STAPHYLOCOCCI  It is cultivated by adding 7.

5%- 10% NaCl

 Derived from the  Responsible for various skin, wound, and deep tissue
Greek words staphule infections.
means “bunches of
grapes” and kokkos, Related infections and Diseases of S. aureus
which means “berries”. 1. Toxin-induced cases
 These are Gram- a. Scalded skin syndrome (SSS) - it is an extensive
positive cocci that exfoliative dermatitis that occurs primarily in newborn
belong to the family of and previously healthy children.
Staphylococcaceae. b. Toxic shock syndrome (TSS) - rare but potentially
 They are catalase-producing and facultative fatal multisystem disease. It is primarily due to the use
anaerobes except for S. saccharolyticus which is of tampons in women.
obligate anaerobe. 2. Bacteremia and sepsis
 They are non-motile, non-spore forming glucose 3. UTI
fermenters. 4. Acute bacterial endocarditis
 They are normal inhabitants of the skin, mucous 5. Cutaneous infections
membranes and intestines. a. Folliculitis - mild inflammation of the hair follicle or
 It has a spherical cells appear in clusters or sometimes sebaceous gland.
singly under the microscope. b. Furuncles - large, raised, superficial abscesses
 They grow on Blood Agar Plate (BAP). which can be extension of folliculitis.
 They appear creamy, white or light gold or buttery c. Carbuncles - developed from multiple furuncles
looking, other species have gray colonies and some which may advance into the deeper tissues and
species may be B-hemolytic like S. aureus. cause fever and chills leading to systemic infection.
d. Impetigo - it is a superficial cutaneous infection
DIFFERENTIAL TESTS BETWEEN SPATHYLOCOCCI that is commonly seen in children characterized by
AND MICROCOCCI crusty lesions and vesicles surrounded by a red
1. Bacitracin/ Taxo A disk test boarder.
 It uses 0.04 units of bacitracin performed on BAP/MHA e. Purulent abscess
 Result: Micrococci are susceptible and 6. Osteomyelitis
Staphylococci are resistant. 7. Septic arthritis
8. Food poisoning
2. Furazolidone susceptibility test
 It uses 100 ug furazolidone and performed on BAP. Enzymes and toxins produced by Staphylococcus aureus
 Result: Staphylococci are susceptible and 1. Coagulase
micrococci are resistant.  It coagulates fibrinogen in the plasma.
 It promotes the formation of a fibrin layer around
3. Lysostaphin sensitivity test the staphylococcal abscess thereby protecting
 Staphylococci are susceptible because the S. aureus the bacteria from phagocytosis.
is lysed with lysostaphin.  2 types of coagulase:
a. Cell-bound coagulase/Clumping factor - this
4. Modified oxidase/Microdase test is bound to the cell wall and clots human, rabbit,
 The reagent paper used is tetramethyl-p- or pig plasma by directly converting fibrinogen
phenylenediamine in dimethyl sulfoxide. into fibrin.
 (+) Result: Appearance of blue color within two b. Unbound/Free coagulase - it is an
minutes for micrococci. extracellular enzyme that is not bound to the
cell wall and causes clot formation when the
5. Growth on Furoxone-Tween 80- Oil red O agar bacterial cells are incubated with plasma.
 (+) Result is growth of micrococci.
2. Hyaluronidase (spreading-factor enzyme)
 It enhances invasion and survival in the tissue.
6. Acid production from glycerol
 It breaks down the hyaluronic acid that present in
 (+) Reaction for staphylococci.
the connective tissue, resulting in the spread of
bacteria.
7. Oxidase-Fermentation (OF) reaction
 Under anaerobic condition, staphylococci ferment
3. Staphylokinase (Fibrinolysin)
the glucose while micrococci fail to produce acid.
 It causes fibrinolytic activity by dissolving fibrin clots.

Staphylococcus aureus 4. Lipase (fat-splitting enzyme)

 This is the most virulent of all species of staphylococci. It  Produce by both coagulase + and coagulase –
is considered at true coagulase-positive. staphylococci.
 Principal virulence factor is coagulase.  Essential in the survival of bacteria in a sebaceous
 Appearance on BAP: colonies have golden-yellow & areas of the body.
beta hemolytic.

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  Important in the formation in carbuncles, furuncles, DIFFERENTIAL TEST FOR Staphylococcus aureus
and boils. 1. Coagulase test
 It is the best single criterion of pathogenicity of S.
5. Dnase and phosphatase aureus.
 It lowers the viscosity of exudate by giving the  Reagent used is rabbit plasma.
pathogen more mobility.  Anticoagulant used is EDTA.
 It destroys the DNA  2 methods:
a. Slide method
6. ß- lactamase → it is used to screen catalase-positive
 It breaks down penicillin and beta lactam drugs. colonies. It detects cell bound coagulase.
→ (+) Clot or coagulum within 30 seconds.
7. Enterotoxin b. Tube Method
 It acts as neurotoxins that stimulate vomiting → it is considered a sensitive but definitive
through the Vagus nerve. method.
 It is produced by the majority of S.aureus isolates. → it detects extracellular or free coagulase.
 Resistant to hydrolysis by gastric and jejunal → (+) Result is clot formation within 1-4 hrs.
enzymes.
 It is stable to heating at 100°C for 30 mins. 2. Mannitol fermentation test
Contaminated food will not prevent the disease.  It is used to differentiate the pathogenic
 Some examples are: enterotoxins A, B, C1, C2, D, E staphylococci from non-pathogenic staphylococci.
and G to J  Media: MSA
 Enterotoxins A,B,D are responsible for food  Ph indicator: Phenol Red
poisoning. Infected food handlers is the  (+) Result: Yellow-colored S. aureus colonies.
source of contamination. Enterotoxin A is the
most common cause. 3. Tellurite glycine agar
 Enterotoxin B is associated with  This results in jet black colonies of S. aureus.
pseudomembranous enterocolitis and found
in contaminated milk products. 4. Polymyxin sensitivity test
 The S. aureus is resistant to this test.
8. Leukocidin/Panton-Valentine leucocidin (Cytolytic toxin)
 It attacks and kills WBC 5. Voges-Proskauer (VP) test
 It is a pore-forming exotoxin and suppresses  It differentiates S. aureus from S. intermedius.
phagocytosis.  (+) Result: Pink color acetoin (acethylmethyl
 It is responsible for necrotizing skin and soft tissue carbinol)
infections.
 Delta hemolysin. 6. Deoxyribonuclease (Dnase) test
 It is used to identify pathogenic species of
9. Hemolysins (Cytotoxin) staphylococci that produces Dnase.
 It causes anemia and make iron available for  Culture medium used is DNA-methyl green agar.
microbial growth.  (+) Result: clear/colorless zone around the test
 there are 4 types of hemolysin organism
a. Alpha-hemolysin (α) - it is the predominant  S. aureus has positive reaction to this test.
hemolysin that is produced by S. aureus. It
destroys RBC, Platelets, and macrophages. METHICILLIN RESISTENT Staphylococcus aureus (MRSA)
b. Beta-hemolysin (β) - it destroys sphingomyelin  It is a type of S. aureus
and RBC around nerves. strain that is resistant to
c. Gamma-hemolysin (γ) - it causes RBC injury in antibiotics such as
culture and produces edematous lesions. methicillin, nafcillin, and
d. Delta-hemolysin (δ) - it destroys RBC and it is oxacillin.
associated with the PantonValentine leucocidin.  It can acquired through
prolonged stay in hospital
10. Exfoliatin serotypes A and B (Superantigens) (ICU and burn patients)
 It destroys the stratum granulosum.  (+) Chromogenic test:
 It causes SSS ( scalded skin syndrome) Changes in the color of
MRSA colonies within 24-48 hrs using CHROM AGAR
11. Toxic shock syndrome toxin 1 against colorless colonies of nonMRSA.
 It causes almost all cases of menstruation-
associated TSS.

12. Protein A
 An antiphagocytic since it competes with
neutrophils for the Fc portion of specific opsonins.

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  It is an indigenous microbiota of the skin. 2. Culture
 Contaminant of medical instruments, catheters,  BAP, MSA, PEA, CNA, CAP, BHI, Thioglycollate, and
Staphylococcus

CSF shunts and prosthetic heart valves. CHROM agar.

epidermidis

 Culture in BAP colonies appear white, opaque  CNA - for purulent exudates
small-medium sixed pin-heads and are  PEA - enriched wit 5% sheep blood is selective for
nonhemolytic. Gram + bacteria.
 Biochemical test : Coagulase - negative  MSA AND PEA - are used for heavily contaminated
staphylococci; MSA(-) specimens.
 Susceptible to 5ug novobiocin.  CHROM - MRSA
 It is associated with community-acquired UTI in
young, sexually active females. 3. Catalase
Staphylococcus

 It adheres to the epithelial cells that line the  Catalase is a heme enzyme that catalyzes the
saprophyticus

urogenital tract. decomposition of H2O2 to water and oxygen.

 Culture: BAP-colonies appear white, opaque,  H2O2 CATALASE H20 + O2
slightly larger than pin-head and non-hemolytic.  It differentiates staphylococci (catalase +) from
 Urine culture: 10,00- CFU/mL (significant findings) streptococci (catalase -)
 Biochemical test: Coagulase-negative  Reagent: 3% H2O2
staphylococci; (-) MSA  (+) Result: Presence of bubble or effervescence.
 Antimicrobial test: Resistant to 5ug novobiocin  Do not get specimen from BAP because the
 It is CoNS by tube method. presence of peroxidase which creates false
 It can be confused with S. aureus if the slide effervescence.
Staphylococcus

coagulase method is performed. It more  Aerobic catalase test- uses 3% H2O2

lugdunensis

aggressive than other CoNS  Anaerobic catalase test- uses 15% H2O2
 It contains MecA gene that codes for oxacillin
resistance. 4. Coagulase Test
 Some related infections include infective  Reagent: Rabbit plasma
endocarditis, meningitis, septicemia, UTI, and skin,  (+) Result: formation of clot/ coagulum
soft tissue infections.
5. Mannitol fermentation test
NOTE:  (+) Result: Presence of yellow halo around colonies
 Novobiocin-susceptible CoNS species are: of Staphylococci.
 S. epidermidis

 S. capitis 6. Pyrrolidonyl arylamidase (PYR) test

 S. haemolyticus  It detects staphylococci-producing arylamidase
 S. hominis enzyme.
 S. lugdunensis  Reagent: PDAB (p-dimethylaminocinnamaldehyde)
 S. saccharolyticus  (+) Result: cherry red
 S. warneri  (-) Result: No color change on the paper disk
 Novobiocin-resistant CoNS species are:  +PYR: S. lugdunensis, S, schleiferi, S. intermidius
 S. Saprophyticus

 S. Cohnii 7. Voges-Proskauer (VP) test

 S. Kloosi  It differentiates Coagulase-positive staphylococci
 S. xylosus by tube method.
 It detects glucose fermentation through the
Resistant genes produced by Staphylococci formation of acetoin which is the product of 2,3
1. Erythromycin ribosomal methylase (ERM) gene butanediol.
 class od enzyme-inactivating genes.  Culture medium used is VP broth with 0.5% glucose.
 It codes for methylatin of the 23s rRNA which results  Reagents: αlpha naphthol and KOH which is added
to resistance to erythromycin and clindamycin. to VP broth after incubation.
 (+) Result: Deep pink or red acetoin/
2. Methylene sulfoxide reductase (MSR) A gene acetylmethylcarbinol
 It codes for an efflux mechanism which results in  VP (+) : S. aureus, S. lugdunensis, S. haemolyticus
the resistance to erythromycin and susceptible to and S. schleiferi
clindamycin.  VP (-) : S. intermedius

Laboratory diagnosis for Staphylococci 8. β- lactamase test

Specimens: Aspirated secretions( best sample), purulent a) Cephalosporinase
exudates, and joint fluids.  Most useful and widely used β-lactamase test.
 (+) Result: deep pink or red color within 60
1. Gram Stain minutes.
 gram positive spherical cells that appear singly, in
pairs or in clusters.

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 b) Acidimetric method
 The reagent is citrate-buffered penicillin,
 pH indicator: phenol red
 (+) Result: change from red to yellow
c) Iodometric method
 The reagents are phosphate-buffered
penicillin and starch-iodine complex.
 (+) Result: colorless solution
 (-) Result: purple-colored solution

9. Antimicrobial testing

10. Latex agglutination test

 Used to detect the clumping factor and Protein A.
 Useful in detecting altered penicillin-binding
proteins and oxacillinresistant strains.

11. Molecular test - Nucleic acid probes or PCR

amplification
 It is utilized for the detection of MecA gene.
 Gold standard for detecting MRSA

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