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.