Impetigo Furuncles/Boils (Infection
of hair follicles usually in areas that sweat)
Carbuncles (clusters of boils)
Surgical wound infections
Bullous impetigo
STAPHYLOCOCCUS AUREUS: CLINICAL INFECTIONS (CON’T)
Skin and woundScalded skin syndrome= Ritter’s disease
Extensive exfoliative dermatitisYoung children and newborns
Toxic Shock SyndromeMultisystem diseaseCaused by TSST-1Affects women, men, and children
STAPHYLOCOCCUS AUREUS: CLINICAL INFECTIONS
Food poisoningSource is infected food handlerEnterotoxin A the most common causeFoods affected include meat, dairy
products, bakery goods with cream fillings, and salads made with eggs and mayonnaise.
COAGULASE-NEGATIVE STAPHYLOCOCCI
Found as indigenous flora Presence can indicate contamination Seeing an increase due to prosthetic devices,
catheters and immunocompromised Abbreviated CNS or CoNS
COAGULASE-NEGATIVE STAPHYLOCOCCI
Habitat: Skin and mucous membranes
Common human isolatesS. epidermidisS. saprophyticusS. haemolyticus
COAGULASE-NEGATIVE STAPHYLOCOCCI: STAPHYLOCOCCUS EPIDERMIDIS
Predominantly hospital acquired infectionsSkin flora gets introduced by catheters, heart
valves, CSF shuntsProduces a slime layer that helps adherence
to prosthetics and avoidance of phagocytosisUTIs are a common result
COAGULASE-NEGATIVE STAPHYLOCOCCI: STAPHYLOCOCCUS SAPROPHYTICUS
UTIs in young sexually active women Due in part to increased adherence to epithelial cells
lining the urogenital tract Rarely present in other skin areas or mucous
membranes Urine cultures
If present in low amounts, it is still considered significant
COAGULASE-NEGATIVE STAPHYLOCOCCI: STAPHYLOCOCCUS HAEMOLYTICUS
Habitat: skin and mucous membranes
Rarely implicated in infections
Associated with wound infections, bacteremia, and endocarditis
BREAK TIME!!!
LABORATORY DIAGNOSIS: SPECIMEN COLLECTION AND HANDLING
Samples must be taken from the actual site of infection
Prevent delay in transport of collected material from infected sites
Transport in appropriate collection device that would prevent drying and minimize growth of contaminating organisms
LABORATORY DIAGNOSIS: DIRECT SMEAR EXAMINATION
Microscopic Examinationo Gram reaction
o Gram-positive cocci
o Cell arrangement
o Pairs and clusters
o Presence/Absence of PMNs
o Numerous polymorphonuclear cells
(PMNs)
Insert Figure 10-1
LABORATORY DIAGNOSIS: CULTURAL CHARACTERISTICS-
Staphylococcus aureusColony morphology
Smooth, butyrous, white to yellow, creamy
Grow well @ 18-24 hours
S. aureus may produce hemolysis on blood agar
S. aureus
LABORATORY DIAGNOSIS: CULTURAL CHARACTERISTICS
S. epidermidis
Smooth, creamy, white
Small-to medium- sized, usually non-hemolytic
S. saprophyticus
Smooth, creamy, may produce a yellow pigment
IDENTIFICATION TESTS: CATALASE
Principle: tests for enzyme catalase2 H2O2 2 H2O + O2
ProcedureSmear a colony of the organism to a slideDrop H2O2 onto smearObserve
CATALASE TEST: INTERPRETATION
Presence of bubbles PositiveStaphylococci
Absence of bubblesNegativeStreptococci
IDENTIFICATION TEST: SLIDE COAGULASE TEST Differentiates members within the Staphylococci Detects clumping factor found in S. aureus
Procedure Place a drop of sterile water on a slide and emulsify a colony Add a drop of rabbit plasma to the suspension Observe
Agglutination = Positive No agglutination= Negative
IDENTIFICATION TESTS: COAGULASE TEST
•Detects the extracellular enzyme “free coagulase” or staphylocoagulase •Causes a clot to form when bacterial cells are incubated with plasma
•Procedure•Inoculate rabbit plasma with organism and incubate at 35-37 0 C•Observe at 30 minutes for the presence of a clot•Continue for up to 24 hours, if needed
IDENTIFICATION TESTS: RAPID COAGULASE TEST
Latex Agglutination Assays Detects cell-bound “clumping
factor,” protein A or a combination of both
Procedure Varies depending on kit type Positive reaction demonstrated
by agglutination
NOVOBIOCIN SUSCEPTIBILITY TEST
Test to differentiate coagulase-negative staphylococci from S.saprophyticus from urine samples S. saprophyticus is resistant
(top) Other CNS are susceptible
MICROCOCCUS
Rarely produces disease Found in environment and indigenous skin flora Catalase + Coagulase = Produces yellow pigment Microdase disc differentiate between Staph &
Micrococcus
Schematic Diagram for Identifying Staphylococcal Species
ANTIMICROBIAL SUSCEPTIBILITY
For non–beta-lactamase producing S. aureus Use pencillin
Penicillinase-resistant synthetic penicillins (methicillin, nafcillin, oxacillin, dicloxacillin)
Beta-lactamase producers break down the beta-lactam ring of penicillin so it inactivates antibiotic before it acts on bacterial cells
METHICILLIN-RESISTANT STAPHYLOCOCCI MRSA Methicillin-resistant S. epidermidis
MRSE Infection control
Barrier protection Contact isolation Handwashing
Treat with vancomycin Test for susceptibility with cefoxitin disk
METHICILLIN-RESISTANT STAPHYLOCOCCI (CONT’D)
mecA geneEncodes penicillin-binding proteins (PBPs)Causes drug ineffectiveness
Gold standardNucleic acid probe or PCR for the mec A gene
VANCOMYCIN-RESISTANT STAPHYLOCOCCI
VRSA= vancomycin resistant Staphylococcus aureus
VISA= vancomycin intermediate Saphylococcus aureus
Detection Vancomycin screening media
ANTIMICROBIAL SUSCEPTIBILITY
Macrolide Resistance Clindamycin sensitivity often requested by physician to
treat Staph skin infection. Referred to as “D” test Clindamycin resistance is often inducible meaning it
only is detectable when bacteria are also exposed to erythromycin
SUMMARY MICROCOCCACEAE Staph. aureus Staph.
Epidermidis Staph. saprophyticus
Micrococcus
Colony Morphology
Opaque, smooth, raised, entire, white-golden(cream)
Opaque, smooth, raised, entire, gray-white
Opaque, smooth, raised, entire, butyrous, glossy, white-yellow
Opaque, smooth, raised, white, bright yellow
Hemolysis Most are beta hemolytic
Non-hemolytic Non-hemolytic Non-hemolytic
Gram morphology
GPC in clusters, pairs, short chains or singly
GPC in clusters, pairs, short chains or singly
GPC in clusters, pairs, short chains or singly
GPC in pairs and tetrads
Catalase Pos Pos Pos Pos Glucose fermentation
Fermenter Fermenter Fermenter Oxidizer
Modified Oxidase
Neg Neg Neg Pos
Bacitracin susceptibility (Taxo A 0.04U)
Resistant Resistant Resistant Sensitive
Coagulase Production (tube)
Pos Neg Neg N/A
Clumping factor (slide or latex Coagulase test)
Pos Neg Neg Neg
REFERENCES
Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams and Wilkins.
http://archive.microbelibrary.org/ASMOnly/Details.asp?ID=2037 http://brawlinthefamily.keenspot.com/gallery/2009-10-18-breaktime/ http://ericaandkevin.pbworks.com/w/page/5827086/Gram-Stain-and-Other-Tests http://faculty.matcmadison.edu/mljensen/111CourseDocs/111Review/Unit2Reviews/micr
ococcaceae_answers.htm http://jeeves.mmg.uci.edu/immunology/Assays/LatexAgglut.htm Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic
Microbiology (4th ed.). Maryland Heights, MO: Saunders.
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